release of platelet activation factor from activated neutrophils

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 268, No. 5, Issue of February 15, pp. 336443373,1993 Printed in U. S A .

Release of Platelet Activation Factor from Activated Neutrophils TRANSGLUTAMINASE-DEPENDENT ENHANCEMENT OF TRANSBILAYER MOVEMENT ACROSS THE PLASMA MEMBRANE*

(Received for publication, July 6, 1992)

Donna L. Bratton$ From the National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206

Extracellular release of platelet activating factor (PAF) following synthesis in inflammatory cells is variable and modulated by a number of as yet unde- fined cellular mechanisms. Using human neutrophils loaded with the tritiated, nonmetabolizable PAF analog, l-O-alkyl-2-N-methylcarbamyl-sn-glycero-3- phosphocholine (C-PAF), extracellular release was studied by techniques involving an albumin extraction method. Further modeling of plasma membrane events in the neutrophil was accomplished using movement across the membrane of erythrocyte ghosts. The data demonstrate that C-PAF release is dependent on cellular activation and is accompanied by alterations in the physical properties of the plasma membrane as measured by enhancement of merocyanine 540 (MC540) staining, as well as by bulk, nonspecific transbilayer movement of endogenous phospholipids as detected by the procoagulant activity of externalized phosphatidylserine (a phospholipid class usually sequestered in the plasma membrane inner leaflet). The finding that competitive inhibitors of transglutaminase significantly inhibited C-PAF release, enhancement of MC540 staining, and externalization of phosphatidylserine, strongly suggest a role for this enzyme in the enhancement of phospholipid transbilayer movement. Furthermore, the data suggest that recycling of C-PAF in the plasma membrane is likewise transglutaminase dependent and limits the net extracellular release of C-PAF which, like liberation of endogenously produced PAF, is dependent on extracellular “acceptors” and shown to be albumin concentration- and cell density-dependent.

Platelet activating factor (l-O-alkyl-2-acetyl-sn-glycero-3- phosphocholine, PAF)’ is synthesized by numerous inflammatory cells and is an extremely potent mediator of a wide

* This work was supported in part by National Institutes of Health Grant HL34303. The costs of publicat,ion of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should he addressed National Jewish Center for Immunology and Respiratory Medicine, 1400 Jackson St., Denver, CO 80206. Tel.: 303-398-1390; Fax: 303-398-1225.

The abbreviations used are: PAF, 1-0-alkyl-2-acetyl-sn-glycero- 3-phosphocholine (platelet activating factor); C-PAF, l-O-alkyl-2-N- methylcarbamyl-sn-glycero-3-phosphocholine; MC540, merocyanine 540; HSA, human serum albumin; pTG, N-p-tosylglycine; RVV, Russell’s viper venom; PMA, phorbol 12-myristate 13-acetate; MDC, monodansylcadaverine; LPS, lipopolysaccharide; KRPD, Krebs- Ringer phosphate buffer; RBC ghosts, erythrocyte ghosts; W L P , N - formyl-L-methionyl-L-leucyl-L-phenylalanine; PS, phosphatidylserine.

variety of proinflammatory activities. However, only a small proportion of the mediator is actually released extracellularly from most synthesizing cells (Bratton, 1992) and the mechanism of release is poorly understood. The amount of PAF that is released from synthesizing inflammatory cells may be a function of cell type (Elstad et al., 1988) and dependent on agonist (Ludwig et al., 1986; Lynch and Henson, 1986; Elstad et al., 1988), priming (Stewart et al., 1991), “phase of response” (Valone and Epstein, 1988), and pH of the medium (Leyra- vaud and Benveniste, 1989). The more recent demonstration that release is cell density dependent (Leyravaud and Ben- veniste, 1989), markedly enhanced by “dynamic removal” of albumin-containing extracellular media (Cluzel et al., 1989), and saponin treatment (Vallari et al. 1990) suggests that the mediator, under certain conditions, is likely expressed in the outer leaflet of the plasma membrane and may be released into the medium in the presence of appropriate carrier molecules. While the amounts of PAF released from inflammatory cells would preclude micellar release (PAF micellar concentration -1.1 pM, (Kramp et al., 1984; Ludwig et al., 1986), the high affinity of albumin (Ludwig et al., 1985; Clay et al., 1990), and possibly other carrier proteins (Matsumoto and Miwa, 1985) for the mediator makes it likely that under certain conditions, if present in the outer plasma membrane leaflet, removal would be a consequence.

The development of the radiolabeled nonmetabolizable analog, carbamyl PAF (l-0-alkyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine, C-PAF), allows tracking of the molecule intra- and extracellularly without the rapid metabolism that vastly complicates the localization of authentic PAF (O’Flaherty et al., 1986; Vallari et al., 1990). Differing from PAF only in the nonmetabolizable sn-2 methylcarbamyl group, replacing the acetyl group of PAF, C-PAF is recognized by the PAF receptor, although with somewhat less potency (Tessner et al., 1989; O’Flaherty et al., 1987), and behaves identically at the level of the plasma membrane to authentic PAF (Bratton et al., 1991, 1992). Utilizing these properties, neutrophils loaded with C-PAF were used to track release of the mediator to the extracellular milieu.

Since recent studies from this laboratory have shown that PAF uptake into stimulated neutrophils (Bratton et al., 1992) and calcium-treated erythrocyte ghosts (Bratton et al., 1991) is accompanied by nonspecific, bulk transbilayer movement of plasma membrane phospholipids, we asked whether, at the level of the plasma membrane, PAF release from inflammatory cells could also be governed similarly, i.e. that in effect movement is bidirectional. Accordingly, human neutrophils loaded with C-PAF were variously stimulated to release the material extracellularly. Our data strongly support the concept that release of the mediator into the extracellular milieu is accompanied by marked alteration in the physical properties of the plasma membrane and transbilayer movement of

3364

Transglutaminase-dependent Release of PAF from Neutrophils 3365

endogenous phospholipids across the neutrophil plasma membrane. Furthermore, a role for cellular transglutaminase activity is suggested by the finding that competitive inhibitors of this enzyme inhibit both the extracellular release and plasma membrane recycling of C-PAF, as well as the alterations in the plasma membrane distribution of phospholipids. Taken together, the data suggest a multistep model for the release of PAF that may explain much of the variability in release of the mediator from synthesizing inflammatory cells that has been documented to date (Bratton, 1992).

