Leukotrienes and Lipoxins

1.   Leukotrienes

The term ‘leukotriene’ was coined because these important eicosanoids were first discovered by Samuelsson and colleagues 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

Biosynthesis of leukotrienes

The biosynthetic precursor of the leukotrienes is arachidonic acid released from phospholipids, and this is acted upon by enzymes located at the nuclear membrane, each of which has a high stereospecificity, starting with 5-lipoxygenase (5-LOX) and generation of 5S-hydroperxy-6t,8c,11c,14c-eicosatetraenoic acid (5-HPETE) by the incorporation of one molecule of oxygen at the C-5 position (see the Introduction to this series of documents for a discussion of the properties of lipoxygenases in general). In contrast to other lipoxygenases, 5-LOX requires the presence of 5-lipoxygenase activating protein (FLAP), located in the nuclear envelope, to function properly. It is believed that FLAP exists as a trimer, which contains a binding pocket for arachidonic acid, from which the latter can interact with the 5-LOX catalytic domain and enable transfer to the active site. Although its structure has not been fully determined, 5-LOX is believed to contain a catalytic domain and an N-terminal domain, which binds calcium and zwitterionic phosphatidylcholine in membranes (but not cationic phospholipids). These are essential for its activity. In addition, the activity of the enzyme is regulated by phosphorylation at three serine residues by specific kinases.

In humans, 5-LOX is expressed mainly in leukocytes, dendritic cells and in foam cells of atherosclerotic tissue (in other cells synthesis is blocked by DNA methylation). In resting cells, 5-LOX occurs either in the cytosol or in the nucleus as a soluble enzyme, depending on the cell type. It is then believed to co-migrate with phospholipase A2 to the nucleus where the latter liberates arachidonic acid from phospholipids, for transfer by FLAP to 5-LOX for metabolism. 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-HPETE can be released as such and reduced to 5S-hydroxy-eicosatetraenoic acid (5-HETE). However, 5-LOX has a dual function in leukotriene synthesis as in a concerted reaction it 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 leukotriene A4 (LTA4), which is the first of the leukotrienes. The 3- and 5-series leukotrienes have eicosatrienoic and eicosapentaenoic 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 nonenzymic hydrolysis of the epoxide ring into a variety of dihydroxy acids with relatively little biological activity (all four stereoisomers of LTB4).

The enzymic reactions leading to the dihydroxy acid 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 (or LTB4 synthase, a zinc-dependent metallo-protein). 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 in 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. LTB4 is catabolized and its biological activity terminated by ω-oxidation carried out by a specific cytochrome P450 enzyme followed by β-oxidation from the ω-carboxyl position, as well as by the pathway established for prostanoids and lipoxins (below).

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, a ‘cysteinyl leukotriene’). This enzyme is found in mainly in mast cells and eosinophils, although it has also been detected in platelets and epithelial cells. LTA4 can also function as a precursor of the lipoxins. Catabolism and deactivation of LTC4 occurs by sequential peptide cleavage reactions to form first LTD4 and then LTE4 before ω-oxidation.

 

2.   Lipoxins and Related Compounds

Lipoxins are trihydroxy-eicosatetraenoic acids, derived from arachidonic acid with the four double bonds in conjugation, which were the first lipid mediators to be discovered that were involved in the resolution phase of inflammation (like the resolvins). These molecules have structural similarities to the leukotrienes and appear to have some complementary biological activities. They are also formed by trans-cellular pathways, since few cell types have both of the required lipoxygenases.

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 A4 (LXA4) and B4 (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-LOX - see the Introduction to this series of web pages).

Biosynthesis of lipoxins

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, by specific hydrolases to form either 5S,6R,15S-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid (LXA4), or to 5S,14R,15S-trihydroxy-6,10,12-trans-8-cis-eicosatetraenoic acid (LXB4). In both products, the stereochemistry of the carbon 15S hydroxyl group is retained. The precursor 15-HETE is found esterified to phosphatidylinositol, and may be a storage form in the membranes of inflammatory cells, released on stimulation.

In a second mechanism in blood vessels, an interaction between leukocytes and platelets is involved via the same epoxy intermediate as in the first mechanism. The initial step is the action of a 5-lipoxygenase in leukocytes (to form leukotriene A4), before the reaction of a 12-lipoxygenase in platelets (platelets are not able to produce lipoxins on their own). Overall, these reactions also reduce leukotriene formation.

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 (and its chirality) 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. 15(R)-HETE produced by the action of a cytochrome P450 enzyme in the absence of aspirin can also be converted to 15-epi-lipoxins.

