Leukotrienes, Lipoxins and Related Eicosanoids

The oxygenated metabolites or oxylipins derived from arachidonic and related fatty acids are produced through a series of complex interrelated biosynthetic pathways often termed the 'eicosanoid cascade'. Here, the di-substituted eicosanoids derived from arachidonic acid are described, including the leukotrienes, lipoxins, eoxins and hepoxilins, together with their manifold biological activities. Of these, the most studied are the leukotrienes, a group of inflammatory mediators that are synthesised primarily by leukocytes in response to a variety of stimuli, including antigens, immune complexes, complement, cytokines, osmotic challenges and pollutants. The lipoxins differ from the others in that they support the resolution of inflammation.

The prostanoids (prostaglandins, thromboxanes and prostacyclins) have distinctive ring structures in the centre of the molecule and are discussed on their own web page, as are the linear mono-hydroxyeicosatetraenes (HETE) and related lipids. Any discussion of these oxylipins must take into account the other specialized pro-resolving mediators, i.e., the resolvins, protectins and maresins, derived from the (n-3) family of polyunsaturated fatty acids, but for practical reasons these have their own web page. The balance between these various oxylipins can be critical for health.

1.   Leukotrienes

The term ‘leukotriene’ was coined because these eicosanoids, first discovered by Samuelsson and colleagues in the white blood cells derived from bone marrow, i.e., the leukocytes, have three double bonds in conjugation (though they have four in total), resulting in characteristic absorbance peaks in their UV spectra (at 270, 280 and 290 nm). They are produced from arachidonic acid mainly by immune cells, and they are known to exhibit a wide range of activities, most of which involve some form of signalling like that of short-lived paracrine reagents. Leukotrienes can be classified according to whether or not they are linked covalently to cysteine, and the structures and basic mechanism for biosynthesis of these oxylipins are described below. The 3- and 5‑series leukotrienes are less studied and have 5,8,11-eicosatrienoic and 5,8,11,14,17-eicosapentaenoic acids, respectively, as the precursors.

Certain types of reaction are common to the synthesis from essential fatty acids (EFA) of both the pro-inflammatory lipid mediators, such as the leukotrienes and those with pro-resolution properties, such as the specialized pro-resolving mediators. First, there is an antarafacial hydrogen abstraction at the C3 position in a cis,cis-1,4-diene moieties. This is followed by a stereo-selective insertion of molecular oxygen to form a carbon-oxygen bond in a hydroperoxide intermediate, and then there is a second hydrogen abstraction with an intramolecular nucleophilic attack by the oxygen atom in this intermediate to produce an epoxide. The final step involves hydrolase-assisted nucleophilic addition of water to a cis-double bond in the epoxide intermediate (not illustrated).

Common reactions in the production of lipid mediators

5-Lipoxygenase - a key enzyme: The biosynthetic precursor of the leukotrienes is arachidonic acid released mainly from phosphatidylinositol and its phosphorylated forms, by the action of phospholipase A2 (cPLA2α), and this is acted upon by enzymes located at the endoplasmic reticulum (ER) or nuclear membrane, each of which has a high stereospecificity, starting with 5-lipoxygenase (5‑LOX, ALOX5) (see our web page on mono-hydroxyeicosatetraenes for a discussion of lipoxygenases in general).

In humans, 5-LOX is expressed mainly in cells of myeloid origin (neutrophils, eosinophils, monocytes-macrophages, mast cells and 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, but in response to cell activation, it co-migrates with phospholipase A2 to the endoplasmic reticulum and perinuclear membranes where the lipase liberates arachidonic acid from phospholipids for metabolism. 5-LOX is a dioxygenase similar in structure to other lipoxygenases and contains an α‑helix catalytic domain that contains non-haeme iron and an N-terminal domain, which bind to calcium and zwitterionic phosphatidylcholine in membranes (but not cationic phospholipids) and are essential for its activity. In contrast to the prostaglandins, an increase in free arachidonic acid alone is not sufficient to induce leukotriene synthesis. Little leukotriene synthesis occurs in resting cells, but this is stimulated by cellular events that raise the level of calcium ions, while the enzyme is regulated by phosphorylation at three serine residues by kinases. The enzyme is regenerated after each catalytic cycle by either a mono-oxygenase or the LTA4 synthase mechanism.

