Clin Blochem. Vol. 23. pp. 459~168, 1990

0009-9120/90 $3.00 ~ .00 Copyright c 1990 The Canadian Society of Clinical Chemists.

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Biosynthesis and Biological Activity of Leukotriene B4 PIERRE BORGEAT and PAUL H. NACCACHE Inflammation and Immunology-Rheumatology Research Unit, Centre de Recherche du CHUL, Centre Hospitalier de I'Universite Laval, 2705, Boulevard Laurier, Sainte-Foy, Quebec, G1V 4G2, Canada The leukotrienes are a family of biologically active molecules derived from arachidonic acid. While prostaglandins and thromboxanes are products of the cyclooxygenase pathway of arachidonic acid metabolism, the leukotrienes are formed by arachidonate 5-1ipoxygenase, an enzyme present in phagocytes, mast cells, and basophils. Inflammatory stimuli, such as chemotactic peptides, platelet-activating factor, phagocytic particles, and immunological stimuli, which activate phagocytes and mast cells, stimulate leukotriene synthesis. Leukotriene B 4, a dihydroxy derivative of arachidonic acid, has a unique stimulatory activity on important functional responses of phagocytes; leukotriene B4 exerts chemotactic and chemokinetic activity towards phagocytes in vitro and in vivo, and it is a putative mediator of inflammation.

KEY WORDS: leukotriene; prostaglandin; arachidonic acid; lipoxygenase; phagocyte; neutrophil; inflammation.

Introduction

p

olyunsaturated C2ofatty acids undergo a highly complex oxidative metabolism in mammalian cells. The metabolism of arachidonic acid (all-cis5,8,11,14-eicosatetraenoic acid) leads to several groups of highly bioactive substances including hydroperoxyeicosatetraenoic acids (HPETEs), prostaglandins, thromboxanes, and the more recently described leukotrienes. These substances are synthesized by dioxygenases, usually referred to as cyclooxygenase, and the lipoxygenases. The metabolism of arachidonic acid through the cyclooxygenase pathway has been reviewed (1). The formation of 12S-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) in h u m a n platelets was the first lipoxygenase reaction reported in animals (2). More recently, leukotrienes were isolated and found to be the products of a lipoxygenase acting specifically at the C-5 position of arachidonic acid (3,4). The name "leukotriene" was introduced because leukocytes constitute an important source of these substances, which are characterized by the

presence of a conjugated triene unit (5). The three conjugated double bonds of the leukotrienes account for the typical ultraviolet absorption of these substances. Some leukotrienes were identified as the bioactive components of the slow-reacting substance of anaphylaxis (SRS-A) (6). The name SRS-A describes an active principle released by lung tissue in response to immunological challenge (7). The main biological effect of SRS-A is contraction of respiratory tract smooth muscles. SRS-A activity prepared from lung tissue consists of a mixture of leukotrienes C 4, D 4, and E4, and from pharmacological studies with synthetic leukotrienes, it is also clear that SRS-A activity can be expressed by leukotrienes C4, D4, and E 4 independently. From early studies on SRS-A performed more than 20 years ago, there is increasing evidence that leukotrienes are mediators of the bronchoconstriction associated with immediate hypersensitivity reactions, such as, asthma. These pathological responses to exposure to antigens may be due to increased sensitivity to, or excessive production of sulfidopeptide leukotrienes (leukotrienes C4, D4, and E4) induced by the antigens. Among other biological activities, the sulfidopeptide leukotrienes increase vascular permeability in skin, and the same compounds are potent constrictors of skin and lung microvasculature, and coronary arteries (8). Although the challenge of determining the chemical nature of SRS-A was the impetus for work on the leukotrienes, another leukotriene with no SRSA activity, leukotriene B4, has important roles in inflammatory responses. This paper reviews leukotriene synthesis and the biological actions of leukotriene B4 on phagocyte functions.