EXPERIMENTAL PROCEDURES

Materials-C-PAF, PAF, N’,N”dimethylcasein, and ionomycin (calcium salt) from Streptomyces conglobatus were obtained from Calbiochem (San Diego, CA). Radiolabeled C-PAF, l-O-(alkyl-l’,2’- 3H(N), specific activity 54.6 Ci/mmol and PAF, l-O-[hexadecyl-1’,2’- ( ~ x ) - ~ H ] , specific activity 56.7 Ci/mmol, were obtained from Du Pont- New England Nuclear. (3H]Putrescine, specific activity 30 Ci/mmol, was obtained from Amersham Corp. The PAF receptor antagonist, WEB 2086, was generously supplied by Boehringer Ingelheim (Water- ford, NY). “LPS-free” HSA was obtained from Biocell Labs (Carson, CA) and determined by the limulus assay, QCL-1000 (Whittaker Bioproducts, Inc., Walkersville, MD) to have an LPS concentration of 4.8 pg/mg HSA. Sarcosine methyl ester was obtained from Bachem (Philadelphia, PA), merocyanine 540 (MC540) from Fluka (Ronkon- koma, NY), and trypan blue (0.4%) from GIBCO. Trichloroacetic acid was obtained from Fisher. N-p-Tosylglycine, cystamine, methylamine, lyophilized Russell’s viper venom (RVV) from Vipera rus- sellz, the formylated peptide, fMLP, cytochalasin D, A23187, phorbol myristate acetate (PMA), putrescine, phenylmethylsulfonyl fluoride, dithiothreitol, aprotinin, leupeptin, monodansylcadaverine, and Me2S0 were all obtained from Sigma.

Plasticware and reagents used in neutrophil studies were tested for the presence of LPS with the Limulus Amebocyte Lysate Kit (Asso- ciates of Cape Cod, Woods Hole, MA) which detects LPS concentrations of 0.01 ng/ml. No LPS was detected on sterile plastic or in reagents at the concentrations used.

Buffers-Neutrophils were suspended in KRPD, pH 7.35, containing 0.2 M NaCI, 4.8 mM KCl, 0.93 mM CaC12, 1.2 mM MgSO,, 3.1 mM NaH2 PO,, 12.5 mM Na2HP04, 0.2% dextrose to which designated amounts of LPS-free human serum albumin were added. For experiments using monodansylcadaverine, the amine (100 mM) was first dissolved in buffer containing 0.1 N HCI, then diluted to the specified concentration, and the pH adjusted if necessary before addition to cell samples and cell lysates. N-p-Tosylglycine was made up with 0.1% ethanol (final concentration) and run in duplicate with ethanol containing control buffer.

Phosphate-buffered saline (PBS) was made by the method of Williamson et al. (1985) with 137 mM NaCI, 2.7 mM KCl, 10.6 mM Na2HP04, 8.5 mM KH2P0,, pH. 7.4, with additions of 1 mM MgC12 or 1 mM CaC12 where designated.

Preparation of Human Neutrophils-Human neutrophils were isolated by an LPS-free plasma-Percoll method described previously (Haslett et aL, 1985) to a purity of no less than 95%. Neutrophils were 99% viable based on trypan blue exclusion. To assess whether neutrophils became “leaky” under any of the experimental conditions, lactate dehydrogenase release was measured by a standard assay (Wroblewski and La Due, 1955) and found to be between 3-5% regardless of conditions, amine concentration, stimulus, and C-PAF release.

Erythrocyte Ghost Preparation-Erythrocyte (RBC) ghosts were prepared as previously described (Bratton et al., 1991). Briefly, eryth- rocytes were hypotonically lysed at 4 “C a t a concentration of 1:5 cells to 1/50 dilution of PBS with MgC12 (1 mM). After incubating for 60 min, isotonicity was restored with 5-fold concentrated PBS containing MgC12 (1 mM), and ghosts were resealed by warming to 37 “C for 1 h. Such ghosts have been shown to retain native plasma membrane phospholipid asymmetry (Williamson et al., 1985; Bratton e t al., 1991).

Release of C-PAF from Neutrophils and RBC Ghosts-For C-PAF release experiments, C-PAF, both radiolabeled (5% of total lipid) and nonlabeled, with a final specific radioactivity of 2.73 Ci/mmol, were dried down in a glass tube under a stream of nitrogen and then resuspended in KRPD with 0.25% HSA by vigorous vortexing over 30 min. Neutrophils (lo7 cells/ml) were incubated in KRPD with

0.25% HSA and C-PAF at 7.5 x lo-* M (final concentrations) for 45 min at 37 “C in a gently shaking water bath. At the end of the incubation period, the neutrophils were centrifuged at 200 X g for 5 rnin at 4 “C, resuspended in KRPD with 2% HSA and washed twice to remove all loosely adherent and outer leaflet C-PAF. Preliminary studies have shown that two washes are sufficient to remove such material. As in previous studies from this laboratory (Bratton et al., 1991, 1992) and the work of Homma et al. (1987) and Tokumura et al. (1990), we assumed that washing of neutrophils in a concentrated albumin solution extracts C-PAF from the plasma membrane outer leaflet, whereas C-PAF remaining after such extraction is assumed to be internalized to the inner leaflet and intracellular sites. Neutro- phil samples were taken for scintillation counting to determine total amount of lipid internalized during the loading period. AS to whether the amount of C-PAF concentrated in the plasma membranes of loaded neutrophils altered membrane structure, the following calcu- lations from the data, with the stated assumptions, demonstrate that this was unlikely. If 30 k 8 (S.D.)% of the available C-PAF was taken up equally by lo7 neutrophils, a mean (k S.D. of 1.19 X lo6 (+ 0.59 X IO6) molecules were present per cell. This would comprise approx- imately 0.020% of the phospholipid of the entire cell (assuming 6 X lo9 phospholipid molecules/cell (Karnovsky and Wallach, 1961)). Assuming between 16 and 30% of total cellular phospholipid was located in the plasma membrane (Riches et al., 1990), the maximum percent C-PAF achieved in the plasma membrane, assuming all the C-PAF went there (highly unlikely, and not supported by autoradi- ography), would have been 0.07-0.12 mol %. Early studies by our group and others have suggested that a minimum of 1.5 mol % (and in several studies, up to 10 mol %) was required to detect any physical alterations in bilayer structure by fluorescence polarization and dif- ferential scanning calorimetry (Bratton et al., 1988).

To measure stimulated release of C-PAF from loaded neutrophils, the cells were then resuspended in KRPD with 2% HSA at lo7 cells/ ml (unless otherwise designated) in 1.5-ml microfuge vials and stimulated for 15 min with or without the various agonists. At the end of the stimulation period, the cells were rapidly pelleted by centrifuga- tion at 13,000 X g for 30 s and 500 p l of the supernatant removed for scintillation counting to determine the amount of C-PAF released to the extracellular medium. As C-PAF release appeared to be a consistent function when expressed as a percent of C-PAF concentration loaded initially into the neutrophils, release data are expressed as percent of material internalized at the start of the stimulation period. Inhibition of release is expressed as the percent of C-PAF released in the presence of transglutaminase inhibitors compared to release from identically stimulated neutrophils in the absence of inhibitors (designated as 100% of stimulated release).