Leukotriene biosynthesis: In the first step of a two-stage concerted reaction in leukotriene biosynthesis, 5‑LOX embedded in the membrane generates 5S‑hydroperoxy-6t,8c,11c,14c-eicosatetraenoic acid (5‑HPETE) from arachidonic acid by a dioxygenation reaction, i.e., the incorporation of one molecule of oxygen at the C-5 position.

To work properly in the second step, 5-LOX requires the presence of two accessory proteins embedded in the membrane - five‑lipoxygenase activating protein (FLAP) and coactosin-like protein (CLP). FLAP forms a trimer with each monomer consisting of four transmembrane α-helices connected by two loops on each of the cytosolic and luminal sides together with a binding pocket for arachidonic acid, from which the latter can interact with the 5-LOX catalytic domain and enable transfer to this site. It may promote coupling of phospholipase A2 (cPLA2) to 5-LOX at the membrane (both cPLA2 and 5-LOX are Ca2+-dependent). 5‑HPETE can be released as such and reduced to 5S-hydroxy-eicosatetraenoic acid (5‑HETE), but with the aid of FLAP and CLP, 5-LOX is able to catalyse 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. Although LTA4 is highly unstable with a half-life of only a few seconds at pH 7.4 in vitro, it is stabilized to some extent in cells by binding to albumin or other proteins that remove water from the immediate environment of the epoxide structure. While it appears to have no biological functions of its own, it is a pivotal intermediate in the synthesis of other leukotrienes and of lipoxins (see below).

Biosynthesis of leukotrienes

In humans, the enzymic reactions leading to the dihydroxy acid LTB4 or to the peptide- or cysteinyl-leukotrienes, e.g., LTC4, are more important from a system standpoint, and their synthesis is controlled by the location of the enzymes for each product in certain types of cell. Hydrolysis of LTA4 is catalysed by LTA4 hydrolase (LTA4H), a zinc-dependent metallo-protein, which has a dual activity as an aminopeptidase and is widely expressed in almost all mammalian cells. 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. The product is LTB4 or 5S,12R‑dihydroxy-6,8,10,14-(Z,E,E,Z)-eicosatetraenoic acid. LTA4H has a high specificity for its substrate LTA4, and it undergoes suicide inactivation during catalysis. It has yet to be determined how such a labile molecule as LTA4 is transferred from 5-LOX to LTA4H and how the product LTB4 is transported to the plasma membrane for export, but it is presumed to be protected in a hydrophobic pocket in a protein such as albumin. LTB4 and its synthesising enzymes (5-LOX, FLAP and LTA4H) are located in intracellular multivesicular bodies, which release their content as exosomes upon stimulation.

Scottish thistleThe alternative pathway for LTA4 metabolism is prominent in cells expressing the enzyme LTC4 synthase (LTC4S 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, 5(S),6(R)-S-glutathionyl-7,9,11,14-(E,E,Z,Z)-eicosatetraenoic acid), the first of the 'cysteinyl leukotrienes'; FLAP is again essential to the reaction. Although this synthase is found in mainly in immune cells, such as mast cells, eosinophils and monocytes, it is also present in platelets and epithelial cells but is suppressed by stimulation of PKC-dependent phosphorylation. LTC4 can be transported out of cells by the multidrug resistance protein 1 (MRP1), which is an ATP-binding cassette (ABC) transporter. A second glutathione-S-transferase (MGST2) is a versatile enzyme that is a LTC4 synthase, a general glutathione transferase and a peroxidase.

A subsequent reaction of LTC4 with γ-glutamyl-transpeptidase (GGT1), attached to the plasma membrane, removes the glutamic acid residue to yield LTD4, and this is acted upon by the ubiquitous dipeptidase (DPEP), which is bound to the plasma membrane and anchored through glycosyl-phosphatidylinositol, to produce LTE4.

A natural isomer of arachidonic acid, 8,11,14,17-eicosatetraenoic acid (20:4(n-3)), undergoes a comparable series of reactions to produce LTB4 analogues, i.e., 8‑hydroxy-9,11,14,17-eicosatetraenoic and 8,15-dihydroxy-9,11,13,17-eicosatetraenoic acids, and peptido-conjugates that are related structurally to the cysteinyl-leukotrienes are produced from protectins, resolvins and maresins.