Biosynthesis of leukotrienes T H E 5-LIPOXYGENASE PATHWAY

Correspondence: Pierre Borgeat, Inflammation and Immunology-Rheumatology Research Unit, Centre de Recherche du CHUL, Centre Hospitalier de l'Universit~ Laval, 2705, Boulevard Laurier, Sainte-Foy, Quebec, G1V 4G2, Canada. Manuscript received February 2, 1990; revised March 16, 1990; accepted March 20, 1990. CLINICAL BIOCHEMISTRY,

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Arachidonic acid is present mainly in ester form in the cells. Its metabolism through the various dioxygenases first involves the action of various lipases, which release the fatty acid in the cellular milieu. The arachidonate 5-1ipoxygenase catalyzes the specific dioxygenation of arachidonic acid at position C-5 and initiates the synthesis of leukotrienes 459

BORGEAT AND NACCACHE

CO2H

~'~ -Ilpoxygenasey

5arachidonate

Arachidonic

acid

OH

O-OH

C02H

COzH

\

5-HPETE

5-HETE

CO2H

/ (LT& hydrolase)

/

~ OH

LTA4

\ (LTC4 synthaso) OH

OH

C02H

CO 2 H

\--/~/~/

LTB4

LTC 4

I

( ~hydroxylase)

Glu

I ('y-glutamyl transferase)

$ OH

~-Cys-Gly

OII

~

OH C02H

CO 2H

\--/~/~/ 03- O H - L T B 4

OH

~

I

S-Cys-Oly

LTD 4 I

cysteinylglycine~'~ dipeptJdase ,J OH

CO 2 H

~

C

OH O

2H

~-Cys 0.)- COOH-LTB 4

LTE 4

Figure 1--Synthesis and enzymatic transformation of LTA4. LT, leukotriene. (Figure 1). Arachidonic acid is first transformed into (5S) - 5 - hydroperoxy - (E,Z,Z,Z)-6,8,11,14 - eicosa satetraenoic acid (5-HPETE). Intact neutrophils rapidly metabolize 5-HPETE into (5S)-5-hydroxy(E,Z,Z,Z)-6,8,11,14-eicosatetraenoic acid (5-HETE), 460

or (5S,6S) - 5(6) - oxido - (E,E,Z,Z) - 7,9,11,14 -eicosatetraenoic acid (leukotriene A4) (3,4,9,10). 5-HETE is probably the product of the reduction of 5-HPETE. The enzyme responsible for this particular reaction has not been fully characterized, but gluthatione CLINICAL BIOCHEMISTRY, VOLUME 23, OCTOBER 1990

BIOSYNTHESIS AND BIOLOGICALACTIVITY OF LEUKOTRIENE B4

C02H LTA4

Nonenzyma~tic

~"~hydrolysis'

/

~ HO

OH

~ ~ H O H C02H

6-trans-LTB4 6-trans-12-epi-LTB

5,6 diHETEs

C02H

4

Figure 2--Nonenzymatic transformation of LTA4. LT, leukotriene. transferase may be involved in this process (11). The formation of leukotriene A 4 from 5-HPETE is enzymatic and involves the stereospeciflc removal of the 10D(R) hydrogen atom (of 5-HPETE} and loss of water (12}. The biosynthesis of leukotriene A 4 in neutrophils is followed by rapid enzymatic and nonenzymatic conversions into various more polar compounds (Figures 1 and 2) (4). As expected for an allylic epoxide, leukotriene A4 is highly unstable and undergoes facile nucleophilic substitution (hydrolysis products are described below); in aqueous buffer at pH 7.4 and 25 °C, the time for 50% decomposition is less than 10 s; leukotriene A 4 instantaneously hydrolyzes at acidic pH; leukotriene A 4 is stabilized under alkaline conditions and upon binding to serum albumin (13). The formation of leukotriene A 4 in biological systems can be demonstrated by trapping with methanol, and measurement of the stable methanolysis products (5S,12SR)-5-hydroxy - 12 - O - methyl - (E,E,E,Z) - 6,8,10,14- eicosatetraenoic acids (epimers at C-12) (4). The (5S,12R)-5,12-dihydroxy-(E,E,E,Z)-6,8,10,14eicosatetraenoic acid (6-trans-leukotriene B 4) and (5S,12S)-5,12-dihydroxy-(E,E,E,Z)-6,8,10,14-eicosatetraenoic acid (6-trans-12-epi-leukotriene B 4) are produced from the nonenzymatic hydrolysis of leukotriene A 4, which occurs spontaneously in aqueous medium. These compounds are by-products in leukotriene A4 biosynthesis (3,4). Hydrolysis (nonenzymatic) of leukotriene A4 also results in the formation of two isomeric 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acids (5,6-diHETEs). The mechanism of formation of these dihydroxy acids, is similar to that of the 5,12-dihydroxy acids with reaction at position C-6 of the carbonium ion intermediate (3); thus, these compounds also appear to be by-products in leukotriene A4 biosynthesis. The products of the nonenzymatic hydrolysis of leukotriene A4 show little biological activities (14); CLINICAL BIOCHEMISTRY,VOLUME 23, OCTOBER 1990

