Leukotriene B4

Leukotriene B4 Bing K. Lam* and K. Frank Austen Department of Medicine, Harvard Medical School, 1 Jimmy Fund Way, Rm 628, Boston, MA 02115, USA * corresponding author tel: 617-525-1270, fax: 617-525-1310, e-mail: [email protected] DOI: 10.1006/rwcy.2000.12007.

SUMMARY Leukotriene B4 (LTB4) is a potent lipid proinflammatory mediator. Biosynthesis of LTB4 involves the enzymatic transformation of arachidonic acid by 5-lipoxygenase to form the epoxide intermediate, LTA4, which is then dehydrated by LTA4 hydrolase to yield LTB4. Through binding to its membrane receptors, LTB4 causes leukocyte adhesion and chemotaxis. At higher concentrations, LTB4 induces aggregation, granule enzyme release and superoxide anion generation by neutrophils. The leukocyteactivating effects of LTB4, together with overproduction of LTB4 in certain inflammatory disorders, implicates LTB4 as a lipid proinflammatory mediator.

Further studies revealed that this metabolite was derived from an epoxide intermediate that also contained three conjugated double bonds which they later termed LTA4 (Borgeat and Samuelsson, 1979b); thus, the dihydroxy product was termed LTB4 (Figure 1). LTB4 was later determined to be a potent activator of neutrophil functions in vitro (Ford-Hutchinson et al., 1980) and was implicated in neutrophil-mediated inflammation in in vivo models of its action (Samuelsson, 1983). Figure 1 Biosynthesis of leukotriene B4. 5-HPETE, 5-hydroperoxy-eicosatetraenoic acid; FLAP, 5-lipoxygenase-activating protein. COOH

Arachidonic acid

BACKGROUND

Discovery Leukotriene B4 (LTB4) is a lipid inflammatory mediator with profound biological effects. It was first discovered in 1979 by B. Samuelsson and coworkers at the Karolinska Institute. While studying the metabolism of polyunsaturated fatty acids (more specifically, arachidonic acid or eicosatetraenoic acid) by the lipoxygenase enzyme of rabbit neutrophils, Borgeat and Samuelsson (1979a) found a new metabolite of arachidonic acid when they incubated 14 C-labeled arachidonic acid with rabbit peritoneal neutrophils. This metabolite of arachidonic acid was more polar than the previously identified monohydroxy metabolite 5-hydroxyeicosatetraenoic acid (5-HETE). Structural and ultraviolet spectrum analysis indicated that this metabolite of arachidonic acid contained three conjugated double bonds and two hydroxy groups. Because of the structural characteristics of the metabolite from leukocytes, they termed it a leukotriene.

(FLAP)

5-lipoxygenase

OOH COOH 5-HPETE

(FLAP)

5-lipoxygenase

O

COOH LTA 4

LTA 4 hydrolase

OH

OH

COOH LTB 4

1370 Bing K. Lam and K. Frank Austen

Structure The structure of LTB4 was elucidated by mass spectrometry and nuclear magnetic resonance (NMR) to be 5(S),12(R)-dihydroxy-eicosatetraenoic acid with four unsaturated double bonds, two cis and two trans configurations, but the exact locations of these cis and trans double bonds were not determined. The stereochemistry of the double bonds was later determined with the aid of organic synthesis to be 6,14-cis and 8,10-trans (Corey et al., 1981). When biologically derived and synthetic LTB4 were compared in in vitro and in vivo assays, the structure of the biologically derived LTB4 was confirmed to be 5(S),12(R)-dihydroxy-6,10-cis,8,10-trans-eicosatetraenoic acid (Lewis et al., 1981).

Main activities and pathophysiological roles LTB4 elicits adhesion and accumulation of human neutrophils in vivo and in vitro and stimulates activated neutrophil responses such as neutrophil degranulation with release of lysosomal enzymes and production of superoxide (Figure 2). Hence, LTB4 generation provides autoamplification of neutrophil-mediated inflammation (Hoover et al., 1984). LTB4 also increases vascular permeability. B lymphocytes respond to LTB4 with enhanced activation and immunoglobulin production (Samuelsson and Claesson, 1990). Apart from these cell surface receptor-mediated effects, LTB4 also acts as an intracellular messenger in model systems. LTB4 binds to the peroxisome proliferator-activated receptor 

Figure 2 Effect of leukotriene B4 on leukocyte function. PPAR, peroxisome proliferator-activated receptor . LTB 4 LTB 4

(PPAR), an orphan nuclear receptor that acts as a transcription factor to induce the expression of enzymes involved in fatty acid oxidation (Devchand et al., 1996). Abnormal LTB4 production has been implicated in the pathogenesis of inflammatory bowel disease, psoriasis, rheumatoid arthritis, and gout.