Lysophosphatidylinositol formed when arachidonic acid is released for leukotriene biosynthesis can be reacylated at the endoplasmic reticulum by the lysophospholipid acyltransferase MBOAT7 to enter the phosphatidylinositol cycle, whereby the lipid components are replenished and phosphatidylinositol 4,5‑bisphosphate is synthesised at the plasma membrane. Ultimately, the arachidonic acid content of the phosphatidylinositol in the perinuclear membranes is renewed by this means.

Surprisingly, the red alga Gracilaria vermiculophylla produces a novel oxylipin, (5R,8S)-diHETE, which rearranges via a 1,8-diol-forming mechanism to produce an enantiomeric mixture of LTB4 isomers. The same organism synthesises prostaglandins.

Trans-cellular biosynthesis. It has become apparent that some of these reactions 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 lipid mediator, so mechanisms must exist to transport the eicosanoid intermediate between cells and across phospholipid membrane barriers. As some cell types do not have all the required enzyme systems for production of the full range of LTA4 metabolites, they can synthesise them by such trans-cellular mechanisms. LTA4 synthesised in neutrophils is released to neighbouring acceptor cells such as erythrocytes or platelets that lack 5-LOX but possess the enzyme LTA4 hydrolase and are then able to produce leukotriene LTB4, while LTA4 generated and released from neutrophils is acted upon in acceptor cells such those in the vascular wall by a second LTC4 synthase (microsomal glutathione S-transferase 2 or MGST2) to produce LTC4. Despite the high chemical reactivity of LTA4, these processes can be highly efficient and have the potential to generate elevated concentrations of cysteinyl-leukotrienes at the local level to affect organ function. Trans-cellular mechanisms are likewise critical for the synthesis of lipoxins (see below).

Catabolism. If LTA4 is not metabolized quickly, it can be transformed by non-enzymic hydrolysis of the epoxide ring into dihydroxy acids with relatively little biological activity (all four stereoisomers of LTB4). LTB4 is catabolized in the liver by ω-oxidation carried out by a cytochrome P450 enzyme, followed by β-oxidation from the ω-carboxyl position to produce 18‑carboxy-dinor-LTB4. At this point, the pathway established for prostanoids and lipoxins (below) operates. An alternative pathway in macrophages involves oxidation of the 12‑hydroxyl group to produce inactive 12‑oxo-LTB4 and eventually 10,11-dihydro-LTB4. Catabolism of LTC4 occurs after conversion to LTE4, which can then be subjected to ω-oxidation.

Leukotriene catabolism

Functions: As pro-inflammatory mediators at concentrations in the low nanomolar range, leukotrienes 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 G‑protein coupled receptors. They are strongly implicated in immuno-metabolic disorders (allergies).

Scottish thistleLeukotriene B4 is one of the most potent chemotactic agents known and is important in the inflammatory process as one of the first signals that attract innate immune cells such as leukocytes to a site of insult. Its action is mediated via two cell surface G protein-coupled receptors, BLT1 and BLT2 (chemoattractant-like). Of these, BLT1 is the high-affinity receptor for LTB4 and is expressed primarily in leukocytes. The second receptor BLT2 has a lower affinity for LTB4 and is expressed relatively ubiquitously in human tissues but mainly in mast cells and in epithelial cells of intestine and skin. Both receptors are seen as potential drug targets against inflammatory diseases.

LTB4 causes neutrophils to adhere to vascular endothelial cells and enhances the rate of migration of neutrophils into extra-vascular tissues, while triggering several responses required for host defence, including the secretion of lysosomal enzymes, the activation of NADPH oxidase, nitric oxide formation and phagocytosis. 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. Beneficial properties are evident in that it is involved in the elimination of pathogens, including viruses (including influenza and COVID‑19), bacteria, fungi, protozoan parasites and helminths, and during fungal infections, transcellular biosynthesis of LTB4 orchestrates neutrophil swarming, a defence mechanism triggered by targets larger than the capacity of a single neutrophil to phagocytose.

