Proc. Nati. Acad. Sci. USA Vol. 80, pp. 5425-5429, September 1983 Medical Sciences

Metabolism of A4 by an in blood plasma: A possible leukotactic mechanism (extracellular metabolism/leulkotriene B4///diapedesis) F. FITZPATRICK*, J. HAEGGSTROM, E. GRANSTROM, AND B. SAMUELSSON Department of Physiological Chemistry, Karolinska Institutet, Box 60400, S-104 01 Stockholm, Sweden Communicated by Sune Bergstrom, May 23, 1983 ABSTRACT Cell-free mammalian plasma.transformed leu- Pronase proteolytic enzyme (Calbiochem) were used as re- kotriene A4 into . This. conversion originated from ceived. a soluble enzymatic activity. Heating at 560C or digestion of plasma Mammalian Plasma. Mammalian blood collected over hep- with a proteolytic enzyme eliminated formation of leukotriene B4 arin, 100 international units/ml, was.centrifuged at 250 X g for but not other diastereomeric dihydroxyicosatetraenoic acids formed 30 min at 250C to sediment cells. Plasma was sterilized and small by nonenzymatic hydrolysis of . Plasma from sev- cells were excluded by passage through a 0.22-,um-pore Millex- eral mammals, including guinea pigs, pigs, cows, sheep, rabbits, HA filter (Millipore). For certain experiments plasma was (i) rats, dogs, and humans, exhibited a qualitatively similar activity. centrifuged at 100,000 X g for 60 min at 40C, or (ii) fractionated Maximal production of leukotriene B4 occurred in guinea pig plasma on a column containing Sephadex G-25 me- near a pH of 7.6; metabolic capacity approached saturation at a PD-10 gel filtration substrate concentration of 10 FaM. Leukotriene A4 anion was dium (bed volume 9.1 ml, bed height 5 cm) equilibrated with transformed but not its methyl ester. The results suggest that con- 0.10 M pH 7.4 potassium phosphate buffer, or (iii) separated version of leukotriene A4 by an enzyme in blood plasma of mam- into macromolecule-free (<5%) and macromolecule-rich (>95%) mals may provide local gradients of the chemotactic substance fractions by ultrafiltration through a hydrophilic Amicon YMT leukotriene B4. High interfacial concentrations of leukotriene B4 membrane (Mr cutoff 10,000), or (iv) dialyzed against 0.10 M between the vascular and leukocytes could facilitate pH 7.4 phosphate buffer. Identical samples, heated at 560C for their diapedesis and accumulation at inflammatory sites. 1 hr, were used as controls. Plasma (1.0 ml) diluted with 0.10 M pH 7.4 phosphate buffer (1.0 ml) was also subjected to diges- (LT), a recently discovered class of naturally oc- tion at 370C for 6 hr with Pronase (5 mg). curring lipids that originate from the oxidative metabolism of Procedure. [3H]LTA4 lithium salt (1-15 /Lg) was added to , are involved in immediate hypersensitivity, plasma (1.0 ml) diluted with 0.10 M pH 7.4 phosphate buffer inflammation, and respiratory disorders (1, 2). A labile epoxide, (1.0 ml). After-30 min at 370C the solution was acidified to pH (5S)-5,6-oxido-7,9-trans-11,14-cis-icosatetraenoic acid, or LTA4 3 with 1 M HCL. LTA4 transformation products were isolated (3), is a pivotal intermediate in the cellular enzymatic pathway by percolating acidic, diluted samples (10 ml) through a C18 SEP- that generates the biologically active leukotrienes designated PAK cartridge. Adsorbed products were purified and concen- LTB4 [(5S, 12R)-dihydroxy-6, 14-cis,8, 10-trans-icosatetraenoic trated by consecutive elution with 10 ml each of water, hexane, acid] (4), LTC4 [(5S)-hydroxy-(6R)-S-glutathionyl-7,9-trans-11, 14- methyl formate, and methanol (10). The methyl formate frac- cis-icosatetraenoic acid] (5), LTD4 [(5S)-hydroxy-(6R)-S-cyste- tion contained >95% of the adsorbed radioactive components; inylglycine)-7,9-trans-11, 14-cis-icosatetraenoic acid] (6, 7), and the solvent was evaporated under nitrogen and the residue was