Lipoxins: endogenous regulators of inflammation

【摘要】  Over the past decade, compelling in vivo and in vitro studies have highlighted lipoxins (LXs) and aspirin-triggered LXs (ATLs) as endogenously produced anti-inflammatory eicosanoids. LXs and ATLs elicit distinct anti-inflammatory and proresolution bioactions that include inhibition of leukocyte-mediated injury, stimulation of macrophage clearance of apoptotic neutrophils, repression of proinflammatory cytokine production, modulation of cytokine-stimulated metalloproteinase activity, and inhibition of cell proliferation and migration. An overview of recent advances in LX physiology is provided, with particular emphasis on the cellular and molecular processes involved. These data coupled with in vivo models of inflammatory diseases suggest that LX bioactions may be amenable to pharmacological mimicry for therapeutic gain.

【关键词】  mesangial and tubular epithelial cells lipoxin A receptor

EFFECTIVE HOST DEFENSE REQUIRES an acute inflammatory response. This is frequently subverted in chronic inflammatory diseases. The pathophysiology underlying such conditions includes disturbances of hemodynamic events, abnormal extracellular matrix turnover, loss of tissue architecture, and a disruption of normal tissue functioning. The release and oxygenation of arachidonic acid are held to be a critical event in regulating key processes in host defense, inflammation, and hemodynamics. Oxygenation of arachidonic acid initiates the biosynthesis of potent bioactive compounds known as eicosanoids ( 95 ). These metabolites include the prostaglandins, the thromboxanes, the prostacyclins, the leukotrienes, and the lipoxins (LXs).

LXs were first identified by Serhan and colleagues ( 106 ) from purified fractions of leukocyte suspensions that were coincubated with the ionophore A-23187 and 15-hydroperoxyeicosatetraenoic acid. During the last 20 years, significant efforts have been directed toward identifying the physiological actions of LXs in the inflammatory response. In contrast to proinflammatory eicosanoids, LXs are proposed to act as endogenous "braking signals" in inflammation ( Fig. 1 ) ( 79, 102 ). 5 S,6 R,15 S -trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid (LXA 4 ) ( 110 ) and its positional isomer 5 S,14 R,15 S -trihydroxy-6,10,12-trans-8-cis-eicosatetraenoic acid (LXB 4 ) ( 107 ) are the principal species formed in mammals. The 15-epi-LXs are usually referred to as the aspirin-triggered LXs (ATLs) and are endogenous 15 R -enantiomers of LXA 4 and LXB 4 ( 23 ). Synthetic ligands of LXA 4 and ATLa have facilitated the characterization of distinct receptors for LXA 4 that mediate the anti-inflammatory signal ( 27, 101, 109, 112 ).

Fig. 1. Schematic diagram of acute inflammatory response. During the acute inflammatory process, the proinflammatory cytokines such as IFN- and IL-1 can induce the expression of anti-inflammatory mediators such as lipoxins (LXs) and IL-4, which promote the resolution phase of inflammation. PAF, platelet-activating factor; ATL, aspirin-triggered LX; LT, leukotreine; TGF, transforming growth factor.

UPDATE ON LX BIOSYNTHETIC PATHWAYS

LXs, an acronym for lipoxygenase (LO) interaction products, are typically formed through cell-cell interactions. Biosynthesis of these LO-derived eicosanoids occurs through three distinct biosynthetic pathways depending on the cellular context. One of the earliest pathways identified for LX biosynthesis is via platelet-polymorphonuclear neutrophil (PMN) interaction ( 34, 111 ). This pathway has been highlighted as a major route for LX formation within the vasculature involving the activities of 5-LO, present in cells of myeloid lineage, and 12-LO, present in platelets. The myeloid 5-LO generates leukotriene A 4 (LTA 4 ) from arachidonic acid that is rapidly taken up by platelets. Platelets convert leukotriene A 4 (LTA 4 ) to LXA 4 through the oxygenase activity of their 12-LO ( 111 ). Under these conditions, the 12-LO functions as an LX-synthase when the substrate is LTA 4.

A second route of LX formation is via the action of 15-LO (epithelial cells and monocytes) whereby molecular oxygen is inserted into carbon 15 of C20:4, predominantly in the S configuration ( 106 ). The product of this reaction is 15( S )hydroperoxyeicosatetraenoic acid and can serve as a substrate for neutrophil 5-LO and yield an unstable epoxide intermediate, 5,6-epoxytetraene, that is converted to LXA 4 and LXB 4 through the actions of epoxide hydrolases in leukocytes ( 106 ).

