Immunology 1983 48 27

Conversion of D4 to leukotriene E4 by a dipeptidase released from the specific granule of human polymorphonuclear leucocytes

C. W. LEE, R. A. LEWIS, E. J. COREY & K. F. AUSTEN Department ofMedicine, Harvard Medical School and the Department ofRheumatology andImmunology, Brigham and Women's Hospital, Boston, Massachusetts and Department of Chemistry, Harvard University, Cambridge, Massachusetts, U.S.A.

Acceptedfor publication 15 June 1982

Summary. Leukotriene D4 (LTD4), the most active activity, termed LTD4 dipeptidase, to be localized only spasmogenic leukotriene constituent ofthe slow react- in the granule fraction. There was a time- and ing substance of anaphylaxis, was converted by sus- calcium-dependent extracellular release of LTD4 pended human polymorphonuclear leucocytes dipeptidase in association with lysozyme (r =097, (PMNs) to a single, less polar metabolite which was n= 16, P<0 001), a constituent of both specific and not further catabolized. This product was identified as azurophilic granules, in the absence of release of leukotriene E4 (LTE4) by its retention time during cytoplasmic lactate dehydrogenase (LDH) and of reverse phase-high performance liquid chromato- f,-glucuronidase from the azurophilic granule. Phor- graphy (RP-HPLC) and subsequent bioassay on the bol myristate acetate (PMA), which selectively induces guinea-pig ileum. LTD4 with a retention time of secretion of specific granules, released lysozyme and 21 + 1 6 min (mean + SD) and a contractile activity of the LTD4 dipeptidase in a constant dose-dependent 5-0 + 0 4 u./pmol (mean + SD) was quantitatively con- manner from PMNs (r=0-96, n=8, P<0 001). Cal- verted extracellularly by PMNs to LTE4 with a reten- cium ionophore A23187 at concentrations less than tion time of 26 + 1 8 min and a contractile activity of 10-7 M stimulated the parallel secretion of LTD4 1 2 + 0 3 u./pmol. Subcellular fractionations ofPMNs dipeptidase and lysozyme (r=0 91, n=9, P <0-005), revealed the recovered LTD4-to-LTE4 converting whereas higher concentrations resulted in secretion of f-glucuronidase and additional lysozyme without Correspondence: Dr K. F. Austen, 604 Seeley G. Mudd further release of dipeptidase. Thus, human PMNs Building, 250 Longwood Avenue, Boston, Massachusetts can convert LTD4 to LTE4, a less vasoactive and 02115, U.S.A. spasmogenic leukotriene, via the secretion of a di- Abbreviations: ATP, adenosine triphosphate; EDTA peptidase associated with the specific granules. ethylenediamine tetraacetate; Hepes, 4-(2-hydroxyethyl)-1- piperazine-ethanesulphonic acid; HBSS, Hanks's balanced salt solution; HPLC, high performance liquid chromato- graphy; LDH, lactic dehydrogenase; LTC4, leukotriene C4; INTRODUCTION LTD4, leukotriene D4; LTE4, leukotriene E4; PMA, phorbol myristate acetate; PMNs, polymorphonuclear leucocytes; The chemical constituents of slow reacting substance RP-HI'LC, reverse phase-high performance liquid chroma- tography; SRS-A, slow reacting substance of anaphylaxis; of anaphylaxis (SRS-A) are 5(S)-hydroxy-6(R)-S- y 32p;, y_32p inorganic phosphate. glutathionyl-7,9-trans, 11, 14-cis-eicosatetraenoic acid 00 1 9-2805/83/0100-0027$02.00 [leukotriene C4, (LTC4); Murphy, Hammarstrom & ©) 1983 Blackwell Scientific Publications Samuelsson, 1979; Corey, Clark, Goto, Marfat, 27 28 C. W. Lee et al.