However, hyperactivation of LTB4 can induce acute and chronic inflammation that results in various inflammatory diseases. For example, 5‑lipoxygenase and LTB4 have been implicated in the chronic inflammation that is a part of the pathophysiology of asthma, rheumatoid arthritis, inflammatory bowel disease, metabolic disease, ophthalmic diseases, atherosclerosis and certain cancers. The enzymes and receptors of the 5-LOX pathway are upregulated in adipose tissue, and LTB4 has a key role in adipose tissue inflammation while promoting liver steatosis and insulin resistance in muscle and adipose tissue, factors that are relevant to obesity. Elevated levels of LTB4 and of the enzyme LTA4 hydrolase have been detected in several cancers, suggesting that the latter might be an appropriate therapeutic target, but further complications arise from reports that the BLT1 receptor is an anti-tumour agent while BLT2 accelerates chemotherapy resistance and tumour promotion. Treatment with blockers of the BLT1 receptor reduced rheumatoid arthritis and bronchial asthma in mice, but BLT2 deficiency facilitated several diseases in the small intestine and skin.

In contrast, leukotriene B5 or 5S,12S-dihydroxy-6Z,8E,14Z,17Z-eicosapentaenoic acid, derived from eicosapentaenoic acid (20:5(n-3) or EPA) strongly inhibits the pro-inflammatory effects of LTB4, while 8,15-dihydroxy-9,11,13,17-eicosatetraenoic acid derived from 20:4(n-3) is similarly anti-inflammatory. In relation to atherosclerotic plaques, it has been reported that the deleterious effects of leukotriene LTB4 resulting from an excessive inflammatory response are countered by the presence of specialized proresolving mediators derived from (n-3) polyunsaturated fatty acids, especially resolvin D1 (RvD1), suggesting a new therapeutic approach to promote plaque stability.

Perhaps surprisingly, 12(S)‑hydroxyheptadeca-5Z,8E,10E–trienoic acid (12-HHT), a C17 metabolite of prostaglandin H (PGH2), is a high affinity ligand for BLT2. Relatively large amounts of this oxylipin are produced by activated platelets during skin injury, and activation of the BLT2 receptor on epidermal keratinocytes has several benefits including accelerating skin wound healing by enhancing cell migration. Acting via the receptor BLT1, LTB4 stimulates the migration of dendritic cells into the skin to activate the immune system. It has been suggested that some side effects of non-steroidal anti-inflammatory drugs, such as delayed wound healing, may be caused by reduced 12-HHT production rather than diminished production of prostaglandins. On the other hand, 12-HHT and its oxo-metabolite may induce resistance to a broad spectrum of anti-cancer chemotherapeutic agents.

Formula of LTC4Leukotriene 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 be pro-inflammator, including by constriction of the airways and vascular smooth muscle, increasing plasma exudation and oedema, and enhanced mucus secretion. They are active in asthma, a chronic inflammatory and allergic disease, and in other inflammatory conditions, including atherosclerosis and myocardial infarction, cancer, and gastrointestinal, skin (atopic dermatitis) and immune disorders. The levels of LTB4 and LTC4 in the circulation are elevated in patients with cerebral ischemia, while patients with peripheral artery disease have elevated levels of LTE4 in urine.

They act through receptors, mainly cysteinyl-leukotrienes type 1 to 3 (CysLT1R, CysLT2R and CysLT3R) receptors at the plasma membrane and nuclear membrane, while GPR99 is a high-affinity receptor for LTE4, and a purinergic receptor, P2Y12, mediates LTE4‑dependent pulmonary inflammation. These are quite distinct from the BLT receptors, and 5-lipoxygenase inhibitors and antagonists to cysteinyl-leukotrienes receptors are both proving useful in treatment of asthma and rhinitis. LTE4 has been reported to upregulate the prostaglandin synthase COX-2 through the PPARγ receptor in mast cells. LTD4 acting through CysLT1R is the most potent bronchoconstrictor so is of special importance to asthma, and antagonists to the receptor are reported to be highly effective in treating allergic rhinitis and for long-term control of asthma. Now, more selective drugs are being sought that limit the production of leukotrienes without a concomitant decrease in pro-resolving products of the 5-LOX pathway like the lipoxins, and recent clinical evidence suggests such inhibitors/antagonists may also be of value for the treatment of cardiovascular diseases and possibly for obesity, as the CysLT1 receptor is present in adipose tissue. In atopic and allergic-contact dermatitis, cysteinyl leukotrienes are potent itch inducers as a result of coupling of LTC4 with its receptor CysLT2R in peripheral sensory neurons. Inflammation caused by cysteinyl leukotrienes in the lung are countered by cysteinyl maresins.