so forth (1). Because of its central role in the biosynthesis of reconstituted in mobile phase (0.10 ml) and analyzed by HPLC leukotrienes, metabolic studies were initiated with intact LTA4 as described (11). Products were detected by UV absorption at rather than endogenous or exogenous arachidonic acid. This 270 nm and by liquid scintillation counting of tritium in the cell-free chromatographic fractions. paper describes the metabolism of LTA4 by plasma LTB4 Bioassay. Bioassay for LTB4 was performed on isolated from mammals, especially guinea pigs. guinea pig lung parenchymal strips suspended in a 3-ml per- fusion chamber containing Tyrode's buffer under nonflow con- MATERIALS AND METHODS ditions as described (12). Materials. [14, 15-3H]LTA4 lithium salt, 0.01-0.05 ,tCi/pig Lysozyme Assay. Lysozyme was assayed turbidimetrically = X salt were with suspensions of Micrococcus lysodeikticus (13). (1 Ci 3.7 10'° Bq), and unlabeled LTA4 lithium Instrumental Analysis. Gas chromatography/mass spec- prepared as described (8). The purity of both starting mate- trometry (GC/MS) was done on an LKB 9000 mass spectrom- rials-LTA4 methyl ester and [14,15-3H]LTA4 methyl ester (31 eter equipped with a 2 m X 3 mm inside diameter column of Ci/mmol; New England Nuclear)-and the products was eval- OV-101, 1% wt/vol, on Chromosorb WHP, 100-120 mesh (All- uated by UV spectrophotometry and reversed-phase high-per- tech, Deerfield, IL). The gas chromatograph was operated iso- formance liquid chromatography (RP-HPLC) (9). Synthetic LTB4 thermally at 210°C with a helium flow of 20 ml/min. Ionization (Upjohn); SEP-PAK cartridges (Waters Associates); PD-10 gel current was 60 ,uA. filtration columns (Pharmacia); Centrifree ultrafiltration mem- energy was 22.5 eV; trap branes (Amicon); glass-distilled solvents for HPLC (Rathburn Abbreviations: LT, leukotriene; RP-HPLC, reversed-phase high-per- Chemicals, Walkerburn, Scotland); methyl formate (Merck); and formance liquid chromatography; SP-HPLC, straight-phase high-per- formance liquid chromatography; HPETE, hydroperoxyicosatetraenoic The publication costs of this article were defrayed in part by page charge acid. payment. This article must therefore be hereby marked "advertise- * Permanent address: Experimental Science I, The Upjohn Co., Kala- ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. mazoo, MI 49001. 5425 Downloaded by guest on September 30, 2021 5426 Medical Sciences: Fitzpatrick et al Proc. Natl. Acad. Sci. USA 80 (1983) RESULTS hydrolysis of LTA4. Their liquid chromatographic mobility, their .RP-HPLC analysis detected, five UV-absorbing radioactive UV spectra with triplet peaks at 263, 272, and 284 nm; the C species (I-V) from guinea pig plasma incubated with [3H]LTA4 values 24.0 (peak IV) and 23.9 (peak V) for their methyl ester (Fig. 1). In 0.10 M pH 7.4 phosphate buffer or in buffer con- trimethylsilyl ethers on an OV-101 column, and their mass spectra taining guinea pig albumin (40 mg/ml) only four UV-absorbing with prominent ions at m/z 171, 203 (base peak), 225, 291, 383, radioactive peaks (I, II, IV, and V) appeared; peak III was ab- 404, 463, 479, and 494 were identical to published values (11, sent. Peaks I and II corresponded to a pair of diastereomeric 14) and to those of standards of diastereomeric 5,6-dihydroxy- 5,12-dihydroxyicosatetraenoic acids formed by nonenzymatic 7,9,11,14-icosatetraenoic acid. hydrolysis of LTA4. The following properties were all identical When [3H]LTA4 was incubated with guinea pig plasma, an to published values (11, 14) and to those of authentic standards additional UV-absorbing radioactive component, peak III, ap- of (5S,12R-dihydroxy-6,8,10-trans-14-cis-icosatetraenoic acid peared. Its chromatographic mobility; its UV spectrum, with (peak I) and (5S, 