The Lipid Library

LEUKOTRIENES AND LIPOXINS

Chemistry and Biology

1.   Leukotrienes

The term ‘leukotriene’ was coined because these important eicosanoids were first discovered in the white blood cells derived from bone marrow, i.e. the leukocytes, and they have three double bonds in conjugation (though they have four in total), resulting in specific absorbance peaks in their UV spectra (at 270, 280 and 290 nm). They are known to exhibit a wide range of biological activities, most of which involve some form of signalling function akin to that of short-lived paracrine reagents. The structures and basic mechanism for biosynthesis are illustrated below

The biosynthetic precursor of the leukotrienes is arachidonic acid released from phospholipids, and this is acted upon by a series of enzymes, each of which has a high stereospecificity, starting with 5-lipoxygenase (5-LOX) and generation of 5S-hydroxy-6t,8c,11c,14c-eicosatetraenoic acid (5HPETE) (see the Introduction to this series of documents). Little leukotriene synthesis occurs in resting cells, but it is stimulated by cellular events that raise the level of calcium ions. It has also become apparent that some of these transformations can occur in one cell type (donor cell) before the intermediate is passed to a second cell type (acceptor cell) to complete the conversion into the biologically active mediator. Mechanisms must exist to transport the eicosanoid intermediate between cells and across phospholipid membrane barriers. For example, some cells lack the 5-lipoxygenase, but are able to synthesise leukotrienes by this cooperative process known as trans-cellular biosynthesis.

5-LOX has a dual function in leukotriene synthesis and also catalyses the second step illustrated above, i.e. the transformation of 5-HPETE into 5,6-epoxy-7t,9t,11c,14c-eicosatetraenoic acid or LTA₄, which is the first of the leukotrienes. The 3- and 5-series leukotrienes have eicosatrienoic and pentaenoic acids, respectively, as the precursors. LTA₄ is highly unstable with a half-life of only ten seconds at pH 7.4 in vitro, although it is stabilized to some extent by binding to albumin or by inclusion in phospholipid liposomes. However, if it is not metabolized quickly, it can be transformed by non-enzymic hydrolysis of the epoxide ring into a variety of dihydroxy acids with little biological activity.

5-Lipoxygenase contains a catalytic domain and an N-terminal domain, which binds calcium and zwitterionic phosphatidylcholine but not cationic phospholipids. These are essential for its activity. In addition, the enzyme can be phosphorylated at three sites with profound affects on its activity.

The enzymic reactions leading to LTB₄ and the peptide-leukotrienes, especially LTC₄, are much more important from a biological standpoint and their synthesis is controlled by the location of the enzymes for each product in specific types of cells in humans. LTA₄ synthesised in erythrocytes is the precursor for leukotriene LTB₄ or 5S,12R-dihydroxy-6c,8t,10t,14c-eicosatetraenoic acid, which is synthesised by the action of the enzyme LTA₄ hydrolase (LTB₄ synthase). This has a dual activity as an aminopeptidase and is located mainly in neutrophils. Unlike most other enzymes involved in the ‘leukotriene cascade’, it is present of the cytosol of the cell, so there must be some mechanism to ensure that it is close to the nuclear membrane where the other steps in the process occur.

Alternatively, LTA₄ generated externally is acted upon by LTC₄ synthase or glutathione-S-transferase, which is found on the nuclear envelope of cells and adds the tripeptide glutathione (γ-glutamyl-cysteinyl glycine) to carbon 6 to yield peptido-leukotriene C₄ (LTC₄). This enzyme is found in mainly in mast cells and eosinophils, although it has also been detected in platelets and epithelial cells. Removal of glutamic acid by a γ-glutamyl transpeptidase gives leukotriene D₄, and further hydrolysis by a dipeptidase gives leukotriene E₄ in which cysteine only is linked to the eicosanoid moiety.

2.   Lipoxins and Related Compounds

Lipoxins are trihydroxy-eicosatetraenoic acids, derived from arachidonic acid with the four double bonds in conjugation, which have distinctive anti-inflammatory properties, i.e. they are involved in the resolution phase of inflammation like the resolvins. These molecules have structural similarities to the leukotrienes and also appear to have some complementary biological activities. They are also formed by trans-cellular pathways.