Mioskowski, Samuelsson & Hammarstr6m, 1980a] 500 ng/site (Lewis, Soter, Corey & Austen, 1981). The and its sequential cleavage products, the 6(R)-S-cys- spasmogenic and vasoactive activities that character- teinylglycyl analog [leukotriene D4 (LTD4); Bach, ize SRS-A (Brocklehurst, 1960; Drazen & Austen, Brashler, Hammarstrdm & Samuelsson, 1980; Lewis, 1974; Drazen, Lewis, Wasserman, Orange & Austen, Austen, Drazen, Clark, Marfat, & Corey, 1980a; 1979) are manifested by the entire 6-sulphido-peptide Morris, Taylor, Piper and Tippins, 1980; Orning, class of with some differences in potency, Hammarstrdm and Samuelsson, 1980a] and the suggesting that the conversion from LTC4 to LTD4 to 6(R)-S-cysteinyl analog (leukotriene E4 [LTE4]; Lewis, LTE4 is a cascade rather than a detoxification path- Drazen, Austen, Clark & Corey, 1980b). Based on the way. conventional definition of one unit of SRS-A as the LTC4 is sequentially converted to LTD4 and to amount that elicits a contraction of the guinea-pig LTE4 in vitro by human plasma (Parker, Koch, Huber ileum equal in amplitude to that evoked by 5 ng/ml of & Falkenhein, 1980b; Lewis, Drazen, Figueiredo, histamine, the activities of LTC4 [1 93 + 0 13 u./pmol Corey & Austen, 1982), rat basophilic leukaemia cells (mean + SD)], LTD4 (6 1 +115 u./pmol) and LTE4 (Parker, Falkenhein & Huber, 1980a; drning, Berg- (1l2+0 05 u./pmol) are 200-1000 times more potent strom & Hammarstrom, 1981), rat peritoneal eosino- than histamine on a weight basis (Lewis et al., 1980a, phil extracts (Sok, Pai, Atraches, Kang & Sih, 1981), 1980b). The potency of the leukotrienes as spas- and guinea-pig liver, lung and kidney homogenates mogens for guinea-pig parenchymal strips that con- (Sok, Pai, Atrache & Sih, 1980; Bergstrom & Ham- tain peripheral airway smooth muscles is greater than marstr6m, 1981; Sok et al., 1981), with LTE4 that of histamine by 100 times for LTC4 and LTE4 remaining as a stable end-product. Enzymatic conver- (EC5o= 3 x 10-9 M) and by 1000 times for LTD4 sion of LTC4 to LTD4 and LTC3 to LTD3 by cleavage (EC5o= 1 x 1O-10 M) (Drazen, Austen, Lewis, Clark, of glutamic acid from the S-glutathionyl domain has Goto, Marfat & Corey, 1980; Lewis et al., 1980b). been shown with y-glutamyl-transpeptidase purified LTC4 and LTD4 are 100-1000 times more potent than from kidney (Sok et al., 1980; Hammarstrom, 1981), histamine in constricting human airways in vitro and cleavage of the carboxy terminal glycine from (Dahlen, Hedqvist, Hammarstr6m & Samuelsson, LTD4 to form LTE4 has been demonstrated with 1980; Hanna, Bach, Pare & Schellenberg, 1981). commercial porcine kidney dipeptidase (Hammar- Intravenous administration of 0 5-1 0 ug/kg of LTC4, strom, 1981; Bergstrom & Hammarstr6m, 1981). The LTD4 and LTE4 to guinea-pigs leads to decreased subcellular localization of LTD4-to-LTE4 converting airway conductance and a more persistent and rela- dipeptidase activity is not defined for any tissue or cell tively greater fall in dynamic compliance, especially type. The present study shows that LTD4 dipeptidase with LTC4 and LTD4 (Drazen et al., 1980; Drazen, activity associated with the specific granules of the Venugopalan, Austen, Brion & Corey, 1982). When human polymorphonuclear leucocytes (PMNs) acts administered by aerosol, LTC4 and LTD4 at 2-20 Mg extracellularly to convert LTD4 to LTE4. doses were 3800 times more potent on a molar basis than aerosolized histamine in impairing airflow measured at 30% of vital capacity in the same normal MATERIALS AND METHODS human subjects (Holroyde, Altounyan, Cole, Dixon & Elliot, 1981; Weiss, Drazen, Coles, McFadden, Lewis, Materials Weller, Corey & Austen, 1982a; Weiss, Drazen, Hanks's balanced salt solution (HBSS, Microbiologi- McFadden, Lewis, Weller, Corey and Austen, 1982b). cal Associates, Bethesda, Md); Ficoll-Hypaque, Rats receiving intravenous LTC4, 1-10 pg/kg, mani- macromolecular dextran (Pharmacia Fine Chemicals, fest an increased total peripheral resistance and a