LTD4 and LTE4 are overexpressed in several types of cancer, including cancer of the pancreas, colon, stomach, prostate, ovaries and lung, and they are considered to be tumorigenic. They may modulate the initiation, progression and metastasis of tumours through regulating the proliferation, apoptosis, migration and invasion of cancer cells, while promoting resistance to immunotherapy. As up‑regulation of the expression of their receptors has been observed in several human cancers, there is great interest in drugs that might inhibit these lipids by acting as agonists to their receptors. Apart from leukotriene biosynthesis, 5-LOX can influence gene expression by modulating miRNA processing and transcription factor shuttling in cancers. Cysteinyl-leukotrienes have been implicated in disorders of the central nervous system that include cerebral ischemia, multiple sclerosis, Parkinson's disease and Alzheimer's disease. They play a role in the pathophysiology of the last of these by binding to their receptors in the brain to stimulate the release of cytokines, which lead directly to the formation of insoluble amyloid senile plaques and neuronal damage.

While the general view is that leukotrienes are harmful in relation to the immune system and allergic diseases, such as asthma, there are suggestions that they may 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 required for immune defence. For example, LTB4 can trigger the release of antimicrobial agents.

2.   Lipoxins and Related Compounds

Lipoxins are trihydroxy-eicosatetraenoic acids derived from arachidonic acid by dioxygenation mechanisms with the four double bonds in conjugation, and they were the first lipid mediators to be discovered that were involved in the resolution phase of inflammation. Indeed, they are now considered to be specialized pro-resolving mediators (SPMs) together with the resolvins, protectins and maresins, which are derived from the omega-3 family of polyunsaturated fatty acids. These molecules have structural and biosynthetic similarities to the leukotrienes and appear act in a complementary manner. As few cell types have both the required lipoxygenases, they are formed mainly by trans-cellular pathways.

There are at least three routes to the biosynthesis of lipoxins that differ among cell types, but a common feature is the insertion of molecular oxygen at two sites in arachidonic acid by lipoxygenases. As illustrated for the biosynthesis of those designated lipoxins A4 (LXA4) and B4 (LXB4) in human mucosal cells (airway epithelial cells, gastrointestinal tract and monocytes), the first step is the formation of 15S‑hydroperoxy-5c,8c,11c,13t-eicosatetraenoic acid (15S-HPETE) by a 15‑lipoxygenase (ALOX12, ALOX15, ALOX15B).

Biosynthesis of lipoxins

In the second step, 15S-HPETE or the reduced form 15S-HETE is acted upon by a 5-lipoxygenase in polymorphonuclear neutrophils to form first an epoxy intermediate, i.e., 5S,6S‑epoxy-15S-hydroxy-ETE and then, depending on the cell type, by hydrolases to form either 5S,6R,15S-trihydroxy-7E,9E,11Z,13E-eicosatetraenoic acid (LXA4), or to 5S,14R,15S-trihydroxy-6Z,8E,10Z,12Z-eicosatetraenoic acid (LXB4). The stereochemistry of the 15S‑hydroxyl group is retained in both products. This pathway not only leads to lipoxin biosynthesis but also reduces leukotriene formation, so that there is an inverse relationship in the production of these two classes of lipid mediator in human leukocytes.

In a similar series of reactions in blood vessels, a trans-cellular 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 with the aid of FLAP in leukocytes to form leukotriene A4 (LTA4), which is secreted into the plasma and is available for reaction with a 12‑lipoxygenase (ALOX12) in platelets to form lipoxins (platelets are not able to produce lipoxins directly from arachidonate).