Release of C-PAF to albumin-containing medium following transbilayer movement from the inner to outer plasma membrane leaflet of RBC ghosts was measured as previously described (Bratton et al., 1991). RBC ghosts were prepared as above, loaded with C-PAF M in 1/50 PBS with 0.025% HSA for 60 min), resealed in 5-fold concentrated PBS at 37 “C for 30 min, and washed free of outer leaflet PAF or C-PAF in PBS with 1% HSA. This procedure was found to remove 54.7 + 2.6% (mean k S.E.) of the total loaded labeled material and was assumed to remove outer leaflet lipid (Schneider et al., 1986; Bratton et al., 1991). Ghost samples were then resuspended in PBS a t 37 “C, treated with ionomycin (10 p ~ ) either in the presence of MgCL (1 mM) or CaC12 (amount designated), with or without cystamine or sarcosine methyl ester for designated periods of time. Although in both release and uptake (see below) experiments some calcium effect is seen in the absence of calcium ionophore, likely due to calcium penetration into some ghosts, enhancement of both release and uptake of C-PAF into ghosts was highly significant and highly reproducible in the presence of calcium combined with ionophore treatment. Such combined treatment resulted in a single, brightly positive MC540 population that appeared to have lost its native plasma membrane phospholipid asymmetry, while calcium treatment alone resulted in two MC540 staining populations of ghosts, one that stained brightly and had likely lost native phospholipid asymmetry, and a second population that did not stain brightly and likely retained its native plasma membrane phospholipid asymmetry (Bratton et al., 1991). At the end of the incubation period, duplicate samples were pelleted (1500 X g for 5 min) to determine the amount of ghost- associated PAF or C-PAF (located in both inner and outer leaflet) and washed twice in 4 ml of PBS with 1% HSA to determine the amount of C-PAF extractable to the extracellular medium as opposed to that located in the inner leaflet, and not extractable with albumin. Preliminary experiments demonstrated that the presence or absence

3366 Transglutaminase-dependent Release of PAF from Neutrophils

of either the transglutaminase inhibitors or cations (either magnesium or calcium, 1 mM) in the albumin-containing ''release medium" did not affect release of C-PAF from calcium-treated ghosts. These experiments effectively ruled out potential alterations in C-PAF binding to albumin due to the presence of either the transglutaminase inhibitors or cations. Scintillation counting was performed as previously described after cell lysis with distilled water and bleaching of samples with 30% hydrogen peroxide (Bratton et al., 1991). Inhibition of release is expressed as the percent of C-PAF released in the presence of transglutaminase inhibitors compared to release from identically treated RBC ghosts in the absence of inhibitors (designated as 100% of stimulated release).

Uptake of C-PAF in Neutrophils and RBC Ghosts-Uptake of C- PAF into the plasma membrane inner leaflet and internal sites within neutrophils was performed as previously described (Bratton et al., 1992). C-PAF, labeled (1%) and nonlabeled, with a final specific activity of 546 mCi/mmol, was prepared as for release experiments. Neutrophils resuspended a t 37 "C for 5 min in KRPD (lo6 cells/ml) with or without the amines (at designated concentrations) were stimulated with the various agonists (at designated concentrations) in the presence of C-PAF (7.5 X lo-' M and 0.25% HSA). After 15 min, samples were centrifuged (200 X g for 5 min), the supernatant removed, and the pellet resuspended in 1 ml of KRPD. Half of the sample (500 pl) was removed for scintillation counting to determine total C-PAF hound (absorbed/adherent plus intracellular) whereas the other half was washed in 1 ml of KRPD with 2% HSA to remove absorbed or loosely adherent lipid leaving only internalized lipid. Preliminary experiments demonstrated that washing cells in this manner resulted in optimal removal of C-PAF by albumin (Bratton et al., 1992). The washed cell pellet was then resuspended for scintillation counting to determine lipid uptake. Additional experiments performed using fatty acid and phospholipid-free HSA prepared by the method of Cham and Knowles (1976) revealed no differences in either uptake of C-PAF or efficiency of removal. Inhibition of uptake is expressed as the percent of C-PAF taken up in the presence of amines compared to neutrophils identically stimulated in the absence of amines (designated as 100% of stimulated uptake).

Transbilayer movement of C-PAF or PAF from outer to inner leaflet of RBC ghosts was measured as previously described (Bratton et al., 1991). Radiolabeled and nonradiolabeled C-PAF were dried down as above and resuspended by vigorous vortexing over 30 min. RBC ghosts were incubated with the C-PAF (lo" M) and ionomycin (10 p ~ ) in phosphate-buffered saline with 0.025% HSA at 37 "C a t 4 X 10' ghosts/ml in a shaking water bath. It should be noted that initial outer leaflet association of C-PAF with ghosts was unchanged in the presence or absence of the transglutaminase inhibitors and or the divalent cations, calcium or magnesium (1 mM), ruling out differ- ential effects of these experimental conditions on solvation of C-PAF in the ghost plasma membranes. At specified times, samples were removed for pelleting, and albumin extraction was performed as in RBC ghost release experiments to determine inner and outer leaflet C-PAF content. Uptake of C-PAF into the inner leaflet of RBC ghosts, where it remains nonextractable, is expressed as a percent of the total C-PAF ghost-associated (both inner and outer leaflets). Inhibition of uptake to the inner leaflet is expressed as the percent of C-PAF taken up in the presence of transglutaminase inhibitors compared to uptake in RBC ghosts identically treated in the absence of amines (designated 100% stimulated uptake).

Assay of Transglutaminme Actiuity-Transglutaminase activity was measured by the method of Piacentini et al. (1986) with certain modifications, by detecting the incorporation of [3H]putrescine into N'N'-dimethylcasein. Neutrophils were washed twice and resuspended in KRPD without calcium, then pelleted, and resuspended in homogenization buffer (50 X lo6 cells/100 pl) containing 250 mM sucrose, 1.5 mM MgCI,, 10 mM KC1, 10 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 pg/ml leupeptin, 10 pg/ml aprotinin, and 50 mM Tris, pH 8.3. The cells were sonicated on ice with a Branson microtip probe sonicator set a t 50% power for four cycles of 15 s each, alternating with 15-s cooling periods. To start the reaction, 1 pCi of [3H]putrescine (10 pl) , 100 pl cell lysate, and 5 mM CaCI2 (50 p l ) were added to 140 pl of buffer containing 10 mM dithiothreitol, 30 mM NaC1, 0.75 mg of dimethylcasein, 0.16 mM putrescine, and 50 mM Tris, pH 8.3. To stop incorporation, 5 ml of 5% trichloroacetic acid (4 "C) were added to each sample and the precipitate centrifuged a t 200 X g for 5 min a t 4 "C. The pelleted precipitates were resuspended and washed twice in 5 ml of 5% trichloroacetic acid, after which the precipitates were dissolved in 500 p l of 0.2 M NaOH, transferred to scintillation vials to which scintil-

lation fluid was added, and the samples counted the following day. Control samples containing no neutrophil lysate were processed identically and the counts subtracted as background measurements.