An additional route of LX biosynthesis is via the aspirin-triggered 15-epi-LX pathway ( 23 ). In a cytokine-primed milieu, aspirin-induced acetylation of cylcooxygenase (COX)-2 shifts its activity from an endoperoxide to a LO, thereby promoting conversion of arachidonate to 15-HETE that carries its C15 alcohol in the R configuration. 15( R )-HETE released from endothelial and epithelial cells may be transformed by leukocyte 5-LO to generate 15-epi-LXA 4 or 15-epi-LXB 4 ( 23 ). These ATLs share many of the biological functions of native LXs albeit with greater potency and efficacy ( 27 ). Furthermore, the bioactions of ATLs may account for some of the beneficial effects of the aspirin independence of antithrombotic actions ( 79, 102 ). LXA 4 biosynthesis may also occur when precursors such as 15-HETE accumulate within the cell membrane, facilitating LX generation in the absence of transcellular LO activity ( 13 ). Interestingly, remodeling of PMN phospholipids with 15( S )-HETE stereoselectively inhibits PMN migration across endothelium in response to LTB 4 and other chemoattractants ( 121 ).

The route of LX formation depends on the cells and enzymes present therein and can be subjected to modulation by cytokines ( 51, 53, 105, 113 ). IL-4 and IL-13, which are thought to be negative regulators of the inflammatory response, both increase 15-LO expression and activity, thereby enhancing LX formation ( 82, 83 ). Proinflammatory cytokines [e.g., granulocyte/macrophage colony-stimulating factor (GM-CSF), IL-3, and transforming growth factor- (TGF- )] upregulate 5-LO transcripts, which are central to the formation of both LXs and ATLs ( 92 ). The generation of LXs by both pro- and anti-inflammatory mediators may represent negative feedback inhibition on the inflammatory phenotype and thus protect the host from potentially deleterious PMN-induced responses ( Fig. 1 ).

The biosynthesis of lipid mediators has been observed to be biphasic during the inflammatory response. In vivo analysis of eicosanoid formation in a murine dorsal air pouch model of inflammation has revealed temporally distinct processes ( 70 ). Injection of TNF- into the pouch cavity led to a rapid increase in LTB 4 that preceded infiltration of PMN ( 70 ). PMN accumulation within the dorsal pouch coincided with increased intradorsal PGE 2 levels. A persistent, marked increase in LXA 4 levels was concomitant with a reduction in PMN infiltration and PGE 2 production. It was proposed that PGE 2 induced a switch in lipid mediator synthesis from a predominantly 5-LO activity yielding leukotriene B 4 (LTB 4 ) to a 15-LO activity generating LXA 4, a response paralleled by a reduction in PMN infiltration. In vitro investigations have implicated PGE 2 -mediated LO gene expression via CREB activation in increased LX producion ( 70 ). This has led to the proposal that in acute inflammation lipid mediator biosynthesis is biphasic, invoking a role for eicosanoids in the initiation, propagation, and termination of the inflammatory response. The initial phase is coupled to leukotriene biosynthesis, and subsequent prostaglandin formation is coupled to induction of LO activity and LX biosynthesis, thereby promoting the resolution of inflammation ( 16 ). This biphasic model of inflammation has also been observed in an experimental model of rat allergic edema ( 6 ). Moreover, the profile of eicosanoid generation is altered in transgenic mice overexpressing the human myeloid LXA 4 receptor (ALXR) that includes lowered levels of LTB 4 and increased LXA 4 production compared with nontransgenic mice ( 33 ). The ALXR transgenic mice showed diminished ability to activate the proinflammatory transcription factor NF- B within the local inflammatory milieu ( 33 ).