12S)-dihydroxy-6,8, 10-trans-14-cis~icosate- triplet peaks at 260, 270, and 281 nm; its C value, 23.6; and its ;traenoic acid (peak II): their liquid chromatographic mobility; mass spectrum with fragment ions at m/z = 129 (base peak), their UV spectra with triplet absorbance peaks at 259, 269, and 167, 191, 203, 217, 229, 267, 293, 383, 404, 463, and 479 were 280. nm; the C value (relative retention time), 24.9, for their identical to published values (11, 14) and to those obtained from methyl ester trimethylsilyl ethers on an OV-101 gas chromato- synthetic LTB4. After conversion to a methyl ester, peak III graphic column; and their mass spectra with fragment ions at was homogeneous by SP-HPLC. It eluted coincident with syn- m/z (mass-to-charge ratio) = 129 (base peak), 191, 203, 217, thetic LTB4 methyl ester and contained no detectable (5S, 12S)- 267, 293, 383, 404, 463, and 479. The UV, gas chromatographic, dihydroxy-6,10-trans-8;14-cis-icosatetraenoic acid. The UV, gas and mass spectral properties of the methyl esters isolated by chromatographic, and mass spectral properties of the methyl straight-phase HPLC (SPrHPLC) were identical to those cited ester isolated by SP-HPLC were identical to those cited above. above. Peak III was collected from several experiments to prepare a Peaks IV and V corresponded to a pair of diastereomeric 5,6- solution for comparative analysis by UV spectrophotometry and dihydroxyicosatetraenoic acids, also formed by nonenzymatic bioassay. On guinea pig lung parenchymal strips the sample had an indomethacin-sensitive contractile activity that was indistin- guishable from that of synthetic LTB4 (Fig. 2)..Bioassay and UV spectrophotometry gave comparable values, 1.2 and 1.0 mM, respectively, for the LTB4.concentrations in the samples. Results were qualitatively equivalent for plasma from normal or sensitized guinea pigs. Analyses by either SP-HPLC or RP- HPLC were consistent. Each plasma examined metabolized LTA4 into LTB4, but not into (5S,12S)-dihydroxy-6, 10-trans-8, 14- cis-icosatetraenoic acid. At an initial LTA4 concentration of 5 uM, LTB4 formation approached a maximum within 5 min (Fig. 3). A decline in the amounts of nonenzymatically produced 5,12-dihydroxyicosa- tetraenoic acids, with corresponding increases in LTB4 for- mation, reflected the consumption of LTA4 substrate. Under the conditions described, 8.2 ± 3.6% (mean + SD, n = 8, range Hi 3PM LTB4 0.3pM l 1

peak 1 Hi 3puM 3pl LTC4 InM I

Hi 100puM -KCI 4OjuM Hi 3pM peak I I -Jr II3p [plus INDOMETHACIN 1OPAu > MINUTES FIG. 2. Isometric contraction of guinea pig lung parenchymal strips FiG. 1. RP-HPLC analysis of products formed during the incuba- in response to 3 gM histamine (Hi) and 0.30 ,4M LTB4 standard (top tion of 5 ,AM [3H]LTA4 with guinea pig plasma. Peaks not designated traces) or to 3 ,uM histamine, 3 1.l ofpeak III in buffer, and 1 nM LTC4 with roman numerals correspond to endogenous UV-absorbing com- (middle traces). Perfusion with buffer containing 10 tLM indomethacin ponents of plasma. The UV spectrum of peak' HI (Inset) had peaks at abolished the contractile response due to LTB4 or peak'III, but not his- 260, 270, and 281 nm. tamine (bottom traces). Downloaded by guest on September 30, 2021 Medical Sciences: Fitzpatrick et aL Proc. Natl. Acad. Sci. USA 80 (1983) 5427 Table 1. pH Dependence of LTB4 formation Metabolite formed, nmol pH I II LTB4 6.5 1.12 1.08 0.48 7.1 1.13 1.01 0.48 7.6 0.70 0.70 1.16 LTB4 8.5 0.90 0.81 0.89 Guinea pig plasma was diluted 1:1 with an appropriate 0.10 M phos- phate buffer to obtain the pH tabulated. The initial LTA4 concentra- tion was5 ,M. Simples were treated as described and analyzed by RP- HPLC. I and .11 are A6-trans-LTB4 and A6-trans-12-epi-LTB4, respec- tively, formed by nonenzymatic decay. of LTA4.