their presence in biological material reflects 5-1ipoxygenase activity and leukotriene A 4 synthesis. The structure identification of these four isomeric dihydroxy acids in initial studies on the leukotriene pathway was a key step to the discovery of leukotriene A 4 (3,4). Leukotriene A 4 also undergoes enzymatic conversions leading to various compounds with potent biological properties (Figure 1); (5S,12R)-5,12-dihydroxy-(Z,E,E,Z)-6,8,10,14-eicosatetraenoic acid (leukotriene B 4) is the product of the enzymatic hydrolysis of leukotriene A 4. It is formed, for example, in h u m a n neutrophils incubated with the ionophore A23187 and other neutrophil stimuli, such as, the formylated chemotactic peptides (15,16). The direct addition of synthetic leukotriene A4 to a suspension of h u m a n leukocytes clearly demonstrated that leukotriene A 4 is an intermediate in leukotriene B 4 synthesis (17). The second pathway of leukotriene A 4 metabolism involves a glutathione transferase; (5S,6R)-5-hydroxy-6-S-glutathionyl-(E,E,Z,Z)-7,9,11,14eicosatetraenoic acid (leukotriene C4) is the product of the conjugation of glutathione and leukotriene A4 (18). Leukotriene C4 is formed in h u m a n polymorphonuclear leukocytes (PMNLs), mainly in eosinophils (see below) stimulated with the ionophore A23187 (or other agents), or incubated directly with precursor leukotriene A4 (19); inhibition of glutathione synthesis or trapping of the tripeptide led to decreased leukotriene C4 synthesis in RBL-1 cells and macrophages (20,21). Phagocytes are the major source of leukotrienes in the body. In in vitro experiments, the profiles of 5-1ipoxygenase products vary drastically, according to incubation conditions. This is illustrated by the profiles of products obtained on stimulation of human blood neutrophils with A23187 or the chemotactic peptide fMet-Leu-Phe (16,22). A23187 causes 461

BORGEAT AND NACCACHE a dramatic and persistent influx of calcium into the cells and induces massive arachidonic acid release and leukotriene synthesis, fMet-Leu-Phe is a physiological stimulus acting through activation of membrane receptors; its effect on leukotriene synthesis is of much lower magnitude t h a n t h a t of A23187. This affects not only the quantity but also the profile of 5-1ipoxygenase products obtained. Indeed, in h u m a n neutrophils, the leukotriene A 4 hydrolase limits the synthesis ofleukotriene B 4 (22). In A23187stimulated cells, the 5-1ipoxygenase will produce amounts of leukotriene A 4 t h a t exceed the catalytic capacity of leukotriene A 4 hydrolase; consequently, beside substantial amounts of leukotriene B4, nonenzymatic conversion of leukotriene A 4 will occur (formation of 6-trans-leukotriene B 4, 6-trans-12-epileukotriene B 4 and two 5,6-DiHETEs epimers). Under fMet-Leu-Phe stimulation, the neutrophils produce smaller amounts of leukotriene A 4, which is almost quantitatively transformed into leukotriene B 4 without significant nonenzymic hydrolysis of the epoxy acid. In addition, in h u m a n neutrophils, leukotriene B 4 is rapidly converted into the ~o-oxidation products, ~o-hydroxy-, and ~o-carboxy-leukotriene B4 (Figure 1 ) (23). Given the limited capacity of the leukotriene B 4 u-oxidation system, the proportion of the leukotriene B4 metabolized will be much higher in fMetLeu-Phe stimulated cells than m the A23187-stimulated cells (16). Other factors, such as exogenous arachidonic acid added to incubation media, or the presence of albumin or plasma, will cause major changes in the profile of 5-1ipoxygenase products generated by neutrophils (22,24). THE ENZYMESOF THE 5-LIPOXYGENASEPATHWAY Much progress has been made in the past few years on the isolation and characterization of the enzymes involved in the synthesis of the 5-1ipoxygenase products. The arachidonate 5-1ipoxygenase has long resisted purification; but it has now been isolated from h u m a n and porcine neutrophils, and from RBL-1 cells and mouse mastocytoma cells f25-29). The h u m a n enzyme is a cytosolic enzyme of an apparent molecular weight of approximately 80,000; some investigators found evidence t h a t the 5-1ipoxygenase is associated with the specific granules of the neutrophils (30). It requires Ca 2+ , ATP, a fatty acid hydroperoxide, the presence of two high molecular weight cytosolic factors, and one membranebound factor for full activation; this illustrates the complexity of the regulatory mechanisms of this enzyme (26,28). These recent studies have also demonstrated t h a t the 5-1ipoxygenase and the leukotriene A 4 synthetase activities reside in a single protein as indicated by the copurification of the two activities, parallel suicide inactivation, the same stability, and the same requirements for activation (26,28). More recent studies indicate t h a t the stimulation of neutrophils by A23187 induces a calcium-dependent translocation of the 5-1ipoxygenase from the 462