LXs, as with other autocoids, are rapidly produced in response to various stimuli, act locally, and are then enzymatically inactivated by processes that involve dehydrogenation at C15 and possibly -oxidation at C20 ( 26, 104 ). To circumvent such metabolic inactivation, stable synthetic analogs have been derived in which methyl groups were placed on carbon-15 and carbon-5 of LXA 4 and LXB 4 structures, respectively, to block dehydrogenation ( 109 ). Additional analogs of LXA 4 were synthesized with a phenoxy group bonding to C16 and replacing the -end of the molecule ( 74 ). This arrangement allows 16-phenoxy-LXA 4 to resist dehydrogenation and potential -oxidation in vivo by steric hindrance of the bulky aromatic ring, as will be discussed below ( Fig. 2 ). Stable synthetic analogs of the 15-epi-LXs were also synthesized ( 101 ). These LX and ATL mimetics typically retain the biological activity of native LX or ATL with enhanced stability and improved efficacy in several models of inflammation ( 25, 27, 39, 69, 81, 112 ).

Fig. 2. Structure of LXA 4 ( A ), with 15-epi-LX stable analog ( B ) with LTC 4 ( C ) and LTD 4 ( D ) for comparison. LXs are trihydroxytetraene molecules generated from arachidonic acid. 15-Epi-'26-(para-fluoro)phenoxy-LXA 4 is an aspirin-triggered LX stable analog. LTC 4 and LTD 4 comprise members of the peptidoleukotrienes. LTC 4 is the first in the series of the peptidoleukotrienes and contains 3 conjugated double bonds in trans geometry, a fourth double bond at C14, a hydroxyl group at C5 in the ( S ) configuration, and S -cysteinyl-glycine at C6 in the ( R ) configuration. LTC 4 can be metabolized to LTD 4 by enzymatic elimination of glutamic acid from the glutathione at C-6 by -glutamyl transpeptidase. The basis for a common interaction at the LTD 4 receptor may be in part related to the shared spatial orientation [( S ), ( R )] of the polar substitution at C5 and C6 in all 4 eicosanoids.

Recently, a novel series of 17( S )-hydroxy-containing docosanoids were identified in murine brain and glial cells ( 57 ). Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), the well-known -3 fatty acids present in fish oils, are each converted via independent pathways to potent bioactive local mediators. These di- and trihydroxy-containing compounds were termed "resolvins" because they are formed within the resolution phase of acute inflammation, at least in part, as cell-cell interaction products ( 108 ). Aspirin therapy can lead to the generation of the 17( R )resolvins from DHA ( 57, 108 ). These compounds are potent inhibitors of leukocyte infiltration in vivo and downregulate cytokine expression in glial cells ( 57 ). Other endogenously produced anti-inflammatory agents not discussed here may include the prostaglandin metabolites PG 13,14J 12 and annexin-1, as reviewed recently ( 88 ).

MOLECULAR MECHANISM OF LX ACTION

LXs have diverse bioactions on individual leukocytes, stimulating the activation of monocytes and macrophages while inhibiting the activation of PMNs, eosinophils, and lymphocytes ( 46, 48, 79, 102 ). In addition, LXs have been shown to modulate the activity of cells of nonmyeloid origin, including fibroblast cells ( 113 ), endothelial cells ( 37, 38 ), gastrointestinal epithelial cells ( 45, 51, 62 ), renal mesangial cells ( 78, 80 ), and splenic dendritic cells ( 2, 3 ). Table 1 summarizes the literature to date on the cellular effects of LX and ATL analogs in vitro.

Table 1. Effect of lipoxins and their analogs in vitro

Within the last few years, considerable efforts have been made to identify the LX receptors. Available data suggest that LXs exert their bioactions through distinct G protein-coupled receptors (GPCR). Our current understanding of the mechanism by which LXA 4 and its GPCR (ALXR) activate anti-inflammatory signals remains surprisingly incomplete. The repertoire of LX bioactions is due, in part, to the generation of specific anti-inflammatory signals after ALXR activation but also to competitive antagonism of the proinflammatory leukotrienes at the cysLT receptors ( 4, 52, 78 ).

