Lipoxin

A lipoxin, an acronym for lipoxygenase interaction product, is a bioactive autacoid metabolite of arachidonic acid made by various cell types. They are categorized as nonclassic eicosanoids and members of the specialized pro-resolving mediator family of polyunsaturated fatty acid metabolites. Like other SPMs, LXs form during an inflammatory response and act to resolve it. The first lipoxins identified were lipoxin A₄ and lipoxin B₄, followed by their respective epimers, the epi-lipoxins 15-epi-LXA₄ and 15-epi-LXB₄.

History

LXA₄ and LXB₄ were first described by Charles Serhan, Mats Hamberg, and Bengt Samuelsson in 1984. They reported that human blood neutrophils, when stimulated, make these two lipoxins and that neutrophils, when stimulated by either of the LXs, mounted superoxide anion generation and degranulation responses. Both responses are considered to be pro-inflammatory in that, while aimed at neutralizing invading pathogens and digesting foreign material, can contribute to damaging host tissues and thereby prolonging and promoting further inflammation. Subsequent studies, however, found that these lipoxins, as well as their epimers, epi-LXA₄ and LXB₄, act primarily to dampen and resolve inflammation, i.e. they are anti-inflammatory cell signaling agents.

Biochemistry

Lipoxins are derived enzymatically from arachidonic acid, an ω−6 fatty acid. Structurally, they are defined as arachidonic acid metabolites that contain three hydroxyl residues and four double bonds. This structural definition distinguishes them from other specialized pro-resolving mediators, such as the resolvins, neuroprotectins, and maresins. All of these SPMs have activities and functions similar, although not necessarily identical, to the lipoxins.

Synthesis

Formation of LXs is conserved across a broad range of animal species from fish to humans. Biosynthesis of the LXs requires two separate enzymatic attacks on arachidonic acid. One attack involves attachment of a hydroperoxy residue to carbon 15, conversion of this species to a 14,15-epoxide, and the resolution of this epoxide to form either 14,15-dihydroxy-eicosatetraenoate or 15-hydroxy-eicosatetraenoate products. This step is catalyzed by enzymes with 15-lipoxygenase activity, which in humans includes ALOX15, ALOX12, aspirin-treated cyclooxygenase 2, and cytochrome P450s of the microsomal, mitochondrial, or bacterial subclasses. ALOX15B may also conduct this metabolism. The other enzyme attack point forms a 5,6-epoxide which is resolved to either 5,6-dihydroxy-eicosatetraenoate or 5-hydroxy eicosatetraenoate products; this step catalyzed by 5-lipoxygenase. Accordingly, these double oxygenations yield either 5,6,15-trihydroxy- or 5,14,15-trihydroxy-eicosatetraenoates. The double oxygenations may be conducted within a single cell type which possesses ALOX5 and an enzyme with 15-lipoxygenase activity or, alternatively, by two different cell types, each of which possesses one of these enzyme activities. In the latter transcellular biosynthetic pathway, one cell type forms either the 5,6-dihydroxy-, 5-hydroxy-, 14,15-dihydroxy- or a 15-hydroxy-eicosatetraenoate, and then passes this intermediate to a second cell type, which metabolizes it to the final LX product. For example, LXs are formed by platelets which, lacking ALOX5, cannot synthesize them. Rather, neutrophils form the 5,6-epoxide leukotriene A₄ via ALOX5, and pass it to platelets that then reduce it to a 5,6-dihydroxy-eicosateteraenoate product and further metabolize it through ALOX12 to form the 15-hydroxy product, LXA₄. The two LXs are distinguished from their 15-epi-LTX epimers by their structural formulae:

LxA₄: 5S,6R,15S-trihydroxy-7E,9E,11Z,13E-eicosatetraenoic acid
LxB₄: 5S,14R,15S-trihydroxy-6E,8Z,10E,12E-eicosatetraenoic acid
15-epi-LxA₄: 5S,6R,15R-trihydroxy-7E,9E,11Z,13E-eicosatetraenoic acid
15-epi-LxB₄: 5S,14R,15R-trihydroxy-6E,8Z,10E,12E-eicosatetraenoic acid

Note that the two LXs have their 15-hydroxyl residues in the S chirality configuration because all of the ALOX enzymes form 15S-hydroxy AA products. In contrast, the 15-hydroxy residues of the two epi-LXs are 15R chirality products because they are synthesized by aspirin-treated cyclooxygenase 2 or the microsomal, mitochondrial, or bacterial cytochrome P450s; these enzymes form almost entirely or partly 15R-hydroxy products.
In addition to the pathways cited above, other transcellular metabolic routes have been shown to make LXs. For example, 5-lipoxygenase in neutrophils and 15-lipoxygenase-1 in immature erythrocytes and reticulocytes operate in series to form LxA₄ and LxB₄; this pathway also occurs in serial interactions between neutrophils and eosinophils; between epithelium or M2 macrophages/monocytes and neutrophils; and endothelium or skeletal muscle and neutrophils.

Stimulation of synthesis

The lipoxins commonly form as a consequence of stimulating the production of pro-inflammatory arachidonic acid metabolites. However, certain cytokines such as IFN-γ and IL-1β further increase production of the lipoxins.