cystosol to membrane structure, an event which appears to be directly linked to the activation of the enzyme (31}. Interestingly, a novel, highly specific, and potent inhibitor of leukotriene synthesis, which did not inhibit the activity of the puri.fied 5-1ipoxygenase, blocked the translocation of the enzyme in intact neutrophils activated by the ionophore A23187 (32). Other studies have clearly established the importance of the glutathione status in the neutrophil for the activation of the enzyme (33), an observation which is probably related to the requirement of the enzyme for lipid hydroperoxides. Arachidonic acid is not the exclusive substrate of the 5-1ipoxygenase; the enzyme will react with fatty acids carrying a pair of methylene interrupted cis double bonds at C-5 and C-8 of other u n s a t u r a t e d fatty acids. Therefore, eicosatetraenoic acid (EPA), 5,8,11-eicosatrienoic acid, and even the two hydroxy fatty acids, 12-HETE and 15-HETE are efficiently transformed by the 5-1ipoxygenase into HETEs or diHETEs (34). To date, the presence of 5-1ipoxygenase activity has been documented in neutrophils, eosinophils, basophils, monocytes, macrophages, and mast cells in man and several other species. The enzyme t h a t catalyzes the stereospecific hydrolysis of leukotriene A 4 into leukotriene B4, i.e., the leukotriene A 4 hydrolase, has also been investigated (35,36). The h u m a n neutrophil leukotriene A 4 hydrolase is a cytosolic enzyme with a molecular weight of about 68,000. It is clearly distinct from the epoxide hydrolases described previously in liver. Like the 5-1ipoxygenase, it is an auto-inactivating enzyme. Inactivation occurs following covalent binding of the substrate at the active site, as demonstrated with aH-leukotriene A 4. Leukotriene A~, formed from EPA, was also shown to be a substrate for the leukotriene A4 hydrolase, leading to the formation of leukotriene B 5 (A17Z-LTB4); it is not transformed as efficiently as leukotriene A4; it also leads to enzyme inhibition (37). Leukotriene A3, which can be formed from 5,8,11-eicosatrienoic acid (a fatty acid formed in essential fatty acid deficiency), is a very poor substrate of the enzyme and a very potent inhibitor (covalent binding at the active site} (38). As mentioned previously, leukotriene A 4 hydrolase is a limiting step in the synthesis of leukotriene B4 in h u m a n neutrophils /23). Unlike the 5-1ipoxygenase, the leukotriene A 4 hydrolase has a wide distribution; it has been detected in h u m a n erythrocytes, in h u m a n liver, and in various tissues of guinea pigs in specific activities higher or comparable to those in leukocytes (39-41). However, it is not clear if the enzyme present in various tissues is identical to the neutrophil enzyme. The synthesis of leukotriene C4 involves a glutathione-S-transferase named leukotriene C 4 synthetase. The enzyme has been isolated from rat basophil leukemia cells (RBL) (42,43). Unlike classical glutathione transferases which are soluble enzymes, the leukotriene C 4 synthetase is a microsomal enzyme. Furthermore, it has a different inhibition profile and substrate specificity t h a n the CLINICALBIOCHEMISTRY,VOLUME 23, OCTOBER 1990