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce The biosynthesis of LTB4 is initiated by cell stimulation with appropriate inflammatory stimuli (Figure 3). An increase in intracellular calcium in turn activates cytosolic phospholipase A2 to release arachidonic acid from membrane phospholipids. The released arachidonic acid binds to the integral perinuclear membrane protein, 5-lipoxygenase activating-protein (FLAP) (Mancini et al., 1993). FLAP presents the arachidonic acid to 5-lipoxygenase that translocates to the perinuclear membrane from either cytosol or nucleoplasm (Brock et al., 1995) via a Ca2‡ -binding domain and tyrosine phosphorylation (Lepley et al., 1996). 5-Lipoxygenase first converts arachidonic acid to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and subsequently to LTA4 (Rouzer et al., 1986). Cytosolic LTA4 hydrolase (Radmark et al., 1984) forms LTB4, which is released into the extracellular environment by its specific transporter (Lam et al., 1990). Cells of bone marrow origin, including neutrophils, macrophages, mast cells, basophils, Langerhans cells, and to a lesser extent B cells, express the critical enzyme 5-lipoxygenase and have the intrinsic ability to generate LTB4. Endothelial cells and erythrocytes express LTA4 hydrolase and can synthesize LTB4 through the transcellular metabolism of LTA4 provided by LTA4-generating cells such as neutrophils.

Bound to receptor –

O2 Phagocytosis –

O2 LTB 4 PPAR-α Nucleus Transcription of lipid metabolizing gene

Granule enzyme release

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators The chemotactic tripeptide fMLP, bacterial lysopolysaccharide (LPS), complement factor C5a, calcium ionophore, and crosslinking of IgE receptors by

Leukotriene B4 1371 antigens (mast cells and basophils) can induce the formation of LTB4. Adenosine has been shown to suppress the generation of LTB4. Diets rich in eicosapentaenoic acid (EPA) and docosahexanoic acid (DHA), from sources such as ocean fish or cod liver oil supplements, can suppress the endogenous generation of LTB4. Both EPA and DHA can be incorporated into the membrane phospholipids and, like arachidonic acid, are released by appropriate stimuli. The released EPA and DHA either will inhibit the 5-lipoxygenase or the LTA4 hydrolase or will compete with LTA4 for these enzymes and thus decrease the generation of LTB4 and/or provide an attenuated product, LTB5, which will compete for the LTB4 receptor (Lee et al., 1984).

RECEPTOR UTILIZATION LTB4 exerts its proinflammatory effects through binding to high-affinity and/or low-affinity membrane LTB4 receptors (BLT receptor) (Yokomizo et al., 1997), and may also bind to the intranuclear transcription factor PPAR.

IN VITRO ACTIVITIES

In vitro findings See Table 1.

Figure 3 Cellular biosynthesis of leukotriene B4. AA, arachidonic acid; FLAP, 5-lipoxygenase-activating protein; 5-LO,5-lipoxygenase; cPLA2, cytosolic phospholipase A2; A4H, LTA4 hydrolase; PLs, phospholipids; LysoPLs, lysophospholipids. Agonist bound Ca++ AA

cPLA 2 cPLA 2

5-LO

PL

FLAP

5-LO AA

s

FL

AA

AP

LysoPLs

5-LO

Nucleus

LTB 4 LTB 4

5-LO 5HPETE

T

LTA 4 A4 H LTA 4 LTA4

for transcellular metabolism

Table 1 In vitro and in situ effects of LTB4 Cell types or tissues

Effects

Neutrophils

Chemotaxis, aggregation, granule enzyme release, superoxide anion release

HeLa cells

Activation of PPAR, induction of fatty acid-metabolizing enzymes

B lymphocytes

Immunoglobulin secretion

T lymphocytes

Proliferation, induction of T helper and T suppressor cell functions, and IL-2 production

Alveolar macrophages

Increase in phagocytic and bactericidal activities

Hamster cheek pouch

Increase in vascular permeability

Rat mesenteric venules

Increase in leukocyte adherence to and emigration through endothelial cells, decrease in leukocyte rolling velocity

1372 Bing K. Lam and K. Frank Austen

Regulatory molecules: Inhibitors and enhancers The increase in vascular permeability is enhanced by prostaglandin E2 (PGE2) and is suppressed by lipoxin A4. Pharmacologic agents, such as glucocorticoids, iloprost, and terbutaline, inhibit the vascular permeability response to LTB4.