Identification and Cloning of a Distinct LXA 4 Receptor Binding Site

A specific LX recognition site in human PMN was established using labeled LXA 4 ( 41 ). In subsequent studies, a cDNA that could encode a high-affinity receptor for LXA 4 (later designated ALXR) ( 14, 17 ) was identified and cloned in human PMN ( 40 ). ALXR has been described as a high-affinity receptor for LXA 4 in PMN, with a K d of 0.5 nM [ K d of 1.7 nM when expressed in Chinese hamster ovary (CHO) cells]. The ALXR is expressed in monocytes ( 74 ), basolateral membrane of gastrointestinal epithelial cells ( 51, 62 ), synovial fibroblasts ( 113 ), bronchial epithelial cells ( 7, 8 ), and mesangial cells ( 78, 80 ). The ALXR was the first receptor for LO-derived eicosanoids to be cloned, and this paradigm was subsequently applied to cloning of several other eicosanoid receptors, including the BLT-1 (i.e., LTB 4 ) receptor ( 14, 40, 124 ). Mouse and rat ALXR have been cloned from a spleen cDNA library ( 120 ) and from peripheral blood leukocytes, respectively ( 19 ). The overall homology between human and murine ALXR was 76% nucleotide sequence and between human and rat ALXR was 74% nucleotide sequence ( 19, 120 ). Recently, a second mouse LX GPCR was identified from a macrophage cDNA library ( 123 ) and shares an 89% sequence identity with the murine ALXR homolog gene ( 120 ). Like the previously described murine ALXR ( 120 ), activation of this receptor is coupled to anti-inflammatory responses ( 123 ).

Upregulation of ALXR transcript in response to cytokines including IL-13 and IFN- has been demonstrated ( 51 ). In synovial fibroblasts, IL-1 and, to a lesser extent, LXA 4 upregulate ALXR expression, which may represent feedback inhibition of the IL-1-driven proinflammatory phenotype ( 113 ).

A provisional nomenclature for the LX receptor has recently been proposed ( 14 ). Although LXA 4 and LXB 4 share a similar biological profile, LXB 4 does not activate the ALX receptor but may activate another putative receptor that remains to be identified ( 94 ).

ALXR as the Low-Affinity N-Formyl-Met-Leu-Phe Peptide Receptor

The ALXR was initially identified as an orphan receptor, having a DNA sequence homology similar to that of the formyl peptide receptor like-1 (FPRL-1) ( 40 ). Activation of ALXR by either N -formylated peptides or LXA 4 leads to contrasting biological responses ( 21, 22, 67 ). Stimulation of the myleloid ALXR with N- formylated peptides (at sub-µM concentrations) induces PMN activation, chemotaxis, and aggregation, while binding of LXA 4 inhibits these PMN responses ( 17, 42 ). LXA 4 binds the ALXR with high affinity, the K d for the receptor being 1.7 nM, which exceeds by 1,000-fold that observed for N -formyl-Met-Leu-Phe peptide (fMLP; K d = 5 µM) ( 17 ). Competition binding studies have revealed that LXA 4 (at sub-nM concentrations) causes a significant reduction in [ 3 H]fMLP binding to human neutrophils and, similarly, fMLP (at µM concentrations) competed with [ 3 H]LXA 4 for binding to human PMN ( 60 ). This is interesting given that peptide and lipid ligands usually have different binding domains at GPCRs.

LXA 4 as a Partial Agonist/Antagonist at Other Receptors

One of the well-appreciated mechanisms by which LXs promote the resolution of inflammation is by receptor antagonism of LTs, the proinflammatory eicsosanoids, in many cell types both in vitro ( 4, 9, 52, 67, 68, 80, 85 ) and in vivo ( 4, 20, 27, 36 ). The counterregulatory responses reported for LX are mediated, in part, by competition at the cysLT 1 receptor ( 4, 52, 80 ) in addition to the activation of a specific ALXR ( 6, 9, 51, 74, 78, 80, 113, 120 ). LX antagonism of the LT receptors has been demonstrated in PMN, endothelial, bronchial smooth muscle, intestinal epithelial, and mesangial cells ( 79 ). A recent study using human vascular endothelial cells demonstrates that both aspirin-triggered 15-epi-LXA 4 and LTD 4 bind and compete with equal affinity at cysLT 1 receptors ( 52 ). In contrast, LTD 4 was an ineffective competitive ligand for recombinant ALXR with [ 3 H]ATL analog, suggesting that LX can act at the cysLT 1 receptor but that the reverse does not hold ( 52 ). Recently, a second cysLT receptor has been cloned (designated cysLT 2 ) ( 84 ). LX antagonism of this LT receptor subtype remains to be determined. Taken together, these results indicate that native LXA 4 binds to several distinct GPCRs: ALX and cysLT receptors. The basis for this common interaction at the LTD 4 receptor may be related, in part, to the shared spatial orientation [( S ), ( R )] of the polar substitution at C5 and C6 in both eicosanoids, because reversal of this configuration leads to the loss of inhibitory interactions as well as biological activities for both eicosanoids ( Fig. 2 ) ( 68, 107, 110 ).