Meracyanine (MC) 540 Staining"MC540 staining of neutrophils was performed as previously described (Bratton et al., 1992). Briefly, a stock solution of MC540 was prepared the day before use a t a concentration of 1 mg/ml in 50% ethanol and stored in a foil-covered tube a t 4 "C. Neutrophils were suspended in KRPD with 0.1% HSA and incubated at room temperature with or without cystamine for 10 min prior to stimulus or buffer addition. Following a 3-min incubation period, MC540 (10 p1 of 1/10 dilution) was added to each 10O-pl sample for 3 min followed by KRPD without albumin (900 pl), and the sample immediately analyzed on a Coulter 751 flow cytofluorograph (Coulter Electronics, Hialeah, FL) equipped with a Coherent argon excitation laser tuned to 514 nm. Fluorescence was monitored through a 570-nm long pass filter and forward angle light scatter and side scatter measured from each sample. Data acquisition and analysis were performed using a Cicero-cytomation analyzer system. Identi- cally processed samples were examined on an Olympus fluorescence microscope by the method of McEvoy et al., (1988), with and without the addition of the water soluble membrane impermeant quencher, N,N"di~3-trimethylammoniumpropyl)thiadicarbocyanine tribrom- ide (Molecular Probes, Junction City, OR). In all cases, quenching of MC540 fluorescence indicated that the dye had remained in the external leaflet and had not been internalized during the incubation.

Modified RVV Assay-This assay measures the formation of pro- thrombinase complex composed of factors Va and Xa, calcium ion, and phospholipid as determined by the rate of clot formation measured with a fibrometer. A marked acceleration of clot formation is demonstrated with the exposure of phosphatidylserine in phospholipid membranes (Chiu et al., 1981; Lindhout et al., 1982). RVV and platelet-poor plasma were prepared as previously described (Bratton et al., 1992).

Neutrophils (5 X lo6 cells) were incubated in KRPD with or without transglutaminase inhibitor or control amine for 5 min prior to stimulation with ionomycin (10 PM) for 15 min. The cells were then washed with a 4-fold dilution of KRPD, centrifuged, washed a second time, and the supernatants decanted to remove free transglutaminase inhibitor. Such steps were necessary to remove free inhibitor so as to prevent interference with the clotting assay likely by inhibition of factor Xllla, the plasma transglutaminase. The neutrophils were then resuspended in normal saline (5 X lo6 cells/75 p l ) at 37 "C. For assay of outer leaflet anionic phospholipid, plasma (75 pl) and RVV 1/10,000 (75 pl) were warmed to 37 "C for 1 min to which neutrophils and CaCI2, 25 mM (75 PI), were added simultaneously, the fibrometer started, and the time to clot formation was recorded. Clotting times for neutrophils treated with transglutaminase inhibitors and washed in the above fashion did not differ from untreated neutrophils. To rule out that shortened clotting times simply reflected degranulation, data utilizing cytochalasin D in conjunction with other stimuli to markedly enhance degranulation did not result in enhanced shortening of clotting time, strongly suggesting dissociation of the two events. Additionally, the RBC ghost model of the isolated plasma membrane events was used to parallel the findings in neutrophils precisely.

RBC ghost,s (10') were incubated in normal saline with or without cystamine or sarcosine methyl ester (0-100 mM for 5 min) and then treated with ionomycin (10 pM) and either 1 mM magnesium or 1 mM calcium for 15 min. The ghosts were then washed free of non- incorporated amine with a 4-fold dilution of normal saline, spun down, and resuspended in normal saline. Assay of outer leaflet anionic phospholipid was performed as above for neutrophils. Clotting times for ghosts treated with transglutaminase inhibitors and washed in the above fashion did not differ from untreated ghost controls. Additional control samples containing no cells, with or without stimulus, were also run in duplicate each day and resulted in clotting times of 2100 s.

Statistics-Statistical analysis of data was performed using a univariate two-way repeat measures ANOVA model and a Tukey's multiple comparisons measure at the 5% significance level unless otherwise described in the individual figure legends. All measures were repeated three to six times using cells from different donors. Graphically depicted points and bars represent sample means k S.E. For analysis of MC540 staining, neutrophil populations appeared to he log normally distributed, and the log mean channel fluorescence for control and stimulated populations were compared by one-sample t tests. Pretreatment (no amine, cystamine-, and sarcosine-pretreatment) used as an independent variable was then compared for change

Transglutaminase-dependent Release of PAF from Neutrophils 3367

in staining as the dependent variable by a univariate repeated measures analysis of variance. A Tukey’s multiple comparison procedure was used at the 5% significance level to determine which pairs of groups were significantly different.

RESULTS

Transbilayer Movement of C-PAF upon Neutrophil Stimu- lation-Incubation of human neutrophils with C-PAF (7.5 X

M ) for 45 min at 37 ”C resulted in cellular incorporation of 30 -t 8(S.D.)% of the analog or 1.19 X lo6 f 0.59 X lo6 (S.D.) molecules/cell into intracellular sites where it could not be extracted with repeated washing in 2% HSA (see “Experimental Procedures”). Resuspension of the loaded neutrophils in media containing 2% HSA in the absence of stimulus resulted in minimal release of labeled material to the extracellular medium (3.3 f 0.2% at 15 min, Fig. 1). Cellular activation, however, with a variety of receptor- and nonrecep- tor-mediated stimuli, resulted in significant release of the labeled analog to the extracellular medium (up to 5.83 & 1.04 nmol/liter for PMA, Fig. 1). To address the question of whether the PAF receptor itself could be acting as a transbilayer “flippase” (Devaux, 1988; Herrmann et al., 1990), experiments were conducted in the presence of receptor antago- nism. As shown in Fig. 1, no evidence of PAF receptor dependence was demonstrated for release of C-PAF since this was unaltered in the presence of the PAF receptor antagonist, WEB 2086. Recent studies from this laboratory have similarly demonstrated that uptake of PAF into neutrophils was nonspecific for certain structural features of the PAF molecule and was not dependent on the PAF receptor (Bratton et al., 1992).