Further metabolism

LXs are rapidly metabolized, mainly by macrophages, to inactive products by being oxidized at carbon 15 to form 15-keto LX products by a 15-hydroxyprostaglandin dehydrogenase; 15-oxo-LXA₄ may be further metabolized to 13,14-dihydro-LXA₄ by an oxidoreductase. 15-Epi-LXA₄ and 15-epi-LXB₄ are more resistant to the dehydrogenation enzyme than their LX epimers. In consequence of the operation of this anabolic pathway, LXs have very short half-lives in vivo. The epi-LXs have longer in vivo half-lives and thereby greater potencies than their LX epimers, and synthetic lipoxins that are metabolically resistant to this pathway have been prepared, used in animal models to study LX activities, and tested as potential therapeutic agents in animals and humans.
Similar to various other AA metabolites such as LTA₄ and 5-oxo-eicosatetraenoic acid, cells and tissues may convert LXs to 20-hydroxy products by omega oxidation; they also have been shown to ligate LXA₄ to glutathione to form cysteinyl-lipoxins, initially LXC₄, which is then sequentially metabolized to LXD₄ and LXE₄. The role of these pathways in limiting or contributing to the activity of the LXs has not been fully evaluated.

Endocannabinoid system

The anti-inflammatory lipid lipoxin A₄ is an endogenous allosteric enhancer of the CB1 cannabinoid receptor. Lipoxin A₄ enhances the affinity of anandamide at this receptor to exert cannabimimetic effects in the brain, by allosterically enhancing AEA signaling and thereby potentiating the effects of this endocannabinoid both in vitro and in vivo. In addition to this, LXA₄ displays a CB1 receptor-dependent protective effect against β-amyloid-induced spatial memory impairment in mice.

Lipoxin analogs

Relatively stable, i.e. metabolically resistant, synthetic analogs of LXs and aspirin-triggered 15-epi-LXA₄s can mimic many of the desirable anti-inflammatory, "pro-resolution" actions of native LXs and are being tested for clinical use. Structurally, these LX analogs often mimic the LXs in being or closely resembling a 20-carbon trihydroxy fatty acid, but are resistant to 15-hydroxyprostaglandin dehydrogenase metabolic inactivation by having a bulky or other structural modification near their 15-hydroxy residues. For example, certain analogs simply alter an LX's structure by: replacing a hydrogen atom with a methyl residue at carbon 15 on LXA₄ to form 15-methyl-LXA₄; changing the last 4 carbons of LXA₄ or 15-epi-LXA₄ to a 1-phenoxy residue or 1-phenoxy-4-fluoro residue to form 16-phenoxy-LX₄, 15-epi-15-phenoxy-LXA₄, 16-(para-fluoro-phenoxy-LXA₄, or 15-epi-16-(para-fluoro-phenoxy-LXA₄; and forming a bond between carbon 9 and carbon 14 of LXA₄ to form an internal phenyl ring analog termed aromatic LXA₄; other, more complex structural analogs in development include 15-epi-LXA₄ analogs termed ZK-142 and ZK994.