Evidence for Non-GPCR LXA 4 Receptors

In addition to the complexities of LXA 4 acting at both the ALXR and cysLT receptor, it has also been shown that LXA 4 can act at a third type of receptor, the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor identified in a hepatoma cell line ( 99 ). Once active, the AhR can interact with dioxin-response elements in the regulatory regions of responsive genes to result in their transcription. Genes switched on by the AhR include those implicated in xenobiotic metabolism and degradation of LX ( 99 ).

ALXR Signaling

It may be considered that the bioactions of LX are due to generation of either specific anti-inflammatory intermediates or the aforementioned partial agonism and/or antagonism at the receptors coupled to inflammatory responses. It is probable that a combination of both mechanisms applies. Signal transduction pathways activated on stimulation of the ALXR are still being identified. In monocytes, LXA 4 activation of the ALXR stimulates monocyte adherence, chemotaxis, and Ca 2+ mobilization, and such responses are sensitive to pertussis toxin and an anti-peptide antibody to the ALXR ( 74 ). In contrast, in PMNs it has been reported that LXA 4 does not stimulate Ca 2+ mobilization (at sub-nM LX concentrations) but decreases LTB 4 - and fMLP-induced Ca 2+ mobilization to prevent neutrophil chemotaxis, transmigration, adhesion, degranulation, and superoxide generation ( 28, 40, 42, 53, 59, 67, 85, 119 ). This contrasts with a recent report by He and co-workers ( 55 ) that demonstrates Ca 2+ mobilization at micromolar concentrations of LXA 4 in PMNs. LXA 4 activation of the ALXR (nM concentrations) in human bronchial epithelial cells results in Ca 2+ mobilization through a pertussis toxin-sensitive and PKA-dependent mechanism ( 7 ). ALXR bioactions in macrophages are also coupled through pertussis toxin-sensitive G proteins ( 48 ). In COS-1 cells transfected with murine ALXR homolog cDNA, LXA 4 treatment with various concentrations stimulated phospholipase C activity as assessed by increased inositol-1,4,5,-trisphosphate accumulation ( 123 ). The myeloid ALXR is also coupled to phospholipase A 2 and phospholipase D activation. CHO and HL-60 cells expressing mutant receptors (mutation at serine 236-237 and tyrosine 302) displayed sustained activation of PLA 2 and PLD in contrast to the transient enzyme activity observed with wild-type ALXR ( 60 ). These functional differences between wild-type and mutant ALXR are paralleled by distinct changes in the receptor protein phosphorylation pattern, suggesting differential signaling events ( 60 ).

Evidence in favor of LX promoting the resolution of inflammation is provided by the observation that LX rapid stimulation of the nonphlogistic phagocytosis of apoptotic PMNs by monocyte-derived macrophages is demonstrated both in vitro and in vivo ( 48, 81 ). It is increasingly appreciated that the benefits of such phagocytosis are not restricted merely to clearance of potentially necrotic cells but is associated with the active suppression of inflammation typified by the production of TGF- 1 ( 97 ). Consistent with a role for LX promoting the resolution of inflammation are the observations that LX-stimulated phagocytosis is associated with increased TGF- 1 release from macrophages and a suppression of IL-8 and monocyte chemotactic protein-1 (MCP-1) release ( 81 ). The mechanisms underlying LX-stimulated phagocytosis are coupled to changes in the actin cytoskeleton in macrophages and activation of the monomeric GTPases RhoA and Rac ( 75 ).

Others have shown that ALXR activation inhibits presqualene diphosphate (a negative regulator of PMN signaling) remodeling in response to LTB 4 stimulation, leading to an accumulation of presqualene diphosphate and potent inhibition of both phospholipase D and superoxide anion generation in PMN ( 72 ). In addition, LXA 4 and ATL analogs inhibited LPS-induced ONOO - formation by leukocytes, a response that is associated with attenuation of nuclear accumulation of transcription factors NF- B and AP-1 and the subsequent reduction of IL-8 gene expression and production ( 59 ). The inhibitory action of LXA 4 on ONOO - production is most likely due to inhibition of O 2 - production because others have demonstrated that LXA 4 reduces TNF- -induced O 2 - formation in human PMN ( 53 ).