Recognizing that neutrophils loaded with labeled PAF or C-PAF distribute the label to various intracellular sites (data not shown; O’Flaherty et al., 1986; Riches et al., 1990), release of this material likely involves initial transport to the plasma membrane inner leaflet either by intracellular transport proteins (Lumb et al., 1983; Banks et al., 1988) and/or by incorporation of granule membranes during exocytosis. Therefore, t o better model the plasma membrane events separately from intracellular mobilization to the inner leaflet, release of C- PAF from erythrocyte ghost membranes was studied. Ghosts prepared so as to retain the native distribution of plasma membrane phospholipid asymmetry (Williamson et al., 1985) were loaded with C-PAF in the inner leaflet (see “Experimen- tal Procedures” and Bratton et al., 1991), and time-dependent release of C-PAF to albumin-containing medium was meas-

- 1 no antagonist 30 - 8 m WEB 2086 T

FIG. 1. C-PAF release following 15 min of stimulation with various agents expressed as percent of total labeled material loaded into neutrophils. The concentration of fMLP was M with or without cytochalasin D ( C D ) ( 5 pg/ml), ionomycin ( lono) (10 p ~ ) , and PMA (10 ng/ml). Hatched bars are cells stimulated in the presence of WEB 2086, 30 p ~ ; solid bars, in the absence of WEB 2086. All stimuli caused significant release of C-PAF ( p < 0.05). There was no significant difference between the groups treated with and without WEB 2086.

ured. As shown in Fig. 2 A , release of C-PAF from RBC ghosts to the extracellular medium was dependent on calcium, and in fact, was inhibited in the presence of 1 mM magnesium and 100 ~ L M EGTA (not shown). Virtually identical calcium dependence was also demonstrated for RBC ghost uptake of C- PAF, from outer leaflet to inner leaflet (Fig. 2 B ) . The non- specificity of uptake for various related analogs has previously been demonstrated by this laboratory (Bratton et al., 1991). Taken together, such findings both in the neutrophil and this plasma membrane model support the concept that the activated plasma membrane, but not the resting plasma membrane, will enhance the transbilayer movement of PAF and other phospholipids (see below). The fact that transbilayer movement may occur in either direction, from inner to outer, or outer to inner leaflet of the plasma membrane, likely contributes to the variability in the proportion of the mediator released from synthesizing inflammatory cells (see below).

A. Release

Ca (uM) 2 +

40

20

0 0 2 0 4 0 60

rn in

1 0 0 0 5 0 0

1 0 0

5 0

1 0

0

-

B. Uptake

30

/ 5 0 0

20 1

0 2 0 4 0 6 0 I

rnin

FIG. 2. Time-dependent release (A) and uptake ( B ) of C- PAF in RBC ghosts. A , release of C-PAF from RBC ghosts expressed as percent of total C-PAF loaded into the inner leaflet of RBC ghosts. Open RBC ghosts were loaded with C-PAF, closed, washed free of outer leaflet C-PAF, treated with ionomycin (10 p ~ ) and various amounts of calcium (at time O), and at specified times albumin extracted to remove C-PAF newly appearing in the outer leaflet. B, uptake of C-PAF into the RBC ghosts inner leaflet following calcium and ionophore (10 p ~ ) treatment (at time 0) is expressed as percent of total C-PAF offered.

3368 Transglutaminme-dependent Release of PAF from Neutrophils

Transbilayer Movement Is Dependent on Cellular Transglu- taminase Activity-Current data support the hypothesis that plasma membrane phospholipid transbilayer distributions and movement depend on cytoskeletal-membrane interactions (Haest and Deuticke, 1976; Comfurius et al., 1985; Verhallen et al., 1987, 1988; Middlekoop et al., 1988). While original reports suggested that proteolysis of cytoskeletal and/ or membrane proteins resulted in enhanced transbilayer movement of phospholipids (Verhallen et al., 1987), more recent studies dissociate these events (see “Discussion” and Comfurius et al., 1990; Henseleit et al., 1990)). Based on the observations that transglutaminase-mediated cytoskeletal/ membrane protein cross-linking has been noted in sickle cells (Ballas et al., 1985) and calcium ionophore-treated red cells (Lorand et al., 1983) where enhanced phospholipid transbilayer movement has been noted (Franck et al., 1985; Bratton et al., 1991), we questioned whether transglutaminase activity could govern the enhanced uptake and release of C-PAF. As shown in Fig. 3, A and B, the stimulated release of C-PAF from both loaded neutrophils and RBC ghosts was significantly inhibited by competitive inhibitors of transglutaminase. In Fig. 3A, the presence of monodansylcadaverine resulted in a marked concentration-dependent inhibition of C- PAF release from PMA-stimulated neutrophils. The nonpen- etrating amine, N-p-tosylglycine, is shown as a negative control. Identical results were seen when calcium ionophore and fMLP (with or without cytochalasin D) were used as the release stimuli (data not shown). As in earlier studies of transglutaminase activity (Bungay et al., 1984; Owen et al., 1988), other competitive inhibitors were found to be somewhat less potent with a rank ordering of monodansylcadaverine > cystamine = methylamine (data not shown). To address whether transglutaminase inhibitors merely resulted in inhibition of degranulation and interruption of potential delivery of C-PAF to the plasma membrane, experiments utilizing the stimulus fMLP with cytochalasin D, together a complete secretagogue, revealed that monodansylcadaverine (up to 0.5 mM) had only a minimal inhibitory effect ( 4 5 % ) on secretion of myeloperoxidase, and no effect on lysozyme secretion as measured by standard assays. To better examine the effect of transglutaminase inhibition on transbilayer movement of plasma membrane C-PAF, apart from intracellular shuttling, the erythrocyte ghost model was employed. Similarly, release of C-PAF from calcium-treated ghosts was inhibited by cystamine (= methylamine; monodansylcadaverine is not taken up into RBC ghosts as determined by measurements of cellular fluorescence (data not shown)), but not by the control amine, sarcosine methyl ester, which lacks the primary amine (Fig. 3B). Release of endogenously produced PAF as measured by mass spectrometry was similarly inhibited by 59 +. 9 (S.E.)% at a concentration of 0.2-0.3 PM monodansylcadaverine while synthesis was inhibited by only 9 * 5(S.E.)%.’ In support of the hypothesis that transbilayer movement in either direction across the plasma membrane is similarly controlled in the activated cell, stimulated uptake, i.e. the transbilayer movement from outer leaflet to inner leaflet, was also inhibited by competitive inhibitors of transglutaminase in both neutrophils and RBC ghosts (Fig. 4, A and B ) .