A third mechanism has 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 our web page on prostaglandins, COX-2 is induced in endothelial and epithelial cells in response to a variety of stimuli, and aspirin acetylates the enzyme to switch it 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 (5S,6S,15S-trihydroxy-7E,9E,11Z,13E-eicosatetraenoic acid) and epi-LXB4 (5S,14R,15R-trihydroxy-6E,8Z,10E,12E-eicosatetraenoic acid), the latter with 15R‑stereochemistry. 15(R)-HETE produced by the action of a cytochrome P450 enzyme in the absence of aspirin can be converted to 15‑epi‑lipoxins in the same manner.

Biosynthesis of epi-lipoxins

In contrast to the trans-cellular pathway, both steps can occur in macrophages alone, i.e., in a single cell type. A complex sequence of reactions is involved beginning with activation of toll-like receptor 4 (TLR4), a receptor for endotoxins, which leads to accumulation of the cyclooxygenase-2-derived lipoxin precursor 15-HETE esterified to the membrane phospholipids, especially phosphatidylinositol, a reaction that can be enhanced by aspirin treatment. It may be stored in this inert form until required for anti-inflammatory purposes, when P2X7, a purinergic receptor for extracellular ATP, is activated with the result that efficient hydrolysis of the phospholipid-bound 15-HETE by group IVA cytosolic phospholipase cPLA2α takes place followed by conversion of the unesterified 15-HETE to bioactive lipoxins by 5-lipoxygenase.

Catabolism. Lipoxins are rendered inert 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 two-fold longer half-life than the lipoxins as they are catabolized less efficiently, possibly because of the uncommon 15R-stereochemistry.

Scottish thistleFunctions: Lipoxins were the first eicosanoids to be discovered that are 'switched on' to limit inflammation. As leukotriene LTA4 is the precursor of all other leukotrienes as well as being a direct precursor of lipoxins, it has been suggested that as inflammation advances and LTA4 levels rise, it starts feeding lipoxin synthesis with subsequent initiation of resolution. Together with the protectins, resolvins and maresins, lipoxins control the inflammatory response in such pathogenic conditions as asthma, periodontitis, arthritis, cardiovascular disorders, cancer, and gastrointestinal, periodontal, kidney and pulmonary diseases. They are believed to be neuro-protective in retinal astrocytes and the optic nerve, while dysregulated lipoxin activity is a factor in Alzheimer's and other neurological diseases. Thus, they oppose 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.

The lipoxin structures, which are conserved across species, seem to act at both temporally and spatially distinct sites from other eicosanoids involved in the inflammatory responses. In particular, LXA4 is produced endogenously and is protective mainly via interactions with a G-protein-coupled receptor complex (ALX or ALX/FPR2) and a nuclear transcription factor, although binding to other receptors has been reported. All the observed reactions appear to be highly stereo-selective in terms of double bond geometry and the chirality of the hydroxyl groups. Both the lipoxins and epilipoxins, together with the docosahexaenoic acid metabolite resolvin D1, function by activating the ALX or ALX/FPR2 receptor, and it is now recognized that resolvin and lipoxin metabolism are intimately related. For example, resolvin D1 has been shown to decrease the ratio of proinflammatory leukotriene B4 to proresolving lipoxin A4 via a kinase signalling pathway by means of which the nuclear:cytoplasmic ratio of 5-lipoxygenase, the common enzyme in the biosynthesis of these two eicosanoids, is lowered. This shift in 5-LOX location dampens LTB4 production and enhances LXA4 production.

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. By increasing their cytosolic calcium (Ca2+) levels, the lipoxins are believed to control the entry of neutrophils to sites of inflammation in affected organs and so promote the resolution of inflammation. They are chemo-attractants for monocytes, i.e., cells that are required for wound healing, while LXA4 reduces the production of hydrogen peroxide and of reactive nitrogen species in primary neutrophils. Lipoxins inhibit the production and action of chemokines while simultaneously stimulating anti-inflammatory cytokines via mediation through various receptors. Likewise, by delaying the apoptosis of macrophages, lipoxins can promote resolution of inflammation. 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.