Inc., Piscataway, N.J.); calcium ionophore A23187 decrease in cardiac output due to a direct negative (Calbiochem-Behring Corp., La Jolla, Calif.); inotropic effect (Pfeffer, Pfeffer, Lewis, Braunwald, adenosine 5'-triphosphate (ATP), atropine sul- Corey & Austen, 1982). LTC4, LTD4 and LTE4 phate, histamine diphosphate, ouabain octahydrate, increase vascular permeability in guinea-pig skin phorbol myristate acetate (PMA), phenolphthalein- (Drazen et al., 1980; Dahlen, Bjork, Hedqvist, Arfors, fl-D-glucuronide, y-glutamyl-transpeptidase, and Hammarstrom, Lindgren & Samuelsson, 1981; Peck, Micrococcus Iysodeikticus (Sigma Chemical Co., St. Piper & Williams, 1981), and elicit a wheal and flare Louis, Mo.); 4-(2-hydroxyethyl)-1-piperazine-ethane- response in human skin when injected intradermally at sulphonic acid (Hepes; Boehringer Mannheim, Conversion of LTD4 to LTE4 by human PMNs 29

Indianapolis, Ind.); cytochalasin B (Aldrich Chemical (pellet 1). The 400 g supernatant was centrifuged at Co., Milwaukee, Wisc.); adenosine 5'-triphosphate 10,000 g for 40 min at 40 to sediment the lysosomal tetra (triethyl ammonium salt), [32p], 1000-3000 granules (pellet 2). The 10,000 g supernatant was Ci/mmol (New England Nuclear Co., Boston, Mass.); centrifuged at 225,000g for 30 min at 40 to yield a final and high performance liquid chromatography supernatant (supernatant 3) and a final pellet (pellet (HPLC) grade methanol (Burdick and Jackson 3); pellet 3 was washed and centrifuged at 225,000g for Laboratories, Muskegon, Wisc.) were obtained from 30 min at 4° with 10 mm Hepes, pH 7 5, and then with I the manufacturers. LTC4, LTD4 and LTE4 were mM Hepes, pH 7 5. Pellets 1, 2 and 3 were resuspended prepared as described (Corey et al., 1980a, Lewis et al., in HBSS containing 2 5 mm MgCl2 and 2-5 mm EDTA. 1980b) and stored frozen at a concentration of A portion of the starting post-cavitation suspension, 25 ug/ml in 0-1 M phosphate buffer, pH 68:ethanol pellets 1, 2 and 3 and supernatant 3, were sonicated (4: 1, v/v) under argon until the day of use. [3H]LTC4 (power 4, 50% pulse cycle, 5 pulses, Branson Sonifier, (60 Ci/mmol), prepared by reacting [3H] Model 350, Danbury, Conn.) and assayed for lactate with glutathione (Corey, Clark, Marfat & Goto, dehydrogenase (LDH), lysozyme, fl-glucuronidase, 1980b), was provided by New England Nuclear. ouabain-inhibitable Na+K+-ATPase, protein, and [3H]LTD4 was prepared by reacting [3H]LTC4 with the capacity to convert LTD4 to LTE4. y-glutamyl-transpeptidase and purified by reverse Ten million PMNs in I ml of HBSS in 15 ml phase-HPLC (RP-HPLC) (Orning & Hammarstrom, centrifuge tubes were incubated in duplicate with or 1980). without cytochalasin B (5 pg/ml) for 5 min at 370 before the introduction ofa degranulating stimulus for Purification, subcellular fractionation and lysosomal various time intervals at 370. The release reaction was enzyme release of PMNs stopped by centrifugation at 8000 g for 2 min in a Thirty-millilitre portions of blood from normal Model B Microfuge (Beckman Instruments, Palo donors were each mixed with 4 ml of 0-15 M sodium Alto, Calif.) and the supernatants were aliquoted and citrate, pH 5-2, and 5 ml of 5% dextran in 0 15 M NaCl stored at - 700. Samples were assayed in duplicate for and allowed to sediment at room temperature for 1 hr. LDH, fl-glucuronidase, lysozyme and the capacity to The supernatants containing leucocyte-rich plasma convert LTD4 to LTE4. The extracellular release of were aspirated and centrifuged at 100 g for 10 min at LDH, fl-glucuronidase and lysozyme were each calcu- 25°. Cell pellets were resuspended in 3 ml of0 2% NaCl lated as a percentage of the total enzyme activity for 1 min to lyse residual erythrocytes and 3 ml of 1-6% available after sonication of unstimulated, replicate NaCl were then added to restore isotonicity. The neutrophil suspensions (power 4, 50% pulse cycle, 5 leucocytes were centrifuged at 250 g for 5 min, washed pulses). Because sonicated PMNs exhibited less and