brane with a Mr cutoff of 10,000. No activity appeared in the protein-free-filtrate. (iii) Ultracentrifugation at 100,000 x g for 60 min did not sediment any active particulate fraction from guinea pig plasma. The supernatant retained a heat-sensitive activity. (iv) Activity eluted with the-unretained high molecular weight fraction (Mr . 10,000) when guinea pig plasma (2 ml) MINUTES was chromatographed on a PD-10 gel filtration column. (v) The FIG. 3. Transformation kinetics of 5 ,tM LTA4 into LTB4 (Ill), A6- activity in plasma was susceptible to proteolytic digestion. In- trans-LTB4 (I), and A6-trans-12-epi-LTB4 (II). cubation at 370C for 6 hr with Pronase completely eliminated its capacity to. form LTB4. Control samples incubated under = 3.2-15.2%) of LTA4 was transformed into LTB4 by guinea identical conditions without Pronase retained 95-100% of their pig plasma diluted 1:1 with 0.10 M pH 7.4 phosphate buffer. enzymatic activity.. Activity, though detectable, diminished to .<1% conversion at a 1:10 dilution; activity was undetectable at a 1:100 dilution. In contrast to the anionic form, the methyl ester of LTA4 was cn HUMAN PLASMA not transformed into LTB4 or LTB4 methyl ester. Heating guinea pig plasma at 560C eliminated its ability to. x 4 convert LTA4 into LTB4 (Fig. 4). Spontaneous decay varied among different samples. Plasma held at 40C for 240 hr retained E lzul ( 31-3 I 50-80% of its activity; plasma samples incubated for 6 hr at 37C retained 80-100% of their activity. The activity in plasma was I-- characterized as a soluble, high molecular weight-protein com.- >2p ponent in five ways: (i) guinea pig plasma (5 ml) dialyzed against 0.10 M pH 7.4-phosphate buffer-(250 ml) for 24 hr at 40C still 1 0 converted LTA4 into LTB4. Heating at 560Cinactivated the di- 4 alyzed plasma. No activity was evident in the dialysate. The dialysis.;membrane retained substances with Mr .10,000. (ii) A heat-sensitive activity remained in the* macromolecule-en- riched fraction after ultrafiltration through a hydrophilic mem- 0.00 K E C 0

w z nanometers 4

0.020- &LIK

10 20 30 40 50 MINUTES FIG. 4. Influence of560C heating on formation ofLTB4 (III)byguinea FIG. 5. RP-HPLC analysis of products formed from the incubation pigplasma. SP-HPLC analysis. Iis A6-trans-LTB4; His A6-trans-12-epi- of 10 pM LTA4 with plasma from a human female. The UV spectrum LTB4. of peak III (Inset) had maxima at 260, 270, and 281 nm. Downloaded by guest on September 30, 2021 5428 Medical Sciences: Fitzpatrick et al Proc. Natl. Acad. Sci. USA 80 (1983) Table 2. Characteristics of plasma enzymatic activity that able in the plasma of other mammals, including the rat, rabbit, converts LTA4 into LTB4 cow, dog, pig, sheep, and human. Fig. 5 depicts results from 1. Nondialyzable, Mr >10,000 the RP-HPLC analysis for human plasma incubated with 10 AM 2. Present in macromolecule fraction after ultrafiltration through LTA4 lithium salt. In all cases, enzymatic activity was abolished membranes with Mr cutoff -10,000 by 560C heating or by proteolytic digestion, and it was char- 3. Not retained by Sephadex G-25 medium, activity present in acterized as a soluble, high molecular weight component. The protein fraction, Mr -10,000 amount of LTB4 formed differed among the species; relative 4. Activity destroyed by heating of plasma at 560C production (mean ± SD) for several individual animals was 8.2 5. Activity destroyed by digestion of plasma with Pronase ± 3.6% (n = 8), 2.3 ± 0.5% (n = 3), 1.5 + 0.4% (n = 4), and 6. Soluble component, not sedimented by centrifugation at 100,000 1.2 ± 0.3% (n = 4), respectively for guinea pigs, pigs, cows, x g for 60 min and sheep when plasma (1.0 ml) diluted with 0.10 M pH 7.4 7. pH-Dependent transformation with optimum pH 7.6 in guinea phosphate buffer (1.0 ml) was incubated with 5 ,uM LTA4 as pig plasma described. For human plasma, concentrated 2x by ultrafiltra- 8. Substrate specific for LTA4 anion, not its methyl ester tion, LTB4 production was 1.14 ± 0.40% (n = 6). Table 2 sum- 9. Saturable at substrate concentrations >10 AM marizes the characteristics of the plasma component that con- 10. Activity not correlated with leukocyte lysis verted LTA4 into LTB4. Conversion of LTA4 to LTB4 in guinea pig plasma was pH DISCUSSION dependent, with an optimum near the physiological value (Ta- Our results indicate that cell-free mammalian blood plasma ble 1). The transforming capacity of guinea pig plasma ap- contains a component capable of converting LTA4 into LTB4, proached saturation at an initial substrate concentration of 10 a chemokinetic, chemotactic, myotropic substance that induces ,uM. For instance, LTB4 production was 0.34, 0.59, 1.26, and adhesion, aggregation, and degranulation (15-18). 1.50 ,uM when substrate concentrations were 2.65, 5.3, 10.6, Heat inactivation, proteolytic digestion, dialysis, ultrafiltra- and 21.2 AuM, respectively. tion, gel filtration, ultracentrifugation, pH dependence, sub- The LTA4-metabolizing activity did not originate from leu- strate specificity, and saturability at micromolar substrate con- kocyte lysis during the isolation of plasma. Lysosomal enzyme centrations support the conclusion that LTB4 formation is activity was undetectable in most plasma samples and, com- attributable to an enzymatic activity. pared to two samples with detectable lysozyme corresponding Leukotriene biosynthesis requires that are distrib- to 2% and 4% of the maximal available, there was no correlation uted among different subcellular compartments and it appears between LTB4 formation and leukocyte degranulation. Fur- that their biosynthesis involves movement of intermediates be- thermore, when leukocytes from guinea pig blood (10 ml) were tween these compartments (19). In the case of LTB4 formation isolated and deliberately lysed, the cell-free supernatant con- the action of a soluble cytosolic hydrolase has been proposed taining as much as 10% of the available lysozyme was unable to be the rate-limiting step (19). Consequently, intermediate to transform LTA4 into LTB4. substrates such as 5-hydroperoxyicosatetraenoic acid (5-HPETE) A similar capacity to transform LTA4 into LTB4 was detect- or LTA4 must accumulate, decay via alternate enzymatic and

FIG. 6. Proposed scheme for LTA4-- LTB4 plasma enzymatic activity involvement with neutrophil diapedesis and . AA, arachidonic acid; 5-HETE, 5-hydroxyicosatetraenoic acid. Downloaded by guest on September 30, 2021 Medical Sciences: Fitzpatrick et al. Proc. Nati. Acad. Sci. USA 80 (1983) 5429 nonenzymatic processes, or be released from cells, such as leu- 1. Samuelsson, B. (1983) Science 220, 568-575. kocytes. In the latter instance a plasma enzyme capable of con- 2. Lewis, R. A. & Austen, K. F. (1981) Nature (London) 293, 103- verting extracellular LTA4 seems reasonable from several per- 108. 