Structural formulae of epi-lipoxins

Aspirin thereby has the distinctive property of initiating the resolution of inflammation by stimulating the formation of mediators of the process much earlier than might have been expected. The distinctive lipoxin 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).

Lipoxins are deactivated by the actions of 15-hydroxyprostaglandin dehydrogenase and prostaglandin reductase with production of 13,14-dihydro-15-hydroxy-LXA4 and eventually 15-oxo metabolites. The epi-lipoxins have a twofold longer half-life than the lipoxins as they are catabolized less efficiently, possibly because of the distinctive 15R-stereochemistry.

 

3.   Eoxins

Recently, novel eicosanoids related to the cysteinyl-leukotrienes were characterized as products of the 12/15-lipoxygenase (15-LOX-1) of human eosinophils and mast cells. The primary product of the lipoxygenase, 15-HPETE is believed to react with the enzyme further to produce the 14,15-epoxide, designated eoxin A4, and then by analogy with leukotriene biosynthesis this in turn reacts with glutathione to produce eoxin C4, and thence eoxin D4 (linked to Cys-Gly) and eoxin E4 (linked to Cys only). Like the cysteinyl-leukotrienes, the eoxins are potent pro-inflammatory agents.

Biosynthesis of eoxins

 

4.  Hepoxilins

Hepoxilins are short-lived monohydroxy-epoxy eicosanoids produced in a number of organs or cell types, but especially the epidermis in humans, 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, the latter across the C11-C12 double bond, and unlike the leukotrienes and lipoxins, none of the double bonds are in conjugation. 12S-HPETE either can be reduced to the hydroxy compound (12S-HETE), or 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.

Structural formulae of hepoxilins

Two hepoxilins have been characterized, i.e. 8(S/R)-hydroxy-11S,12S-trans-epoxyeicosa-5c,9t,14c-trienoic acid (hepoxilin A3 or HXA3) and 10(S/R)-hydroxy-11S,12S-trans-epoxyeicosa-5c,9c,14c-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 is labile and can be opened by an epoxide hydrolase to yield trihydroxy metabolites, termed ‘trioxilins’’, which may also have some biological activity. In addition a family of hepoxilins, derived from the action of 15-lipoxygenase, has been identified, including forms linked to cysteinyl residues.

 

5. Biological Activities

As pro-inflammatory mediators, leukotrienes at concentrations in the low nanomolar range stimulate cellular responses that are quick in onset but do not last long, such as smooth muscle contraction, phagocyte chemotaxis, and increased vascular permeability, all of which are mediated via specific G-protein-coupled receptors.

Leukotriene B4 is one of the most potent chemotactic agents known and has an important function in the inflammatory process by its effect on leukocytes mediated via two G-protein-coupled receptors. It causes neutrophils to adhere to vascular endothelial cells and enhances the rate of migration of neutrophils into extravascular tissues, and it triggers several functional responses important for host defence, including the secretion of lysosomal enzymes, the activation of NADPH oxidase activity, nitric oxide formation, and phagocytosis. Also, it activates such intracellular signalling events 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 arthritis and atherosclerosis, for example, and it can promote the growth of certain cancers. In contrast, leukotriene B5 derived from eicosapentaenoic acid strongly inhibits the pro-inflammatory effects of LTB4.

Leukotriene C4, together with LTD4 and LTE4 (the cysteinyl-leukotrienes, which jointly comprise the 'slow-acting substance of anaphylaxis', recognized but not identified in the 1930s), 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, again exerting their effects through three distinct G-protein-coupled receptors. LTD4 and LTE4 are overexpressed in several types of cancer and are considered to be tumourigenic. Up-regulation of the expression of their receptors has been observed in several human cancers, so there is great interest currently in drugs that inhibit the effects of these lipids by funtionng as agonists to their receptors.

While the general view is that leukotrienes produce harmful effects, especially in relation to the immune system and allergic diseases, such as asthma, 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 promoting the expression of mediators and receptors that are important for immune defence. For example, leukotriene B4 can trigger the release of antimicrobial agents.

Eoxins have been implicated in inflammation of the airways in asthma patients, and in those with Hodgkin lymphoma, a malignant disorder with many characteristics of an inflammatory illness.