resuspended in HBSS at 1-2 x 108 cells/ml. Three- LTD4-LTE4 converting activity than the supernatants millilitre cell suspensions were layered on 3 ml Ficoll- obtained with non-cytotoxic degranulation, the Hypaque cushions and centrifuged at 400 g for 20 min release of LTD4 dipeptidase is expressed as total at 250 to yield a PMN pellet (Boyum, 1968) which was extracellular activity and not as percentage release. washed twice and suspended at a concentration of 107 LDH was assayed spectrophotometrically by fol- cells/ml in HBSS. The leucocyte preparation was more lowing the disappearance of NADPH at 340 nm upon than 97% neutrophils with few contaminating erythro- conversion of pyruvate to lactate at 37° (Bergmayer, cytes or . 1963). f,-Glucuronidase activity was also determined One milliard PMNs in 100 ml ofcalcium-free HBSS spectrophotometrically by measuring the release of containing 2-5 mm MgCl2 were equilibrated at 400 phenolphthalein at 552 nm from its ,B-glucuronide p.s.i. of nitrogen for 20 min at 40 in a cavitation-cell after 4 hr of incubation at 370 (Talay, Fishman & disruption chamber (Parr Instrument Co., Moline, Huggins, 1946). Lysozyme activity was quantified by Ill.) and disrupted by the rapid release of pressure the rate of lysis of M. lysodeikticus according to a (Klempner, Mikkelson, Corfman & Andre-Schwartz, turbidometric assay (Litwack, 1955). Ouabain- 1980; Tonnesen, Klempner, Austen & Wintroub, inhibitable Na+K+-ATPase was assessed by measur- 1982). The disrupted cell suspension was collected into ing the hydrolysis ofy-32p inorganic phosphate (y-32p;) ethylenediamine tetraacetate (EDTA) at a final con- from radiolabelled ATP in the presence of 0-01% centration of 2 5 mm and centrifuged at 400 g for 20 deoxycholate (Harlan, DeChatelet, Iverson & McCall, min at 40 to pellet the nuclei and undisrupted cells 1977; Tonneson et al., 1982) and its activity was 30 C. W. Lee et al. calculated as the difference between 32Pi released in the assessed by direct bioassay of samples of cell suspen- absence and presence of 5 mm ouabain. sion-incubation mixtures on the guinea-pig ileum. There was a linear, time-dependent loss of 72+6% Assay ofLTD4 dipeptidase activity (mean + SD) of the spasmogenic activity over the first Samples of 0 25 ml each to be assessed for LTD4 30 min of interaction in four experiments without dipeptidase activity were incubated with 2-0 pg LTD4 further inactivation for up to 120 min (Fig. 1). There for 30 min at 370 in HBSS, pH 7-4, at a final volume of was minimal inactivation of contractile activity when 1 ml. The reactions were stopped by immersion of the 107 PMNs were incubated with 1 jg LTD4 at 0° for 120 tubes in ice and samples were injected directly onto min (Fig. 1) as compared with LTD4 incubated at HPLC for physicochemical assay. Reverse-phase either temperature in buffer without cells. HPLC was performed on a 4 6 x 250 mm C18 Lichro- In four additional experiments the same concentra- sorb column with a 3 2 x 40 mm C18 precolumn using tions of PMNs and LTD4 were incubated together at dual Altex Model IOOA pumps (Altex-Rainin Co., 370 for up to 60 min followed by centrifugation at 400 Berkeley, Calif.) at 1500 p.s.i. and a flow rate of 1 g for 10 min to remove cells so that the supernatants ml/min in an isocratic solvent system of methanol: could be analysed by RP-HPLC for LTD4 and its water: acetic acid (65: 34 9: 0 1) at pH 5 6. Absorbance metabolite(s). There was a time-dependent decrease in at 280 nm (A280) of column effluent was continuously the A280 peak corresponding to the LTD4 retention monitored with an on-line Model 100-40 spectro- time of 21 + 1 6 min with quantifiable conversion to a photometer (Hitachi Ltd, Tokyo, Japan). The column single new peak eluting at 26 + 18 min and corres- was calibrated with synthetic LTD4 and LTE4 for ponding to the retention time of synthetic LTE4 (Fig. retention times (mean+SD) of 21-8+2-1 min and 2). The ultraviolet spectrum for this new peak, pooled 25-7+2-1 min