3. Borgeat, P. & Samuelsson, B. (1979) Proc. Natl. Acad. Sci. USA 76, spectives. Physiologically, it could produce high interfacial 3213-3217. concentrations of LTB4 between vascular endothelial surfaces 4. Borgeat, P. & Samuelsson, B. (1979)J. Biol. Chem. 254, 2643-2646. and leukocytes. Such local gradients of a highly chemotactic 5. Murphy, R., Hammarstrom, S. & Samuelsson, B. (1979) Proc. Natl. agent could facilitate adhesion of leukocytes to the endothelium Acad. Sci. USA 76, 4275-4279. and their diapedesis between cells with subsequent accumu- 6. Orning, L., Hammarstr6m, S. & Samuelsson, B. (1980) Proc. Nati. lation in the extravascular space. It is interesting to consider our Acad. Sci. USA 77, 2014-2017. results and their proposed significance in the context of known 7. Lewis, R., Austen, K. F., Drazen, J., Clark, D., Marfat, A. & in vivo Namely, Corey, E. J. (1980) Proc. Nati. Acad. Sci. USA 77, 3710-3716. characteristics of granulocyte adherence (20). 8. Corey, E. J., Clark, D. A., Goto, G., Marfat, A., Samuelsson, B. inflammatory stimuli increase the pool of marginating neutro- & Hammerstrom, S. (1981)J. Am. Chem. Soc. 102, 1436-1439, and phils; adherence involves no visible specifically adhesive sub- correction (1981) 102, 3663. stance between neutrophils and endothelium; adherence is a 9. Wynalda, M., Morton, D., Kelly, R. & Fitzpatrick, F. (1982) Anal. local phenomenon (displaced neutrophils do not adhere else- Chem. 54, 1079-1082. where and newly arriving neutrophils will adhere to vacant sites); 10. Powell, W. S. (1980) 20, 947-957. and anticoagulants or fibrinolytic substances do not inhibit ad- 11. Borgeat, P. & Samuelsson, B. (1979)J. Biol. Chem. 254, 7865-7869. in contrast to corticosteroids, divalent cation chela- 12. Dahlen, S.-E., Hedqvist, P., Granstrom, E., Lindgren, J. A. & herence, Westlund, P. (1983) Acta Physiol. Scand., in press. tors, or channel blockers (20). Furthermore, one or more non- 13. Smolelis, A. N. & Hartsell, S. E. (1949)J. Bacteriol. 58, 731-736. dialyzable, 56TC heat sensitive factors in plasma have a dem- 14. RAdmark, O., Malmsten, C., Samuelsson, B., Clark, D., Goto, onstrated ability to modulate granulocyte adherence in vivo (21). G., Marfat, A. & Corey, E. (1980) Biochem. Biophys. Res. Com- Leukocyte chemotaxis and diapedesis is a complex process that mun. 92, 954-961. involves several molecular and cellular mechanisms (22), some 15. Ford-Hutchinson, A. W., Bray, M., Doig, M., Shipley, M. & of which remain unknown (23). Our results suggest that extra- Smith, M. (1980) Nature (London) 286, 264. cellular enzymatic transformation of LTA4 into a potent chemo- 16. Malmsten, C., Palmblad, J., Uden, A. M., RAdmark, O., Eng- stedt, L. & Samuelsson, B. (1980) Acta Physiol. Scand. 110, 449- taxin, LTB4, may be involved. Because plasma exudation is an- 451. other prominent feature of inflammation and because LTB4 17. Serhan, C., Radin, A., Smolen, J., Korchak, H., Samuelsson, B. induces not only neutrophil adhesion and locomotion but also & Weissmann, G. (1982) Biochem. Biophys. Res. Commun. 107, aggregation, degranulation, and superoxide generation (15-18), 1006-1012. the plasma enzymatic activity responsible for its production may 18. Dahlen, S.-E., Bjork, J., Hedqvist, P., Arfors, K.-E., Hammar- play a general role in modulating the inflammatory response. strom, S., Lindgren, J. A & Samuelsson, B. (1981) Proc. Nati. Acad. The physiological significance of our results depends upon Sci. USA 78, 3887-3891. 