BIOSYNTHESIS AND BIOLOGICALACTIVITY OF LEUKOTRIENE B4 liver glutathione transferases; consequently, it is a unique enzyme. The leukotriene C4 synthetase activity is present in h u m a n eosinophils, monocytes, peritoneal macrophagaes, basophils, and mast cells; these cell types also contain the 5-1ipoxygenase, and therefore, possess the enzymatic activities to produce leukotriene C4 from arachidonic acid. Several cell types which do not contain the 5-1ipoxygenase had leukotriene A 4 hydrolase or leukotriene C4 synthetase activities, and it was shown that transcellular metabolism of leukotriene A 4 generated by neutrophils could lead to the formation of leukotriene B 4 by erythrocytes, lymphocytes (44,45), or leukotriene C4 by platelets or endothelial cells (46,47).

activities. However, during differentiation, the 5lipoxygenase, the 15-1ipoxygenase, and the cyclooxygenase activities appear (53-55) with the responsiveness to leukotriene B4 (56). Note that the profile of arachidonic acid products in differentiated (granulocytic) HL-60 cells is very different from the profile obtained from normal blood neutrophils. HL-60 cells, unlike blood neutrophils, have considerable cyclooxygenase activity. In addition, HL-60 cells produce leukotriene C4 and leukotriene D4 in larger amounts than leukotriene B4; the metabolism of leukotriene B 4 (~o-oxidation) is weak in comparison to that of h u m a n blood neutrophils. MODULATIONOF LEUKOTRIENESYNTHESIS

LIPOXYGENASEPRODUCTPROFILESIN HUMAN PHAGOCYTES H u m a n PMNLs are frequently assumed to be neutrophil suspensions, because eosinophils and basophils represent very few cells in such preparations obtained from a normal donor. In the past few years, it has been suggested that among PMNLs, eosinophils are the major source of leukotriene C4. More recently, using highly purified neutrophil and eosinophil preparations from normal human blood, it was clearly demonstrated that eosinophils produce large amounts of leukotriene C 4 and little leukotriene B4, whereas neutrophils produce mainly leukotriene B 4 and little, if any leukotriene C4. The same studies also demonstrated that the 15-1ipoxygenase was largely an eosinophil enzyme, and that unlike neutrophils, eosinophils do not metabolize leukotriene B4 through the omega-oxidation pathway (48-50). Therefore, the metabolism of arachidonic acid in neutrophils and eosinophils shows striking differences. Because of the difficulties in obtaining h u m a n blood basophils, there are very few studies on the lipoxygenase-mediated transformation of arachidonic acid in these cells. However, it seems that h u m a n basophils produce leukotriene C4 (51,52); present studies do not permit detailed comparisons with neutrophils and eosinophils. The metabolism of arachidonic acid through the 5-1ipoxygenase pathway was also studied in h u m a n mononuclear phagocytes; there were striking differences between blood monocytes and alveolar macrophages. While the former produced leukotrienes B4 and C4, the latter produced leukotriene B 4 and little leukotriene C4; neither cell type metabolized leukotriene B 4 through the ~o-oxidation pathway, a very active catabolic process in the neutrophils. The profile of products generated by h u m a n peritoneal macrophages resembles the formation of leukotrienes B 4 and C4 in monocytes (leukotrienes D4 and E 4 were also formed from leukotriene C4). The h u m a n promyelocytic cell line HL-60 has recently been used for various investigations. These cells can be induced to differentiate into granulocytic or monocytic cells. Before differentiation, the cells show little lipoxygenase and cyclooxygenase CLINICAL BIOCHEMISTRY,VOLUME 23, OCTOBER 1990