and A280 recoveries (mean+SD) of and concentrated from four experiments, showed an 81 3 + 1088% and 81 1 + 16%, respectively (n = 3). absorbance maximum at 280 nm, with shoulders at For quantifiable determination of LTD4 dipepti- 270 and 290 nm, indicating a conjugated triene dase activity A280 peaks of LTD4 and LTE4, resolved structure. by RP-HPLC, were converted to micrograms, based In order to examine the distribution of leukotrienes on established A280 recoveries of synthetic LTD4 and between supernatants and cells, replicate reaction LTE4 standards. LTD4 dipeptidase activity was mixtures of 107 PMNs, 100,000 c.p.m. of [3H]LTD4, expressed, based on the amount of LTE4 generated and 1 0 jg of cold LTD4 were incubated for 30 min at from 2 pg LTD4 (4-5 nmol), with 1 unit of activity 370 or 00. In two experiments 85% and 96% of starting being defined as generating 1 nmol of LTE4 (mol. wt radioactivity were recovered from the supernatants, 439) after 30 min ofincubation at 37°. The activity was and 6% and 3% from the cell pellets, with total multiplied by the initial dilution factor to standardize the activity ofthe fraction being assessed per millilitre. The leukotrienes with or without resolu- bioassay of 100 tion by RP-HPLC was conducted with the guinea-pig ileum suspended in Tyrode's buffer containing 1 jiM atropine (Brocklehurst, 1960). A contractile response 2 80 to LTD4 equal to that of 5 ng of histamine was defined as 1 unit of spasmogenic activity. The bioassay served b 60 to establish that each reference leuikotriene prep- aration had the expected functional activity and to £40 define the activity per picomole of the conversion product. c 20 _ 370 II 0 30 60 90 120 RESULTS Incubation (min) Conversion of LTD4 to LTE4 by human PMNs Figure 1. Time course of inactivation of the spasmogenic activity of LTD4 for the guinea-pig ileum by suspensions of The metabolism of I jg LTD4 by I07 human PMNs in human PMNs at 37° (0) and 0° (0). Each time point 1 ml of HBSS at 370 for periods of 0-120 min was represents the mean + SD of four experiments. Conversion ofLTD4 to LTE4 by human PMNs 31

with 1 pg cold LTE4, the radiolabel co-eluted with the A280 peak. No peaks of radioactivity or of A280 were detected after RP-HPLC of an 80% ethanol extract of the entire cell pellet obtained after either 0° or 370 incubation. Thus, [3H]LTE4 was the only recognized 80 product formed from [3H]LTD4 by human PMNs. 60 The LTD4 metabolite was further characterized in terms ofits biological activity. After the incubation of 40) 1 pg LTD4 and I07 PMNs for 60 min at 370, RP-HPLC of the supernatant yielded a single metabolite eluting in the position of LTE4. In three consecutive experi- 0420 ments the metabolite had an activity of 1-2+0-3 u./pmol which is identical to LTE4 (Lewis et al., 1980b), as compared with an activity of 5-0+004 u./pmol for the LTD4; calculation of mass was based 0 10 20 30 upon A280 and a molar extinction coefficient, 6280, of Incubation (min ) 40,000 cm-1 M- 1. Thus, the single metabolite formed Figure 2. Time course ofconversion of LTD4 (0) to LTE4 (0) from LTD4 by human PMNs was LTE4, both as by human PMNs at 370 as assessed in the supernatants after defined by retention time on RP-HPLC and by RP-HPLC to resolve the A280 peaks. Each time point spasmogenic activity per unit mass. represents the mean + SD of four experiments in which the percentage ofeach leukotriene recovered is expressed relative to total recovery which was set at 100%. The actual total recovery ofthe leukotrienes relative to A280 units applied was Subcellular localization of LTD4 dipeptidase 72 + 9% (mean + SD). To localize the LTD4 dipeptidase activity in the PMNs, nitrogen-cavitated PMNs were separated into recoveries of91% and 99%. Resolution ofthe superna- granule, membrane and cytosol fractions by differen- tant by RP-HPLC revealed that [3H]LTD4 was con- tial centrifugation in three experiments. As summar- verted to a single radiolabelled peak eluting at 25-27 ized in Table 