Lipoxins 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 treatments both by oral administration and topical application, and they appear to act via their own receptor, although this has yet to be identified. The possibilities for therapeutic intervention with lipoxins to reduce the adverse effects of inflammation in many different disease states are being explored with animals and humans, although lipoxins per se are not yet available in sufficient quantity for clinical study. A synthetic lipoxin mimetic with greatly improved chemical stability, i.e., a benzo-LXA4, is undergoing clinical trials for human periodontal diseases, and it is believed to have potential benefits in diseases of the kidney. Other lipoxin analogues are under development.

Hemiketal eicosanoids: Two isomeric hemiketals, designated HKE2 (illustrated) and HKD2, are formed in epithelial cells by reaction of 5S‑hydroxytetraenoic acid (5S-HETE) with COX-2 with the reaction proceeding via an unstable di-endoperoxide. HKE2 promotes angiogenesis by enhancing activation of the vascular endothelial growth factor receptor 2 (VEGFR2).

Biosynthesis of hemiketal HKE2

3.   Eoxins

Eoxins are the C14,15 counterparts of the cysteinyl-leukotrienes and are products of the 12/15‑lipoxygenase (15-LOX-1, ALOX15) of human eosinophils and mast cells. The primary metabolite 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 is conjugated with glutathione to produce eoxin C4 and thence eoxin D4 (linked to Cys-Glc) and eoxin E4 (linked to Cys only).

Biosynthesis of eoxins

Like the cysteinyl-leukotrienes, the eoxins are potent pro-inflammatory agents. They 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. Ethanolamides of EXC4 and EXD4, termed 'eoxamides', and analogues of anandamide are produced in human cell preparations in vitro.

4.  Hepoxilins

Hepoxilins are short-lived monohydroxy-epoxy eicosanoids produced in several organs or cell types but mainly the epidermis in humans. They are derived from unesterified arachidonic acid first by the action of a 12‑lipoxygenase, of which the spinal eLOX3 isoform is most important, followed by two divergent pathways to produce 12S‑hydroperoxy-5c,8c,10t,14c-eicosatetraenoic acid (12S‑HPETE), which can either be reduced to the hydroxy compound (12S-HETE) or can be acted upon by a hepoxilin synthase (isomerase) to cause isomerization of the hydroperoxide group to produce an epoxide. The relative rates of the two pathways are controlled by the reducing potential of the cell.

Structural formulae of hepoxilins

Hepoxilins 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. Two 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,9c14c-trienoic acid (hepoxilin B3 or HXB3), but only HXA3 is known to be active in tissues. The epoxide ring is labile and can be opened by an epoxide hydrolase to yield trihydroxy metabolites termed ‘trioxilins’, which may have some activity. Analogous compounds derived from eicosapentaenoic and docosahexaenoic acids have been described.

In skin, the epidermal lipoxygenase 12R-LOX generates the fatty acid hydroperoxide R-HPODE from linoleate in esterified form (complex ceramides), before eLOX3 acting as a hydroperoxide isomerase produces hepoxilin-like compounds such as hepoxilin A illustrated. Hydrolysis of the epoxide ring produces octadec-9R,10S,13R-trihydroxy,11E-enoate (tri-HODE), while the action of an NAD+-dependent dehydrogenase generates the highly reactive octadec-9,10-trans-epoxy,13-oxo,11E-enoate. These are believed to induce changes to the unique structural ceramides in this tissue by promoting covalent linkages to epidermal proteins (see our web page on skin ceramides for further discussion).

Formation of hepoxilin-like compounds from linoleate in epidermis

Hepoxilins have pro-inflammatory properties in the skin, especially in psoriatic lesions, but they are anti-inflammatory in neutrophils. Most of observations suggest an association with mobilization of calcium and potassium ions within cells or across membranes. In addition, hepoxilin A3 is now known to be a regulator of mucosal inflammation in response to infection by bacterial pathogens, such as that responsible for Lyme disease. Although oxylipin production by lipoxygenases in brain tissues tends to be low, there is significant biosynthesis of hepoxilins in the pineal g land, which may be involved in the regulation of melatonin production. It contributes to increased sensitivity to pain during inflammation by interaction with TRPV1 and TRPA1 receptors. Stable synthetic analogues of hepoxilins are useful treatments in animal models of lung fibrosis, cancer, thrombosis and diabetes.

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