There are at least three routes to the biosynthesis of lipoxins that differ among cell types. However, a common feature is the insertion of molecular oxygen at two sites in arachidonic acid by distinct lipoxygenases. For example as illustrated for the biosynthesis of the lipoxins designated LXA₄ and LXB₄ by one of the recognized mechanisms (in airway epithelial cells and monocytes), the first step is the formation of 15S-hydroperoxy-5c,8c,11c,13t-eicosatetraenoic acid by a 15-lipoxygenase (15-LO - see the Introduction to this series of web pages).

This or the reduced form 15S-HETE is then acted upon by a 5-lipoxygenase to form first an epoxy intermediate, i.e. 5S,6S-epoxy-15S-hydroxy-ETE and then, depending on the cell type, either the trihydroxy-tetraene lipoxin A₄ (LXA₄) or lipoxin B₄ (LXB₄). In both products, the stereochemistry of the carbon 15S hydroxyl group is retained. In a second mechanism in leukocytes and platelets via the same epoxy intermediate, the initial step is the action of a 5-lipoxygenase (to form leukotriene A₄), before the reaction of the 15-lipoxygenase.

An important third mechanism has recently been discovered that produces lipoxins of different stereochemistry, i.e. the epi-lipoxins, sometimes termed the aspirin-triggered lipoxins (‘ATL’), as the reaction is initiated by aspirin and requires the cyclooxygenase COX-2 in the first step. As discussed in the Introduction to these pages, COX-2 is induced in endothelial and epithelial cells in response to a variety of stimuli. The effect of aspirin is to acetylate the enzyme, switching its catalytic activity from prostanoid biosynthesis to production of 15R-HETE rather than the S-enantiomer. This is in turn converted to 5S,6S-epoxy-15R-hydroxy-ETE, as described above for lipoxins, by the action of the 5-lipoxygenase in leukocytes and thence to epi-lipoxins, i.e. epi-LXA₄ and epi-LXB₄ with 15R-stereochemistry.

These distinctive structures, which are conserved across species, are formed via cell-cell interactions, and they seem to act at both temporally and spatially distinct sites from other eicosanoids involved in the inflammatory responses (see below). Their importance is only now being recognized.

3.  Hepoxilins

Hepoxilins are monohydroxy-epoxy eicosanoids produced in a number of organs or cell types and derived mainly from the product of 12-lipoxygenase action on arachidonic acid, i.e. 12S-hydroperoxy-5c,8c,10t,14c-eicosatetraenoic acid (12S-HpETE). They contain both hydroxyl and epoxy groups, and unlike the leukotrienes and lipoxins, none of the double bonds are in conjugation.

12S-HpETE can either be reduced to the hydroxy compound (12S-HETE), or it can enter the hepoxilin pathway where it is acted upon by a hepoxilin synthase, which effects isomerization of the hydroperoxide group. The enzyme in skin is distinct from that in other tissues. Two hepoxilins have been characterized, i.e. 8(S/R)-hydroxy-11S,12S-trans-epoxyeicosa-5c,9t14c-trienoic acid (hepoxilin A₃ or HXA₃) and 10(S/R)-hydroxy-11S,12S-trans-epoxyeicosa-5c,9c14c-trienoic acid (hepoxilin B₃ or HXB₃). Only HXA₃ is biologically active. Unlike the leukotrienes and lipoxins, none of the double bonds are in conjugation. The epoxide ring in HXA₃ can be opened by an epoxide hydrolase to yield trihydroxy metabolites, termed ‘trioxilins’’, which may also have some biological activity.

4. Biological Activities

Leukotriene B₄ has an important function in the inflammatory process by its effect on leukocytes mediated via G-protein-coupled receptors. It causes neutrophils to adhere to vascular endothelial cells and enhances the rate of migration of neutrophils into extra-vascular tissues. Also, it activates various intracellular signalling events such as the mobilization of calcium, activation of phospholipases, the production of diacylglycerols and phosphoinositides, and the release of either anti- or pro-inflammatory agents, depending on circumstances. 5-Lipoxygenase and LTB₄ especially have been implicated in the chronic inflammation that is a part of the pathophysiology of atherosclerosis.

Leukotriene C₄, together with LTD₄ and LTE₄ (the cysteinyl-leukotrienes), are known to exert a range of pro-inflammatory effects, including constriction of the airways and vascular smooth muscle, increasing plasma exudation and oedema, and enhanced mucus secretion. They are important mediators in asthma especially, but also in other inflammatory conditions, including cardiovascular disease, cancer, and gastrointestinal, skin, and immune disorders, exerting their effects through G-protein coupled receptors. Some consider that it is the over-production of leukotrienes that is harmful rather than production per se. However, there is great interest currently in drugs that inhibit the effects of these lipids by functioning as agonists to their receptors.