1, LTD4 dipeptidase was found in the min. In the two experiments, conversion was quantifi- 10,000g pellet (pellet 2) which contained the lysosomal able in that the applied counts eluted as LTD4 at 20-22 granules as indicated by the presence of,B-glucuroni- min with 43% and 68% recoveries after the 00 incuba- dase and lysozyme. Within each experiment the per- tion and as a single peak at 25-27 min, with 40% and centages of the LTD4 dipeptidase, lysozyme and 64% recoveries after the 370 incubation. On rechroma- fl-glucuronidase in the granule pellet were internally tography ofthe 3H-labelled metabolite obtained at 370 consistent, ranging from 37 5% to 46%, 55% to 61 5%

Table 1. Distribution of marker enzymes and LTD4 dipeptidase in subcellular fractionations from 109 polymorphonuclear leukocytes

Na+K+- ATPase (Ouabain- LTD4 Protein LDH inhibitable) fl-Glucuronidase Lysozyme dipeptidase (mg) (%) (%) (%) (%) (%) Starting cavitate* 50 3 100 100 100 100 100 Pellet 1 6-8 13 21 26 25 30 Pellet 2 3-6 0 0 45 46 37-5 Pellet 3 1-0 0 62 3 0 0 Supernatant 3 30 3 76 0 2-8 4 0 Total recovery 41-7 89 83 76-8 75 67 5

* The activity ofeach marker enzyme and of LTD4 dipeptidase in the starting cavitate was considered 100%. Values shown were obtained from a single representative experiment. 32 C. W. Lee et al. and 15 8% to 310%. The remaining recovered activities Human PMNs (107/ml) were stimulated with vary- were almost entirely in pellet 1, representing unbroken ing concentrations of PMA, a specific granule-releas- cells. ing agent (White & Estensen, 1974), for 30 min at 37°. The maximum effect of PMA on both lysozyme and Association of LTD4 dipeptidase activity with specific LTD4 dipeptidase release was achieved at concentra- granules tions of 20-50 ng/ml without further significant secretion of either enzyme at higher concentrations. When 107 PMNs were incubated in 1 ml ofHBSS with There was a constant relationship (r = 0-96, n =8, or without 1-8 mm calcium for 0, 15, 30 and 60 min, at P<0001) between the release of LTD4 370 and centrifuged at 8000 g for 2 min at 40, the dipeptidase and lysozyme in response to PMA time-dependent and calcium-related appearance of (Fig. 4). In two experiments PMA (50 ng/ml) lysozyme in the supernatant occurred in a constant stimulation of relationship with LTD4 dipeptidase (Fig. 3, r=0 97, PMNs (107/ml) pretreated with cytochalasin B (5 ug/ml) for 10 min resulted in 36% and 76% lysozyme n = 16, P < 0001). Secretion of both lysozyme and release and 22 6% and 28% release LTD4 dipeptidase was significantly reduced in parallel fl-glucuronidase throughout the incubation period in the absence of after 30 min, as compared with 36% and 41% lysozyme extracellular calcium. The addition of calcium to the release and 2-7% and 6 0% f,-glucuronidase release supernatants from the calcium-free PMN incubations without pretreatment with cytochalasin B. LDH did not result in increased conversion of LTD4 to release was 2-4% and 7-8% in the presence and 1.2% LTE4. Release of the azurophil granule marker, and 2 8% in the absence ofcytochalasin B. Secretion of azurophilic granules by introducing cytochalasin B fl-glucuronidase, and of cytoplasmic LDH was mini- (Yurewicz & Zimmerman, 1977) contributed no addi- mal for the entire 60 min ofincubation with or without tional LTD4 dipeptidase activity over 5-1 units and 7-8 the presence of calcium. The selective secretion of units, respectively, released from the specific granules human PMN-specific granules, as assessed by lyso- by PMA alone. zyme release, is calcium-dependent (Goldstein, Horn, Kaplan & Weissmann, 1974; Goldstein, Weissmann, That LTD4 dipeptidase activity was associated only Dunham & Soberman, 1975; Wright & Gallin, 1979), with the specific granules was further shown by using and hence the associated release of the LTD4 dipepti- incremental concentrations of calcium ionophore dase (Fig. 3) suggested its origin from specific A23187 to stimulate the selective release of specific granules.