Because of the putative roles of leukotrienes in h u m a n diseases, the regulation of leukotriene synthesis is presently the subject of extensive investigations. The available data suggest that the regulation of the 5-1ipoxygenase pathway is more complex than that of other arachidonic acid metabolic pathways. The formation of the various arachidonic acid metabolites is dependent on substrate availability. The concentration of free arachidonic acid in cells under normal conditions is low, compared to the total amount of the fatty acid present in the form of esters. Consequently, the formation of cyclooxygenase and lipoxygenase products is initially controlled by the activity of the various lipases which make arachidonic acid available to metabolizing enzymes. The effects of the ionophore A23187, which unspecifically stimulates the release of arachidonic acid through stimulation of Ca2+-dependent phospholipase A 2 and the synthesis of cyclooxygenase and lipoxygenase products in a variety of systems, support this concept (57). The synthesis of 5-HETE and leukotrienes also depends on substrate availability. However, in many cell types, such as in h u m a n blood neutrophils, eosinophils and monocytes, substrate availability is not the sole condition for synthesis of products, as the 5-1ipoxygenase will not readily transform exogenous arachidonic acid (or only to a small extent). However, incubation of these cells in the presence of A23187 leads to synthesis of substantial amounts of 5-HETE and leukotrienes from endogenous (or exogenous) arachidonic acid (15,34). These observations were of primary importance because they clearly indicated that the ionophore causes the release of arachidonic acid and activates the 5-1ipoxygenase (a Ca2+-dependent enzyme) involved in the further transformation of the fatty acid. Thus, most cells produce little 5-1ipoxygenase products in response to exogenous arachidonic acid; in addition to substrate, the enzyme requires activation. This is a major difference between the 5-1ipoxygenase and other dioxygenases, and it implies the existence of additional regulatory mechanisms for the synthesis of leukotrienes. It also implies that the 5-1ipoxygenase exists in two different forms, which reflect 463

BORGEAT AND NACCACHE different states of activation. The mechanisms responsible for the activation of the 5-1ipoxygenase are not yet fully understood. On the one hand, it is now well recognized t h a t agents t h a t can activate phagocytes, basophils and mast cells, such as the chemotactic peptide fMetLeu-Phe, platelet-activating factor (PAF), opsonized zymosan (phagocytosis), IgE (monomeric or aggregated) or immune complexes, through interactions with surface receptors, induce the synthesis of 5lipoxygenase products from endogenous substrate. On the other hand, it is also generally accepted that the 5-1ipoxygenase is a Ca2÷-dependent enzyme. It is therefore possible t h a t alterations of Ca 2+ metabolism induced by cell activation (Ca 2÷ influx or release from intracellular pools) are involved in the activation of the 5-1ipoxygenase. Other regulatory mechanisms have been shown recently to affect directly the activity of the 5-1ipoxygenase (see previous text). Other factors t h a t can modulate the synthesis of leukotrienes in neutrophils include: the various HETEs, mainly 15-HETE, which is an inhibitor of LT synthesis (IC50 ~ 5 #xm) (58), interaction with activated platelets, which can release arachidonic acid, and diets where arachidonic acid is partially replaced by EPA (marine diet or fish oil supplement), or 5,8,11-eicosatrienoic acid (essential fatty acid deficiency) in cellular lipids (59,60). Study of the priming effects of some growth factors, mainly the granulocyte/macrophage-colonystimulating factor (GM-CSF), on several neutrophil functions has revealed a novel, and probably important, mechanism of regulation of leukotriene synthesis. GMCSF, a cytokine derived from activated T-lymphocytes, macrophages, fibroblasts, and endothelial cells, initially described as a glycoprotein regulating the proliferation and differentiation of the progenitor cells of the immune system (61), has actions on mature phagocytes. Among other effects on various cellular functions, recombinant GM-CSF, at subnanomolar concentrations, increased the synthesis of leukotrienes in eosinophils and neutrophils stimulated with A23187, the chemotactic peptides (62,63), or PAF (64); this suggests an important role for the cytokine as a modulator of inflammatory responses. Stimulation of neutrophil functional responses by l e u k o t r i e n e B 4