Biological activity

Cellular studies

In the initial phases of many acute inflammatory responses, damaged tissues, invading pathogens, and other local events cause nearby cells to make and release arachidonic acid-derived pro-inflammatory metabolites such as: leukotrienes, e.g. LTB₄, LTC₄, LTD₄, and LTE₄; hydroxyeicosatetraenoic acids, e.g. 5-HETE and 12-HETE; and oxoeicosanoids, e.g. 5-oxo-eicosatetraenoic acid and 12-oxo-ETE. These metabolites proceed to act directly or indirectly to recruit circulating leukocytes, tissue macrophages, and tissue dendritic cells to the disturbed tissue site. The consequential congregation of the various cell types promotes transcellular pathways in forming specialized pro-resolving mediators, including the LXs, which then proceed to stimulate cellular and tissue responses that trend to reverse the actions of the pro-inflammatory mediators, dampen and reverse the inflammatory response, and initiate tissue repair.
LXA₄ and 15-epi-LXA₄ are high-affinity receptor ligands for and activators of the FPR2 receptor. FPR2, which is now termed the ALX, ALX/FPR, or ALX/FPR2 receptor, is a G protein coupled receptor initially identified as a receptor for the leukocyte chemotactic factor, N-formylmethionine-leucyl-phenylalanine, based on its amino acid sequence similarity to the known FMLP receptor, FPR1. At least six homologues of this receptor are found in mice. ALX/FPR is a promiscuous various N-formyl oligopeptides that, like FMLP, are either released by microbes and mitochondria or are analogs of those released by microbes and mitochondria; b) microbe-derived non-formyl oligopeptides; c) certain polypeptides that are associated with the development of chronic amyloidosis and/or inflammation including serum amyloid A proteins, a 42-amino acid peptide form amyloid beta termed Aβ42, humanin, and a cleaved soluble fragment other SPMs including resolvins RvD1, RvD2, RvD5, AT-RvD1, and RvD3.
LXA₄ and 15-epi-LXA₄ inhibit chemotaxis, transmigration, superoxide generation, NF-κB activation, and/or generation of pro-inflammatory cytokines by neutrophils, eosinophils, monocytes, innate lymphoid cells, and/or macrophages, as well as suppress proliferation and production of IgM and IgG antibodies by B lymphocytes. These actions appear to involve stimulating anti-inflammatory signaling pathways, but also blocking the actions of other ALX/FPR ligands which simulate pro-inflammatory pathways. Transgenic mice made to overexpress ALX/FPR exhibit markedly reduced inflammatory responses to diverse insults. LXA₄ and 15-epi-LXA₄, when introduced by intrathecal administration into rodents, suppress the perception of inflammatory pain; this action may involve the ALX/FPR receptor shown to be present on the spinal astrocytes of test animal and, based on studies using 15-epi-LXA, inhibition of the NALP1 inflammasome signaling complex.
By mechanisms yet to be clearly identified, the two LXs also: a) stimulate the bacteria-killing capacity of leukocytes and airway epithelial cells; b) block production of the pro-inflammatory cytokine, TNFα, while increasing production of the anti-inflammatory cytokine, CCR5 by T lymphocytes; c)' enhance the ability of monocytes and macrophages to phagocytos and thereby remove potentially injurious apoptotic neutrophils and eosinophils from inflammatory sites cause various cell types to reduce production of pro-inflammatory reactive oxygen species and expression of cell adhesion molecules and increase production of the platelet inhibitor, PGI2 and the vasodilator, nitric oxide; e) inhibit production of pro-inflammatory cytokines by mesangial cells, fibroblasts, and other pro-inflammatory cell types; and f) reduce perception of pain due to inflammation.
LXA₄ and 15-epi-LXA₄ also act by mobilizing transcription factors that regulate expression of various inflammation-regulating genes. LXA₄ stimulates various cell types to promote the entry of Nrf2 into the nucleus and thereby to increase the expression of genes such as heme oxygenase-1, which increases production of the anti-inflammatory gaseous signaling agent, carbon monoxide, and genes involved in the synthesis of glutathione, a product which neutralizes oxidative stress and oxidant-induced tissue damage. Metabolically resistant structural analogs of LXB₄ and 15-epi-LXA₄ inhibit formation of peroxynitrite to attenuate the mobilization of NFκB and AP-1 transcription factors by reducing their accumulation in the nucleus of neutrophils, monocytes, and lymphocytes; NFκB and AP-1 increase expression of pro-inflammatory genes. The two LXBs also trigger activation of Suppressor of cytokine signaling proteins which, in turn, inhibit activation of STAT protein transcription factors which up-regulate many genes making pro-inflammatory products.
LXA₄ and 15-epi-LXA₄ are also high-affinity antagonists of the cysteinyl leukotriene receptor 1 for which leukotrienes LTC₄, LTD₄, and LTE₄ are agonists, i.e. the three leukotrienes bind to and thereby stimulate smooth muscle contraction, eosinophil chemotactaxis, mucous gland secretion, and various other pro-allergic responses in the cells of lung, skin, and other tissues. The ability of these LXs to block the actions of the three LTs may contribute to their ability to resolve allergic reactions; for example, LXA4 relaxes the smooth muscle contraction caused by the cysteinyl leukotrienes in the hamster cheek pouch assay and a metabolically resistant 15-epi-LXAA₄ analog potently inhibits allergen-driven airway hypersensitivity and inflammation in a mouse model.
At higher concentrations, LXA₄ binds to AHR, the arylhydrocarbon receptor; following this binding, AHR enters the nucleus, where it joins with AhR nuclear translocator. The AHR/ARNT complex binds to xenobiotic response elements to activate transcription of genes, most of which are involved primarily in xenobiotic metabolism. These genes include SOCS2, CYP1A1, CYP1A2, CYP1B1, glutathione S-transferase Ya subunit, quinone oxidoreductase, UDP-glucuronosyltransferase and aldehyde dehydrogenase 3 family, member A1. This LXA₄ activity has been demonstrated only in murine cells.
LXA₄ binds to and activates estrogen receptor alpha, with an IC50 of 46nM. LXA₄ and ATLa were shown to activate transcriptional and functional responses via ERa in human endometrial epithelial cells in vitro and in mouse uterine tissue in vivo. Interestingly, LXA₄ also demonstrated antiestrogenic potential, significantly attenuating E2-induced activity. In a mouse model of endometriosis physiologically relevant concentrations of ATLa caused a reduction in lesion size and impacted the production of inflammatory mediators. Molecules regulated via ERa were also impacted, implying that Lipoxin A₄ and analogues, inhibiting both proliferative and inflammatory pathways, might be considered as potential therapeutics.
The actions of LXB₄ and 15-epi-LXB₄ have been far less well defined than those of their LXA₄ analogs. Their mechanism of stimulating target cells is not known. One or both of these analogs have been shown to inhibit the recruitment of neutrophils to sites of inflammation, inhibit the cytotoxicity of NK cells, stimulate the recruitment of monocytes to inflammatory sites, enhance macrophage phagocytosis, and suppress the perception of inflammatory pain in rodents.