Using a standard assay of transglutaminase activity, the incorporation of radiolabeled putrescine into dimethylcasein (Schroff et al., 1981; Piacentini et al., 1986) was demonstrated by lysates of neutrophils (Fig. 5). Dependence of transglutaminase activity on the presence of divalent cations, either calcium or magnesium, was also demonstrated (data not shown) as has been previously shown for lysates of murine

K. L. Clay, personal communication.

A. Neutrophils

PTG

0.0 0.1 0.2 0.3 0.4

amine (mM)

B. Erythrocyte ghosts I 1

SARC

50 7511

CYST

0 . 5

-I 0 20 4 0 6 0

amine (mM)

FIG. 3. Inhibition of C-PAF release in neutrophils ( A ) and RBC ghosts ( B ) by transglutaminase inhibitors. A , concentration-dependent inhibition of stimulated C-PAF release from neutrophils by monodansylcadaverine ( M D C ) but not with the control amine, N-p-tosylglycine (pTG) . Results are expressed as percent of stimulated C-PAF release attained from neutrophils treated for 15 min with 10 ng/ml PMA in the absence of amine pretreatment. Significant inhibition of C-PAF release ( p < 0.05) was seen at all concentrations of MDC as determined by paired t test. B, inhibition of C-PAF release from calcium (1 mM)/ionophore (10 pM)-treated RBC ghosts by cystamine ( C Y S T ) ( p < 0.001) but not the control amine, sarcosine methyl ester (SARC). Results are expressed as percent of C-PAF release attained from ionophore-treated RBC ghosts incubated for 30 min with calcium in the absence of amine pretreatment.

cultured and peritoneal exudate macrophages (Schroff et al., 1981). Significant inhibition of transglutaminase activity was demonstrated in the presence of either monodansylcadaverine or cystamine, but not the control amine, sarcosine methyl ester (Fig. 5). Brief stimulation of neutrophils with either calcium ionophore or fMLP prior to homogenization did not result in heightened activity (data not shown) suggesting that,

Transglutaminuse-dependent Release of PAF from Neutrophils

A. Neutrophils

0.0 0.1 0.2 0.3

amine (mM)

B. Erythrocyte ghosts

loo

75

50

25

n

0 2 5 5 0 7 5 100

1000 9 CYST 0

3369

amine (mM)

FIG. 4. Inhibition of C-PAF uptake in neutrophils ( A ) and RBC ghosts ( B ) by transglutaminase inhibitors. A, concentration-dependent inhibition of stimulated C-PAF uptake into neutrophils by monodansylcadaverine ( M D C ) but not the control amine, N-p-tosylglycine (pTG) . Results are expressed as percent of C-PAF uptake attained from neutrophils stimulated for 15 min with 10 ng/ ml PMA in the absence of amine pretreatment. B, concentration- dependent inhibition of C-PAF uptake into the plasma membrane inner leaflet in calcium (1 mM)/ionophore (10 pM)-treated RBC ghosts by cystamine (CYST) but not the control amine, sarcosine methyl ester (SARC). Results are expressed as percent of C-PAF uptake attained from ionophore-treated RBC ghosts incubated for 30 min with calcium in the absence of amine treatment.

while such stimulation of intact neutrophils may enhance transglutaminase activity (perhaps by altering intracellular calcium levels), it does not apparently result in stable modi- fication of existing enzyme.

Stimulation of Neutrophils Results in Bulk Nonspecific Transbilayer Movement Qf Phospholipids-As shown in Table I, stimulation of neutrophils resulted in significant enhancement of staining with MC540, a fluorescent dye which inter-

0.4

m i n FIG. 5. Neutrophil transglutaminase activity determined by

the incorporation of [3H]putrescine into dimethylcasein over time in the presence of lysate from 50 X 10’ neutrophils. Also shown is the significant inhibition ( p < 0.05 by t test) of incorporation in the presence of transglutaminase inhibitors cystamine (CYST, 10 mM) and monodansylcadaverine (MDC, 0.5 mM), but not the control amine, sarcosine methyl ester (SARC, 10 mM).

TABLE I Cystamine inhibition of MC540 staining enhancement accompanying

neutrophil activation MC540 staining (change in log mean channel

Stimulus“ Pretreatment‘ fluorescence)*

Noned Cvstamine‘ Sarcosine’

fMLP (Io-’ M) +20.4 i 3.8 -1.5 % 4.3 +18.3 & 4.1 PMA (2 ng/ml) +26.2 f 6.4 +12.4 ? 5.4 +26.0 f 6.6 Ionomvcin (0.5 uM) +34.5 k 3.8 +20.2 & 2.7 +33.3 f 4.2 a Following pretreatment, samples were incubated for 3 min with

or without stimulus, then stained with MC540 for 3 min and immediately examined in the cytofluorograph.

* Enhanced MC540 staining compared to control, unstimulated neutrophils is shown as a positive change in log mean channel fluorescence & S.E.

‘ Neutrophils were pretreated for 10 min at room temperature with or without the amines, cystamine or sarcosine methyl ester (50-100 mM).

d”Stati~tical significance: significant enhancement of MC540 staining was seen with all stimuli ( W L P , p < 0.01; PMA, p < 0.05; ionomycin,p < 0.001), without amine pretreatment or with sarcosine methyl ester pretreatment when compared with unstimulated control cells. Cystamine pretreatment resulted in complete inhibition of enhancement in MC540 staining for WLP-stimulated cells ( p < 0.05) and partial inhibition for other stimuli (PMA, p = 0.06 ionomycin, p < 0.05). Cystamine and sarcosine methyl ester pretreatment alone did not alter MC540 staining in unstimulated cells (data not shown). N = five to six donors.

calates in hydrophobic or loosely packed membranes (Wil- liamson et al., 1983) and in some cells indicates a loss of plasma membrane phospholipid asymmetry (see “Discus- sion’’) (Williamson et al., 1985; Bratton et al. 1991). If such membrane physical changes are attributable to transglutaminase activity, then competitive inhibition of transglutaminase should inhibit the enhancement of MC540 staining. En- hancement of MC540 staining was completely inhibited in fMLP-stimulated neutrophils and partially inhibited with the other stimuli, when cells were pretreated with the transglutaminase inhibitor, cystamine (Table I). No such decrease in staining enhancement was seen using the control amine, sarcosine methyl ester. The inhibition of MC540 staining enhancement by cystamine was transient (largely decaying by 7-8 min following stimulation). Unfortunately, monodansylcadaverine (the most potent inhibitor of transbilayer movement), because of intrinsic fluorescence, could not be tested.

Stimulation of neutrophils (Bratton et al., 1992) and RBC ghosts (Henseleit et al., 1990; Bratton et al., 1991) can also result in the nonspecific bulk transbilayer movement of other

3370 Transglutaminase-dependent Release of PAF from Neutrophils

plasma membrane phospholipids. Utilizing the Russell’s viper venom clotting assay to detect the outer leaflet expression of the procoagulant phospholipid, phosphatidylserine, calcium and ionophore treatment of both neutrophils (Fig. 6 A ) and RBC ghosts (Fig. 6B) resulted in significant shortening of the clotting time, i.e. increased expression of PS. It should be noted that the other neutrophil stimuli also result in altered clotting times when neutrophils are coincubated with NaF to inhibit the aminophospholipid translocase, a calcium-inhib- itable and energy-dependent translocase that maintains native sequestration of phosphatidylserine to the plasma membrane inner leaflet (Bratton et al., 1992). I t is hypothesized that if transbilayer movement of PS is attributable to transglutaminase activity, then competitive inhibition of transglutaminase should inhibit such outer leaflet expression of PS. Pretreatment of both neutrophils and RBC ghosts with cystamine (but not sarcosine methyl ester) resulted in concentration-dependent diminished expression of phosphatidylserine (50 mM shown in Fig. 6, A and B ) . Similar inhibition of PS expression was seen following monodansylcadaverine pretreatment of calcium ionophore-stimulated neutrophils (data not shown.)