and the formylated oligopeptides (68,69). Leukotriene B 4 has chemokinetic and chemotactic activity (70). The locomotory activity of leukotriene B 4 is three or four orders of magnitude t h a t of monoHETEs (71), 100 times that ofleukotriene A 4 (72,73), and 10 times that of leukotriene Bs (74). Modifications of the stereochemistry of the hydroxyl groups at C-5 and C-12, of the double bonds in the triene portion, and of the length of the carbon chain separating the hydroxyl and the carboxyl groups, greatly reduce chemotactic potency (68,71,75). The methyl ester of leukotriene B 4 has been reported to be as active as native leukotriene B 4 (68), or to have greatly reduced activity (71). Though ~o-OH-leukotriene B4 is essentially as active an in vitro chemoattractant as leukotriene B 4, its physiological role is unclear in view of recent reports t h a t ~-oxidation of leukotriene B 4 occurs to a much more limited extent in whole blood t h a n in isolated neutrophil suspensions (24). The ~o-carboxy derivative of leukotriene B 4, however, elicits a significantly smaller locomotory response from the neutrophils than does leukotriene B 4 (71,75). The chemotactic activity of leukotriene B 4 was also demonstrated in several systems in vivo. Intraperitoneal injection of leukotriene B 4 caused the accumulation of macrophages and PMNL in the rat peritoneal cavity (76). Injection of leukotriene B 4 into the interior chamber of rabbit eye and intradermal injections produced neutrophil accumulation at the site of administration (77,78). Similar effects of leukotriene B4 were observed using skin chamber techniques on the rabbit back and the h u m a n forearm (79). Elegant experiments were performed to directly observe (by microscopy) the effects of leukotriene B 4 on leukocytes and microvasculature in hamster cheek pouch or rabbit mesentery preparations. Topical applications of nanomolar solutions of leukotriene B 4 rapidly caused the adherence of PMNL to vascular cells in exposed postcapillary venules, and their progressive migration (diapedesis) into extravascular tissues (79-81). The adhesion of PMNL to endothelial cells was rapid and reversible (within minutes) upon withdrawal of leukotriene B 4 stimulation; it lasted longer if a higher dose of leukotriene B4 was used (79). The migration of PMNL into interstitial spaces was apparent 20 min after treatment of rabbit mesentery with leukotriene B 4 (79).

LOCOMOTIONAND CHEMOTAXIS

ADHERENCE TO ARTIFICIALSURFACESAND TO ENDOTHELIUM

The stimulation of neutrophil locomotion by leukotriene B4 represents the first reported biological activity of this compound (65,66); it may be the most relevant one for these cells. Leukotriene B 4 is a potent chemoattractant for h u m a n and rabbit neutrophils (67) active at subnanomolar concentrations. Its activity rivals on a concentration basis the effects of peptidic chemotactic factors such as C5a

Neutrophil locomotion is a composite reaction t h a t depends on a balance between adherence to the substratum and the development of contractile forces. Leukotriene B4 has been shown to influence the adherence of neutrophils to nylon wool (82), Sephadex G-25 (83), and endothelial cells (84,85). The stimulation of the latter interactions is the result of effects of leukotriene B4 on the endothelial cells, and not on the neutrophils (85).

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CLINICALBIOCHEMISTRY,VOLUME 23, OCTOBER 1990

BIOSYNTHESIS AND BIOLOGICALACTIVITYOF LEUKOTRIENEB4 AGGREGATION Neutrophil aggregation in vitro has been related (86,87) to the margination of the neutrophils t h a t accompanies the intravenous injection of chemotactic factors (88,89). Leukotriene B4, and to a lesser extent, ¢o-OH-leukotriene B4, ¢o-COOH-leukotriene B 4 (14,65), and the 5- and 12-HETEs (90) induce an aggregation response from the neutrophils. The neutrophil aggregatory properties of arachidonic acid are also generally ascribed to its lipoxygenase metabolites (91,92).

of a role of leukotriene B 4 in inflammation. Within the next few years, leukotriene synthesis inhibitors and antagonists should facilitate the assessment of the role of leukotrienes in defense mechanisms and in several inflammatory diseases. In particular, it will be interesting to define the importance of leukotrienes relative to other mediators, such as PAF. The discovery of leukotrienes in the late 70s has certainly generated hope for the development of new classes of anti-inflammatory drugs and improved treatments for inflammatory diseases.

DEGRANULATION

References

Several lipoxygenase products possess secretory activity towards the neutrophils, including leukotriene B4 (72,93). The magnitude of the exocytosis induced by leukotriene B4 is significantly smaller than t h a t observed with fMet-Leu-Phe; in contrast to the sigmoidal shape of the secretory response of the latter stimulus, it appears to be directly related to the dose of the fatty acid used (72).