Lipoxins were the first eicosanoids to be discovered with a role in the resolution of inflammation, i.e. they are 'switched on' to limit the effects of inflammation. Indeed together with the resolvins and protectins, they control the inflammatory response in such pathogenic conditions as asthma, arthritis, cardiovascular disorders, cancer, and gastrointestinal, periodontal, kidney and pulmonary diseases. Thus, they have opposing effect to LTC4 and inhibit bronchial spasms. Like lipoxins, the aspirin-triggered epi-lipoxins have potent anti-inflammatory actions, and 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. In particular, LXA4 is produced endogenously and evokes protective effects via interactions with specific G-protein-coupled receptors and a nuclear transcription factor. All of the observed reactions appear to be highly stereoselective in terms of double bond geometry and chirality of the hydroxyl groups. Lipoxins also have a regulatory role in the immune response to infection by parasitic pathogens, such as Toxoplasma gondii and Mycobacterium tuberculosis. LXB4 and epi-LXB4 are effective both by oral administration and topical application, and they appear to function via their own receptor, although this has yet to be identified.

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 and protectins 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 of inflammation by controlling the entry of neutrophils to sites of inflammation and the affected organs. They are chemo-attractants for monocytes, i.e. cells that are 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 mobilization of calcium and potassium within cells or across membranes. In addition, hepoxilin A3 is now known to be an important regulator of mucosal inflammation in response to infection by bacterial pathogens. Although lipoxygenase activity in brain tissues tends to be low, there is significant biosynthesis of hepoxilins in the pineal gland, which may be involved in the regulation of melatonin production.

 

Recommended Reading

  • Bannenberg, G. and Serhan, C.N. Specialized pro-resolving lipid mediators in the inflammatory response: An update. Biochim. Biophys. Acta, 1801, 1260-1273 (2010) (DOI: 10.1016/j.bbalip.2010.08.002).
  • Buczynski, M.W., Dumlao, D.S. and Dennis, E.A. Proteomics. An integrated omics analysis of eicosanoid biology. J. Lipid Res., 50, 1015-1038 (2009) (DOI: 10.1194/jlr.R900004-JLR200).
  • Claesson, H.-E. On the biosynthesis and biological role of eoxins and 15-lipoxygenase-1 in airway inflammation and Hodgkin lymphoma. Prostaglandins Other Lipid Mediat., 89, 120-125 (2009) (DOI: 10.1016/j.prostaglandins.2008.12.003).
  • 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) (DOI: 10.1007/s00018-007-7228-2).
  • Haeggström, J.Z. and Funk, C.D. Lipoxygenase and leukotriene pathways: biochemistry, biology, and roles in disease. Chem. Rev., 111, 5866–5898 (2011) (DOI: 10.1021/cr200246d).
  • Maderna, P. and Godson, C. Lipoxins: resolutionary road. Brit. J. Pharm., 158, 947-959 (2009) (DOI: 10.1111/j.1476-5381.2009.00386.x).
  • Murphy, R.C. and Gijón, M.A. Biosynthesis and metabolism of leukotrienes. Biochem. J., 405, 379-395 (2007) (DOI: 10.1042/BJ20070289).
  • Nakamura, M. and Shimizu, T. Leukotriene receptors. Chem. Rev., 111, 6231–6298 (2011) (DOI: 10.1021/cr100392s).
  • Pace-Asciak, C.R. The hepoxilins and some analogues: a review of their biology. Brit. J. Pharm., 158, 972-981 (2009) (DOI: 10.1111/j.1476-5381.2009.00168.x).
  • Rådmark, O. and Samuelsson, B. 5-Lipoxygenase: mechanisms of regulation. J. Lipid Res., 50, S40-S45 (2009) (DOI: 10.1194/jlr.R800062-JLR200).
  • Sala, A., Folco, G. and Murphy, R.C. Transcellular biosynthesis of eicosanoids. Pharmacol. Rep., 62, 503-510 (2010).
  • Savari, S., Vinnakota, K., Zhang, Y. and Sjölander, A. Cysteinyl leukotrienes and their receptors: Bridging inflammation and colorectal cancer. World J. Gastroenterol.,20,968-977 (2014) (DOI: 10.3748/wjg.v20.i4.698).
  • Smith, M.L. and Murphy, R.C. The eicosanoids: cyclooxygenase, lipoxygenase and epoxygenase pathways. In: Biochemistry of Lipids, Lipoproteins and Membranes. 5th Edition. pp. 331-362 (Vance, D.E. and Vance, J. (editors), Elsevier, Amsterdam) (2008).
  • Werz, O. and Steinhilber, D. Therapeutic options for 5-lipoxygenase inhibitors. Pharmacol. Therapeut., 112, 701-718 (2006) (DOI: 10.1016/j.pharmthera.2006.05.009).

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Updated May 26, 2014