There is a general impression is that leukotrienes produce harmful effects, especially in relation to the immune system and allergic diseases, such as asthma. However, there are suggestions that they may also be beneficial in that they stimulate the body’s innate immunity against pathogens, including bacterial, fungal and viral infections, by promote the expression of mediators and receptors that are important for immune defense. For example, leukotriene B₄ can trigger the release of antimicrobial agents.

Interestingly, lipoxins have opposing effect to LTC₄ and inhibit bronchial spasms. In addition, they have anti-inflammatory properties in such pathogenic conditions as asthma, arthritis, cardiovascular disorders, and gastrointestinal, periodontal, kidney and pulmonary diseases. Like lipoxins, the aspirin-triggered epi-lipoxins also have potent anti-inflammatory actions. Indeed, this may provide further explanation for the efficacy of aspirin as a drug. It not only inhibits the synthesis of pro-inflammatory mediators but also induces the synthesis of anti-inflammatory ones. The arachidonate-derived lipoxins and epi-lipoxins appear to be defining members of the first class of endogenous lipid mediators that are ‘switched on’ in the resolution phase of an inflammatory response to limit the effects of inflammation. In particular, LXA₄ is produced endogenously and evokes protective effects in many physiological conditions via interactions with specific G protein-coupled receptors. All of the observed reactions appear to be highly stereo-selective in terms of double bond geometry and chirality of the hydroxyl groups.

In the initial phase of inflammation, prostaglandin PGE₂ and other pro-inflammatory prostaglandins are produced. The signals that lead to the synthesis of such molecules in turn stimulate the transcription of enzymes required for the generation of lipoxins from arachidonate and the resolvins from fatty acids of the omega-3 family of fatty acids, which also have anti-inflammatory properties. The lipoxins are believed to function in promoting resolution by controlling the entry of neutrophils to sites of inflammation and the affected organs. They are also chemo-attractants for monocytes, i.e. cells that appear to be required for wound healing. In effect, it appears that leukocytes are programmed to progress from pro- to anti-inflammatory responses, utilizing metabolites derived from both omega-6 and omega-3 fatty acids in the process. The possibilities for therapeutic intervention with such lipids to reduce the adverse effects of inflammation in various disease states are being actively explored.

Hepoxilins have pro-inflammatory properties in the skin, but anti-inflammatory in neutrophils. Most of the observed activities are associated with calcium mobilization within cells or in the transport of calcium across membranes. In addition, hepoxilin A₃ is now known to be an important regulator of mucosal inflammation in response to infection by bacterial pathogens.

Recommended Reading

• Fiore, S. Leukotrienes and lipoxins. In: Bioactive Lipids. pp. 223-243 (edited by A. Nicolaou and G. Kokotos, Oily Press, Bridgwater) (2004).
• Flamand, N., Mancuso, P., Serezani, C.H.C. and Brock, T.G. Leukotrienes: mediators that have been typecast as villains. Cell. Mol. Life Sci., 64, 2657-2670 (2007).
• Folco, G. and Murphy, R.C. Eicosanoid transcellular biosynthesis: From cell-cell interactions to in vivo tissue responses. Pharmacol. Rev., 58, 375-388 (2006).
• Nigam, S., Zafiriou, M.-P., Deva, R., Ciccoli, R. and Roux-Van der Merwe, R. Structure, biochemistry and biology of hepoxilins. FEBS J., 274, 3503-3512 (2007).
• Rådmark, O. and Samuelsson, B. 5-Lipoxygenase: Regulation and possible involvement in atherosclerosis. Prostaglandins Other Lipid Mediators, 83, 162-174 (2007).
• Serhan, C.N. Lipoxins and aspirin-triggered 15-epi-lipoxins are the first lipid mediators of endogenous anti-inflammation and resolution. Prostaglandins, Leukotrienes Essential Fatty Acids, 73, 141-162 (2005).
• Smith, M.L. and Murphy, R.C. The eicosanoids: cyclooxygenase, lipoxygenase and epoxygenase pathways. In: Biochemistry of Lipids, Lipoproteins and Membranes. pp. 341-371 (Vance, D.E. and Vance, J. (editors), Elsevier, Amsterdam) (2002).

Updated: 13/2/2008 Credits/disclaimer	Scottish Crop Research Institute (and MRS Lipid Analysis Unit), Invergowrie, Dundee (DD2 5DA), Scotland