5- c 4- _P

4 - 0 03 Q) 0 0 3 af)Q 2 2-

0 10 20 30 40 Lysozyme release (%) 0 10 20 30 40 50 Lysozyme release (%) Figure 3. Relationship between release oflysozyme and LTD4 dipeptidase at 0, 15, 30 and 60 min at 37° in the presence (@) Figure 4. Relationship between release oflysozyme and LTD4 and absence (0) ofcalcium from two experiments. Release of dipeptidase in response to PMA 0, 10, 25 and 50 ng/ml. P-glucuronidase and LDH ranged from 1% to 8% with Release of,-glucuronidase and LDH ranged from 1% to 7% calcium present, and from 2% to 6% without calcium. and 2% to 6%, respectively, from two experiments. y=0 3197+0 13x, r== 0967, n= 16. y=0-068+0 12x, r=0 96, n=8. Conversion ofLTD4 to LTE4 by human PMNs 33 that the PMN suspensions quantitatively converted radiolabelled LTD4 to a single radiolabelled metabo- lite with the retention time of LTE4 on RP-HPLC. Because the conversion of LTD4 by human PMN 60 6 suspensions yielded a single metabolic product that co-chromatographed with LTE4 and had the identical spasmogenic activity, the responsible PMN-derived 0 to indicate that 240 2 enzyme was termed a dipeptidase 0Y E 40L 4o glycine had been cleaved from the 6(R)-S-cysteinyl- glycyl side-chain of LTD4 substrate. That the neutrophil dipeptidase functioning in the 0 1fF8 10-7 Ic6 10-5 extracellular conversion of LTD4 to LTE4 was derived A23187 concentration (M) from the granular compartment is indicated both by with subcellular fractionation of cells Figure 5. Dose-related percentage release of lysozyme (0), its localization ,B-glucuronidase (es) and LDH (A) and of LTD4 dipeptidase and its extracellular appearance after stimulus- activity (-) in response to A23 187. Each point represents the induced secretion. With subcellular fractionation the mean + SD of three experiments, except for LDH which was recovered dipeptidase activity was consistently assessed in only one. detected in the fraction containing the granule markers fl-glucuronidase and lysozyme (Table 1), with granules at low concentrations and both types of the remaining granular material being almost entirely granules at high concentrations (Goldstein et al., confined to the pellet containing cellular debris and 1975). Ten million PMNs incubated with A23187 at unbroken cells. As specific granules are known to concentrations less than 10-7 M for 30 min showed secrete into the extracellular milieu in relation to its concentration-dependent release of LTD4 dipeptidase calcium concentration (Goldstein et al., 1974, 1975; and specific granule-associated lysozyme (Fig. 5). At Wright & Gallin, 1979), it seemed likely that the 10-6 M and 10-5 M A23187 there was a release of extracellular dipeptidase obtained from apparently glucuronidase and LDH along with incremental unstimulated PMNs might be derived from this com- release of lysozyme, but without additional LTD4 partment. The established components of the specific dipeptidase. granules of the human PMNs include lysozyme (Spitz- nagel, Dalldorf, Leffel, Folds, Welsh, Cooney & Martin, 1974), lactoferrin (Leffel & Spitznagel, 1972; DISCUSSION Bentwood & Henson, 1980) and vitamin B12 binding protein (Kane & Peters, 1975). The exact granular Human PMNs diminish the spasmogenic activity of localization of a metallo-proteinase, termed colla- LTD4 due to the extracellular action of a dipeptidase genase, that degrades acid-soluble collagen (Lazarus, released from the specific granules. That the time- Daniels, Brown, Bladen & Fullmer, 1965) has been dependent inactivation of LTD4 by PMNs (Fig. 1) is controversial (Ohlsson & Olsson, 1973; Murphy, extracellular is indicated by the progressive quantifi- Reynolds, Bretz & Baggiolini, 1977), perhaps due to its able conversion of LTD4 to a single metabolite (Fig. 2) existence as a complex of a 65,600-67,000 mol. wt in the supernatant with the physical characteristics of proteinase and a 20,000-25,000 mol. wt inhibitor LTE4 as assessed by resolution by RP-HPLC. The (Tschesche & Macartney, 1981). single conversion product formed extracellularly in Even though lysozyme resides in both specific and the presence of cells and then isolated from the azurophilic granules, the convenience of its measure- supernatant not only exhibited the retention time of ment and the existence of techniques for achieving synthetic LTE4 on RP-HPLC but also had spasmo- non-cytotoxic release of specific granule-associated genic activity for the guinea-pig ileum of 1 