Bioassays used Biological activities of LTB4 are measured by chemotactic assay, granule enzyme release assay, superoxide generation, hamster cheek pouch vascular permeability assay, rat mesenteric venule leukocyte adherence and emigration assay, lymphocytes proliferation assay, and leukocyte adhesion assay.

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Normal physiological roles Because the critical biological activities of LTB4 are geared toward peripheral blood leukocytes, including activation of neutrophils and lymphocytes, its role is predominantly in inflammation and secondarily in immune regulation at inflammatory sites (Figure 4). Its physiologic role relates directly to innate and adaptive host immune defense that depend on a controlled inflammatory response.

Figure 4 Chemotactic effect of leukotriene B4. LTB4

B4 LT

LTB 4

LTB 4

Leukocyte emigration

LTB 4

Species differences The biological effects of LTB4 on leukocytes are similar in different animal species such as human, dog, rat, and mouse.

Knockout mouse phenotypes There is no known knockout mouse for LTA4 hydrolase. However, the 5-lipoxygenase knockout mice, which lack both LTB4 and cysteinyl leukotrienes, showed reduced inflammatory and immune responses to infection. These leukotriene-deficient mice manifest enhanced lethality from Klebsiella pneumoniae in association with decreased alveolar macrophage phagocytic and bactericidal activities; these effects can be reversed by the administration of LTB4 (Bailie et al., 1996). The effect of chronic overproduction of LTB4 in animals is not known because there are no transgenic animals for LTB4. As the 5-lipoxygenase and FLAP gene-disrupted mice, respectively, exhibit impaired IgE biosynthesis, influx of cells (especially CD4+ T cells and eosinophils) at antigen-sensitized and -challenged sites, the global effect of LTB4 and LTC4 deficiency is the attenuated extravascular localization of various cell types. This effect indicates a proinflammatory role of the intact system.

Pharmacological effects The topical application of LTB4 to the skin of normal individuals or on the noninvolved skin of individuals with psoriasis induces pathologic effects that resemble psoriasis, such as edema and intraepidermal neutrophil microabscesses (Soter et al., 1983).

Interactions with cytokine network LTB4 induces the production of IL-1 and TNF by human monocytes and enhances the production of IL-2 by CD4+ T cells in the presence of IL-1 (RolaPleszczynski et al., 1988).

Endogenous inhibitors and enhancers

Leukocyte adhesion LT B 4

LTB 4

There is no known endogenous inhibitor or enhancer for the leukocyte activation effect of LTB4. PGE2 enhances the vascular permeability effect of LTB4,

Leukotriene B4 1373 whereas lipoxins antagonize this effect in the hamster cheek pouch. Lipoxins also inhibit LTB4-induced chemotaxis (Takano et al., 1998).

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Normal levels and effects Because LTB4 is generated mostly by activated neutrophils and monocytes/macrophages, very little LTB4 is formed under normal physiologic conditions. The pathway is part of the physiologic innate and host immune defense system and is most likely to function subclinically in the tissue microenvironment.

Role in experiments of nature and disease states Because LTB4 is a potent neutrophil activator and is synthesized mostly by neutrophils, the level of LTB4 increases in certain pathological conditions. LTB4 has been shown in animal studies to play an important role in promoting leukocyte infiltration and degranulation in -globulin-induced glomerulonephritis and in nephrotoxic serum glomerulonephritis induced by high-dose antiglomerular basement membrane immunoglobulin. LTB4 levels are higher in the blood and the synovial fluid of patients with rheumatoid arthritis, psoriasis, and inflammatory bowel disease. 5-Lipoxygenase inhibitors, FLAP inhibitors, LTB4 receptor antagonists and other pharmacological agents that reduce the cellular ability to generate LTB4 could prove to be beneficial in treating these disorders.

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