Taken together, these data demonstrate that stimulation of neutrophils results in marked changes of the plasma membrane that allow for bulk, nonspecific, transbilayer movement of phospholipids including C-PAF. A role for transglutaminase is strongly suggested in that competitive inhibition of this calcium-activated enzyme inhibits release of C-PAF, the en-

A. Neutrophils

No C a 2 + Ca z + CYST SARC C a 2 + Ca 2 +

B. Erythrocyte ghosts 80

No C a 2 + C a 2 + CYST SARC Ca2+ Can+

FIG. 6. Cystamine inhibition of enhanced RVV-induced clotting time in neutrophils ( A ) and RBC ghosts ( B ) . The significant enhancement of clotting time seen with ionomycin (IO p ~ ) and calcium (1 mM) treat.ment was inhibited by cystamine (50 mM) pretreatment but not by sarcosine methyl ester (50 mM) pretreatment, when compared to control cells treated with ionomycin in the absence of calcium ( p < 0.001 by univariate repeated measures ANOVA with the Dunnett’s multiple comparisons procedure at the 5% significance level).

hanced staining with MC540, and the appearance in the outer leaflet of phosphatidylserine.

Ultimate Net Release of C-PAF Depends on Plasma Mem- brane Recycling and Partitioning to Extracellular “Accep- tors”-One proposed explanation for the limited release of PAF from inflammatory cells is recycling at the level of the plasma membrane where the PAF is taken back into the synthesizing cell (Bratton et al. 1992; Cluzel et al., 1989,1991). This hypothesis is supported by the finding that uptake into the plasma membrane inner leaflet was markedly enhanced in both stimulated neutrophils (Bratton et al. 1992) and calcium-treated RBC ghosts (Fig. 2B; Bratton et al., 1991). PAF release has been reported to be dependent on the concentration of cell suspension with more concentrated suspen- sions yielding less proportionate release (Betz and Henson, 1980; Betz et al., 1980; Leyravaud and Benveniste, 1989). We suggest that recycling of the PAF synthesized by a given cell and or recycling by adjacent cells is a likely explanation for the finding of cell density dependence of release. As shown in Fig. 7, release to the extracellular media was inhibited by increasing cell density where albumin is likely excluded from cell aggregates. As would also be predicted, direct uptake of PAF from albumin-containing medium was similarly decreased with increasing cell density (data not shown). Parti- tioning of PAF from cells to albumin (Clay et al., 1990) or other plasma proteins (Matsumoto and Miwa, 1985) has been previously described (Cluzel et al., 1989), and albumin dependence of C-PAF release was similarly demonstrated for loaded neutrophils (Fig. 8). It is proposed that both availabil- ity of albumin (or other acceptors) to the plasma membrane and the activation state of nearby inflammatory cells will likely contribute to the noted variability in the proportion of the mediator released to the extracellular milieu in a given inflammatory process.

DISCUSSION

Recent data from several laboratories strongly suggest that there is an asymmetric distribution of the phospholipid classes across the plasma membrane bilayer in circulating blood cells (Schlegel and Williamson, 1987). Although phosphatidylcho- line and phosphatidylethanolamine appear to be distributed

15 min

cel l density (neutrophilslrnl)

FIG. 7. Cell density dependence of C-PAF release. Neutro- phils were loaded with C-PAF, resuspended a t different densities in KRPD with 2% HSA, and stimulated for release with PMA (10 ng/ ml) for 15 and 60 min.

Transglutaminuse-dependent Release of PAF from Neutrophils 3371

0 0.1 0.5 1 2 4

HSA (Yo) FIG. 8. Albumin dependence of C-PAF release. Neutrophils

were loaded with C-PAF, resuspended in KRPD with different amounts of HSA, and stimulated for release with 2.5 wg/ml A23187 (solid bars) for 15 min. Nonstimulated controls are shown by hatched bars.

in both plasma membrane leaflets, phosphatidylserine ap- pears to be sequestered almost entirely in the inner leaflet, and sphingomyelin, in the outer leaflet (Devaux, 1991). Main- tenance of this phospholipid asymmetry is not fully understood but is thought to involve stabilization of the plasma membrane by the cytoskeleton (Haest and Deuticke, 1976; Comfurius et al., 1985; Verhallen et al., 1987, 1988; Middel- koop et al., 1988) and activity of an aminophospholipid translocase which specifically transfers the aminophospholipids from the outer to inner leaflet of the plasma membrane (Daleke and Huestis, 1985; Sune et al., 1987; Zachowski et al., 1987). Notably, the equilibrium distribution across the plasma membrane of exogenously added phospholipid of each class closely mirrors that of endogenous phospholipids, suggesting that transbilayer movement is controlled by the same factors governing plasma membrane phospholipid asymmetry (Pa- gano and Sleight, 1985; Middelkoop et al., 1989; Sune et al., 1987). Loss of plasma membrane asymmetry is best demonstrated in the activated platelet (Bevers et al., 1983,1989) and calcium-treated erythrocyte (Henseleit et al., 1990) where a more random distribution of the phospholipid classes is achieved. Additionally, detectable outer leaflet expression of PS likely involves a second event in addition to heightened flip/flop, namely, calcium-dependent inhibition of the aminophospholipid translocase (Middelkoop et al., 1988; Bevers et al., 1989). The data presented here using human neutrophils, and further explored in the RBC ghost model (Figs. 3, 4, and 6), as well as that presented previously (Bratton et al. 1991, 1992), support the hypothesis that cellular activation markedly enhances transbilayer movement of phospholipids of several classes including the choline-containing phospholipid, PAF.