1. WolfeLS. Eicosanoids, prostaglandins, thromboxanes, leukotrienes, and other derivatives of carbon-20 unsaturated fatty acids. J Neurochem 1982; 38: 1-14. 2. Hamberg M, Samuelsson B. Prostaglandin endoperoxides. Novel transformations of arachidonic acid in human platelets. Proc Natl Acad Sci USA 1974; 71: 3400-4. 3. Borgeat P, Samuelsson B. Metabolism of arachidonic acid in polymorphonuclear leukocytes: structural analysis of novel hydroxylated compounds, d Biol Chem 1979; 254: 7865-9. 4. Borgeat P, Samuelsson B. Metabolism of arachidonic acid in polymorphonuclear leukocytes: unstable intermediate in formation of dihydroxy acids. Proc Natl Aca4 Sci USA 1979; 76: 3213-7. 5. Samuelsson B, Borgeat P, HammarstrSm S, Murphy RC. Introduction of a nomenclature: leukotrienes. Prostaglandins 1979; 17: 785-7. 6. Murphy RC, HammarstrSm S, Samuelsson B. Leukotriene C: a slow-reacting substance from murine mastocytoma cells. Proc Natl Acad Sci USA 1979; 76: 4275-9. 7. Sirois P. Pharmacology of the leukotrienes. Adv Lipid Res 1985; 21: 79-101. 8. Samuelsson B. Leukotrienes, mediators of immediate hypersensitivity reactions and inflammation. Science 1983; 220: 568-75. 9. Franson R, Waite M. Relationship between calcium requirement, substrate charge and rabbit polymorphonuclear leukocyte phospholipase A2 activity. Biochemistry 1978; 17: 4029-33. 10. Borgeat P, Hamberg M, Samuelsson B. Transformation of arachidonic acid and homo-gamma-linolenic acid in rabbit polymorphonuclear leukocytes. J Biol Chem 1976; 251: 7816-20. 11. Skoog MT, Nichols JS, Harrison BL, Wiseman JS. Specificity of an HPETE peroxidase from rat PMN. Prostaglandins 1988; 36: 373-84. 12. Maas RL, Ingram CD, Taber DF, Oates JA, Brash AR. Stereospecific removal of the Dr hydrogen atom at the 10-carbon of arachidonic acid in the biosynthesis of leukotriene A4 by human leukocytes. J Biol Chem 1982; 257: 13515-9. 13. Fitzpatrick FA, Morton DR, Wynalda MA. Albumin stabilizes leukotriene A 4. J Biol Chem 1982; 257: 4680-3. 14. Naccache PH, Molski TFP, Becker EL, et al. Specificity of the effect of lipoxygenase metabolites of arachidonic acid on calcium homeostasis in neutrophils: correlation with functional activity. J Biol Chem

OXIDATIVE METABOLISM The lipoxygenase metabolites of arachidonic acid are poor activators of the respiratory burst of the neutrophils. Superoxide production and increased chemiluminescence in response to leukotriene B 4 have, however, been reported in some (94,95), but not all (70,96) studies. The magnitudes of these effects are two to five times smaller than those produced by fMet-Leu-Phe; the doses required are higher than those affecting neutrophil locomotion. Leukotriene B 4, however, potentiates the chemotactic peptide-stimulated oxidative metabolism of the neutrophils (96).

Conclusion Evidence supports the role of leukotriene B4 in inflammation. First, leukotriene B 4 is a potent agonist of phagocytes and activates functional responses in these cells. In particular, leukotriene B4 is strongly chemotactic for phagocytes; this biological activity is specific to leukotriene B 4 and may be its most important property with regard to its putative role as a mediator of inflammation. Second, leukotriene B4 is synthesized by the cells actively involved in defense mechanisms and inflammation, the PMNLs, monocytes, and macrophages. Third, inflammatory stimuli, such as PAF, chemotactic peptides, and phagocytic particles trigger leukotriene B4 synthesis in phagocytes. Furthermore, data now support increased formation of leukotrienes in inflammatory diseases in man and animal models (97). Although well documented, these observations on the biosynthesis and bioactivity of leukotriene B4 constitute only circumstantial evidence in support

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