19. Jakschik, B. & Kuo, C. (1983) in Advances in , cellular release of LTA4, which must remain in an intact form, and Leukotriene Research, eds. Samuelsson, B., at least momentarily, for eventual presentation to and conver- Ramwell, P. & Paoletti, R. (Raven, New York), Vol. 11, pp. 141- sion by the enzymatic activity reported here. Several prece- 145. dents indicate that this is reasonable. First, washed, viable neu- 20. MacGregor, R. (1980) in The Cell Biology of Inflammation, ed. trophil suspensions convert exogenously added LTA4 into various Weissmann, G. (Elsevier/North-Holland, Amsterdam), pp. 267- leukotrienes in vitro (14, 24) and intact LTA4 has been isolated 298. from neutrophils stimulated by ionophore A23187 (25). These 21. Lentnek, A. L., Schreiber, A. D. & MacGregor, R. R. (1976)J. results indicate that LTA4 can traverse cell membranes. Sec- Clin. Invest. 57, 1098-1103. 22. Snyderman, R. & Goetzel, E. J. (1981) Science 213, 830-837. ond, another plasma component, albumin, can stabilize LTA4 23. Vinegar, R., Truax, J., Selph, J. & Voelker, F. (1982) Fed. Proc. to a significant degree in vitro (26). Third, transcellular me- Fed. Am. Soc. Exp. Biol. 41, 2588-2594. tabolism occurs along the /endothelium (27), platelet/ 24. Radmark, O., Malmsten, C. & Samuelsson, B. (1980) Biochem. neutrophil (28, 29), and the lymphocyte/ axes (30). Biophys. Res. Commun. 96, 1679-1687. It is axiomatic that this related process, transcellular metabo- 25. RAdmark, O., Malmsten, C., Samuelsson, B., Goto, G., Marfat, lism, involves release and transfer of substrate from a donor cell A. & Corey, E. J. (1980)J. Biol. Chem. 255, 11828-11831. for assimilation and metabolism by a recipient cell. Labile sub- 26. Fitzpatrick, F., Morton, D. & Wynalda, M. (1982)J. Biol. Chem. undergo this 257, 4680-4683. strates, such as prostaglandin endoperoxide, can 27. Marcus, A., Weksler, B., Jaffe, E. & Broekman, M. (1980)J. Clin. process (27). Extracellular conversion, as described here for Invest. 66, 979-986. LTA4, is an unusual enzymatic process in the arachidonic acid 28. Marcus, A., Broekman, M., Safier, L., Ullman, H., Islam, N., cascade. All previously described enzymatic transformations of Serhan, C., Rutherford, L., Korchak, H. & Weissmann, G. (1982) LTA4, or its counterparts, the prostaglandin endoperoxides, have Biochem. Biophys. Res. Commun. 109, 130-137. originated exclusively from cell-associated microsomal (31-33) 29. Maclouf, J., Fruteau de Laclos, B. & Borgeat, P. (1982) Proc. Natl. or cytosolic enzymes (34, 35). Acad. Sci. USA 79, 6042-6046. 30. Goldyne, M. & Stobo, J. (1982) Prostaglandins 24, 623-630. Fig. 6 depicts our proposed scheme for one physiological role 31. Wlodawer, P. & Hammarstrom, S. (1979) FEBS Lett. 97, 32-36. involving extracellular conversion of LTA4 into LTB4. This 32. Hammarstrom, S. & Falardeau, P. (1977) Proc. Natl. Acad. Sci. USA scheme is notable because it accommodates certain paradoxical 74, 3691-3695. aspects of diapedesis and inflammation such as the obligate 33. Jakschik, B. A., Harper, T. & Murphy, R. (1982)J Biol. Chem. 257, presence of neutrophils to mediate LTB4-induced increases in 5346-5349. vascular permeability (36). 34. Nugteren, D. & Hazelhof, E. (1973) Biochim. Biophys. Acta 326, 448-461. We thank Dr. S. E. Dahlen for LTB4 bioassay. This work was sup- 35. Maycock, A., Anderson, N., DeSousa, D. & Kuehl, F. (1982)J. ported by the Swedish Medical Research Council (projects 03X-217 and Biol. Chem. 257, 13911-13914. 03X-5915). The sabbatical of F.A. F. was financed by the Upjohn Com- 36. Bjork, J., Hedqvist, P. & Arfors, K.-E. (1982) Inflammation 6, 189- pany. 200. Downloaded by guest on September 30, 2021