Except in the case of inward movement of the aminophospholipids via the aminophospholipid translocase (Daleke and Huestis, 1985; Sune et al., 1987; Zachowski et al., 1987), the actual mechanism of phospholipid flip/flop across the plasma membrane is not well understood (Bitbol and Devaux, 1988; Devaux, 1991). In contrast to phospholipid vesicles lacking membrane proteins where transbilayer movement of phospholipids is extremely slow (on the order of hours to days), the introduction of certain proteins, effectively working as “flippases” can markedly and nonspecifically enhance transbilayer movement of phospholipids of the various classes (Gerritsen et al., 1980; Homan and Pownall, 1988; Devaux, 1991). The mere presence of native plasma membrane proteins, however, does not result in significant transbilayer

movement as seen by unstimulated neutrophils (Fig. 1; Brat- ton et al., 1992) and RBC ghosts prepared in the absence of calcium so as to retain native erythrocyte plasma membrane asymmetry (Fig. 2). In contrast, activation of the neutrophil (Fig. 1) or calcium treatment of RBC ghosts markedly enhanced transbilayer movement of C-PAF, either from inner to outer leaflet or outer to inner leaflet (Fig. 2; Bratton et al., 1991). Considering the presumed role of the cytoskeletal- membrane interactions in the maintenance of asymmetry (Haest and Deuticke, 1976; Comfurius et al., 1985; Verhallen et al., 1987, 1988; Middelkoop et al., 1988), it is likely that activation-induced alterations in such proteins are responsible for enhanced transbilayer movement. Either the creation of new flippases or flip sites and/or untethering the plasma membrane, perhaps allowing phospholipids access to flip sites along the membrane, are obvious possibilities. Notably, data from several laboratories suggest that proteolytic cleavage of cytoskeletal elements is likely not responsible for enhanced transbilayer movement. Cytoskeletal proteolysis has been dis- sociated from enhanced phospholipid movement in platelets and red cells (Comfurius et al., 1990; Henseleit et al., 1990), both in their respective cation dependence and following inhibition of calpain. In keeping with those observations, leupeptin, in our hands, also had no effect on either uptake or release of C-PAF (data not shown). Similarly, trypsiniza- tion of RBC ghosts, while resulting in extensive proteolysis of various cytoskeletal/membrane proteins, did not result in enhanced transbilayer flip/flop of phospholipids (Henseleit et al., 1990).

To date, the role of transglutaminase-mediated cytoskeletal cross-linking in the enhanced phospholipid transbilayer movement accompanying cellular activation has not been thoroughly explored. Previous studies of calcium ionophore- treated red cells (Lorand et al., 1983) and sickle cells (Ballas et al., 1985) have suggested that transglutaminase activity may be accompanied by enhanced movement of phospholipid probes from outer to inner plasma membrane leaflet and enhanced outer leaflet PS expression (Franck et al., 1985). We have shown that competitive inhibitors of transglutaminase profoundly inhibited transbilayer movement of C-PAF (both release and uptake) and outer leaflet expression of PS in both the neutrophil and RBC ghost model (Figs. 3, 4, and 6). Additionally, the finding that competitive inhibitors of transglutaminase also inhibit the enhanced MC540 staining that accompanies neutrophil activation implicates the activity of transglutaminase in the plasma membrane physical alteration that occurs with activation (Table I).

Transglutaminases have been identified in a variety of cells and in plasma where they catalyze the cross-linking of sus- ceptible y-carboxamide groups of glutamine residues with either free amines or polypeptide-bound lysine residues to render highly stable y-glutamyl polyamine or y-glutamyl-c- lysine bonds (Greenberg et al., 1991). To date, transglutaminase activity has been described in circulating and inflammatory cells including platelets (Puszkin and Raghuraman, 1985; Lorand et al., 1987; Greenberg et al., 1991), red cells (Lorand et al., 1987), lymphocytes (Gunzler et al., 1982), macrophages and monocytes (Fbiis et al., 1984; Murtaugh et al., 1984; Henriksson et al., 1985), and neutrophils (Giinzler et al., 1982). Both the demonstration of transglutaminase activity in neutrophil lysates and concentration-dependent inhibition by known competitive inhibitors (Fig. 5) lend further support to the hypothesis that this enzyme likely plays a pivotal role in the enhancement of transbilayer movement of phospholipids. Further controls governing neutrophil transglutaminase ac-

3372 Transglutaminme-dependent Release of PAF from Neutrophils

tivity have yet to be defined and will be the focus of future studies.

The data suggest that PAF is transported to the outer leaflet of the neutrophil in a nonspecific, bulk fashion along with other phospholipids. Also recognized is the potential for plasma membrane recycling of PAF (and other phospholipids) from outer leaflet to inner leaflet. Such reciprocal, bidirectional movement (illustrated in Fig. 2, A and B ) is likely required to maintain cellular integrity by preventing inappro- priate expansion of either leaflet (Devaux et al., 1991). Actual release of PAF from the outer leaflet to the extracellular milieu likely occurs because of decreased hydrophobicity, a characteristic of phospholipids bearing a short chained sn-2 group (Mohandas et al., 1982; Tokumura et al., 1990), and the high affinity for PAF exhibited by albumin (Tokumura et al., 1987; Clay et al., 1990). The dependence of release upon the presence of albumin has been seen by others (Benveniste et al., 1972; Ludwig et al., 1985) as well as demonstrated here for the PAF analog (Fig. 8). It is proposed that net release of the mediator is determined by actual recycling of the mediator balanced by partitioning to albumin or other acceptors including perhaps contiguous cell membranes. Both the greater yield of released PAF with "dynamic removal" of the media from synthesizing neutrophils as shown by Cluzel et al. (1989), and lesser yield to media when cells are suspended at high density (Fig. 7; Leyravaud and Benveniste, 1989) is entirely consistent with modulation of extracellular release by such cellular recycling, Furthermore, cellular recycling may have biological significance as proposed by Ludwig et al. (1985) who noted the dependence of sustained synthesis on PAF release, and suggested that such coupling is due to negative feedback inhibition of retained PAF on further PAF biosyn- thesis.

Taken together, the current data along with previous findings would suggest that at least three steps are involved in the release of PAF from a PAF-synthesizing inflammatory cell: 1) transport of the mediator from an intracellular site(s) of synthesis to the plasma membrane inner leaflet (Lumb et al., 1983; Banks et al., 1988); 2) transglutaminase-dependent transbilayer movement from the inner to the outer leaflet of the plasma membrane bilayer; and 3) partitioning from the outer leaflet to extracellular acceptors such as albumin (Clay et al., 1990) or other plasma proteins (Matsumoto and Miwa, 1985). This last step is likely dependent on the extent of recycling of outer leaflet PAF to the plasma membrane inner leaflet. The mechanism(s) by which transglutaminase activity leads to enhanced phospholipid transbilayer movement and accompanies the release of PAF from activated inflammatory cells will be the focus of future studies.

Acknowledgments-I t h a n k J e n a i M. Kailey and Elisabeth Dreyer for technical assistance, Dr. Peter M. Henson for many s t imula t ing discussions and thoughtful review of the manuscr ip t , Dr. L y n n Ack- erson for assistance with statist ical analysis, and Georgia Wheeler for p repara t ion of the manuscript .

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