2+0-3 lysozyme prompted measurement of this enzyme as a u./pmol, which is identical to that of synthetic LTE4 marker for the specific granule. The time- and calcium and substantially less than the 5 0 + 04 u./pmol ion-dependent release of LTD4 dipeptidase and lyso- characteristic of the synthetic LTD4. The possible zyme from neutrophils was linear (Fig. 3; r= 097, presence of additional metabolic products lacking the n = 16, P < 0-001). There was no incremental release of triene structure was minimized by the demonstration cytoplasmic LDH or the azurophilic granule marker, 34 C. W. Lee et al. fl-glucuronidase, supporting the view that although combining centrifugation and sedimentation at 1 g. lysozyme resides in both specific and azurophilic Scand. J. clin. Lab. Invest. 21 (Suppl. 97), 77. limited calcium-dependent lysozyme secre- BROCKLEHURST W.E. (1960) The release of histamine and granules, formation of a slow reacting substance of anaphylaxis tion is derived selectively from the former. With (SRS-A) during anaphylactic shock. J. Physiol. 151, 416. limited concentrations of calcium ionophore, 10-8 COREY E.J., CLARK D.A., GoTo G., MARFAT A., Mios- and 10-I M, accelerated secretion of LTD4 dipeptidase KOWSKI C., SAMUELSSON B. & HAMMARSTROM S. (1980a) Stereospecific total synthesis of a 'slow reacting sub- and lysozyme occurred with minimal release of fi-glu- stance' ofanaphylaxis, leukotriene C-1. J. Am. chem. Soc. curonidase or LDH (Fig. 5). PMA, a selective stimulus 108, 1436 and correction 108, 3663. for specific granule secretion (White & Estensen, 1974; COREY E.J., CLARK D.A., MARFAT A. & GOTo G. (1980b) Goldstein et al., 1975; Wright, Bralove & Gallin, Total synthesis of slow reacting substance (SRS), 'leuko- 1977), also released LTD4 dipeptidase and lysozyme in triene C-2' (1 1-trans-leukotriene C) and leukotriene D. Tetrahedron Lett. 21, 3143. a linear relationship (Fig. 4; r = 096, n = 8, P < 0-001) DAHLEN S.E., BWORK J., HEDQVIST P., ARFORS K.E., HAM- without the concomitant extracellular appearance of MARSTROM S., LINDGREN J.A., & SAMUELSSON B. (1981) LDH or P-glucuronidase. In the presence of cyto- Leukotrienes promote plasma leakage and leukocyte chalasin B, PMA elicited additional non-cytotoxic adhesion in postcapillary response. Proc. natn. Acad. Sci. but not of LTD4 dipep- 78, 3887. release of f3-glucuronidase DAHLEN S.E., HEDQVIST P., HAMMARSTROM S. & SAMUELSSON tidase. Thus, the LTD4 dipeptidase recognized to B. (1980) Leukotrienes are potent constrictors of human reside in the neutrophil granules by subcellular frac- bronchi. Nature (Lond.), 288, 184. tionations is established as a resident of the specific DRAZEN J.M. & AUSTEN K.F. (1974) Effects of intravenous granule by its appearance in relation to lysozyme and administration ofslow reacting substance ofanaphylaxis, the histamine, bradykinin, and F2 on pulmon- not fl-glucuronidase during selective stimulation of ary mechanics in the guinea pig. J. clin. Invest. 53, 1679. cell with extracellular calcium, limited concentrations DRAZEN J.M., AUSTEN K.F., LEWIS R.A., CLARK D.A., GoTo of the calcium ionophore, or PMA in the absence of G., MARFAT A. & COREY E.J. (1980) Comparative airway cytochalasin B. and vascular activities ofleukotrienes C and D in vivo and in vitro. Proc. natn. Acad. Sci. 77, 4354. ACKNOWLEDGMENTS DRAZEN J.M., LEWIS R.A., WASSERMAN S.I., ORANGE R.P. & AUSTEN K.F. (1979) Differential effects of a partially AI-07722, purified preparation of slow reacting substance of ana- This work was supported in part by Grants phylaxis on guinea pig tracheal spirals and parenchymal AI-10356, HL-17382, HL-19777, and RR-05669 from strips. J. clin. Invest. 63, 1. the National Institutes of Health, and in part by DRAZEN J.M., VENUGOPALAN C.S., AUSTEN K.F., BRION F. & Grants from the Lilla Babbitt Hyde Foundation and COREY E.J. (1982) Effects of leukotriene E on pulmonary Dr mechanics in the guinea pig. Am. Rev. respir. Dis. 125, the New England Home for Crippled Children. 290. C. W. Lee is a postdoctoral trainee supported by GOLDSTEIN I.M., HOFFSTEIN S.T. & WEISSMANN G. (1975) Grant AI-07167 from the National Institutes of Mechanisms of lysosomal enzyme release from human Health. Dr R. A. Lewis is a recipient of an Allergic polymorphonuclear leukocytes. Effect of phorbol myris- Diseases Academic Award (AI-00399) from the tate acetate. J. Cell Biol. 66, 647. 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