In gastrointestinal epithelial cells, LXA 4 analog reduced NF- B-mediated transcriptional activation and inhibited degradation of I B ( 45 ). These studies are noteworthy given the importance of NF- B in proinflammatory signaling pathways and the subsequent loss of gastrointestinal epithelial barrier function in response to the gastroenteritis-causing pathogen Salmonella typhimurium ( 45 ). Interestingly, in endothelial cells, LXA 4 activation of tissue factor expression was not associated with I B degradation and may suggest that these responses are cell specific or mediated through distinct receptor subtypes ( 76 ).

In primary cultures of human renal mesangial cells, LXA 4 inhibits PDGF and LTD 4 mitogenic responses ( 78, 80 ). Activation of the mesangial PDGF receptor- (PDGFR- ) by PDGF-BB induces a potent proliferative response in human mesangial cells and has been linked to the etiology of glomerulonephritis ( 1, 29 ). Specific inhibition of PDGFR- tyrosine kinase activity or antagonism of PDGFR- ameliorates mesangial proliferation and renal scarring in an experimental model of Thy-1.1 glomerulonephritis ( 47 ). Interestingly, LTD 4 proliferative responses are mediated via transactivation (tyrosine phosphorylation) of the PDGFR- and p60 c-Src (c-Src) recruitment to the PDGFR- in human mesangial cells ( 78 ). PDGF-BB and LTD 4 activation of the PDGFR- was inhibited by LXA 4 ( Fig. 3 ) ( 78 ). LXA 4 also inhibits LTD 4 -induced phosphoinositide 3-kinase (PI3-K) activation in these cells, a response mediated in part by the specific activation of the ALXR ( 80 ) and not by previously described partial agonism at the cysLT 1 receptor ( 4 ).

Fig. 3. Graphic depiction of LXA 4 receptor (ALXR) cross talk with the PDGF receptor- (PDGFR- ) tyrosine kinase in human renal mesangial cells. LXA 4 inhibits tyrosine phosphorylation of the PDGFR- and mesangial cell proliferation by PDGF. Interestingly, there is differential activation of the MAP kinases by both ligands. The potential involvement of Src phosphatase homology in this pathway remains to be determined. PI3-K, phosphatidylinositol 3-kinase.

Interestingly, PDGF-BB, LTD 4, and LXA 4 all activate mesangial cell MAPKs, albeit with different kinetics ( 80 ). The transient activation observed in response to LTD 4 and PDGF was consistent with a proliferative stimulus, whereas the prolonged activation induced by LXA 4 typifies alternative responses that may include inhibition of DNA synthesis and expression of cdk inhibitor proteins ( 80, 122 ). Interestingly, in PMNs, LXA 4 at micromolar concentrations did not stimulate ERK activation but did generate limited p38 activation ( 55 ). It was also demonstrated in PMNs that LXA 4 inhibited ERK activation induced by serum amyloid A (SAA) ( 55 ). Therefore, it can be deduced that the conditional LXA 4 activation of the MAPKs is dose and cell dependent.

The antiproliferative responses of LX may not only be confined to growth factor stimuli such as PDGF-BB. The stable synthetic analog 15-epi-16-para-fluoro-phenoxy-lipoxin A 4 was also found to inhibit VEGF-induced endothelial cell proliferation and migration; however, the effects of LXA 4 on the activation of the VEGF receptor were not assessed in this study ( 38 ). In an in vivo model of cutaneous inflammation, ATL analog blocked epidermal hyperproliferation ( 101 ). Thus an antiproliferative effect can add to the repertoire of LX bioactions in the inflammatory milieu. These data highlight complex cross talk that exists between multiple receptors to control proliferation and provide unique insight into the mechanisms whereby LXA 4 can exert its anti-inflammatory actions ( Fig. 3 ).

ALXR as a Promiscuous Receptor

On the basis of nucleotide sequence, the ALXR is a member of the chemokine receptor superfamily, and consistent with this is its ability to bind pleiotropic ligands (both lipid and peptide) ( 17, 21, 22, 42, 119 ). The peptide ligands include synthetic peptides ( 21, 22, 31 ); several N -formylated hexapeptides ( 17 ); SAA protein ( 55, 119 ); amyloid (A 42 ) ( 64 ); HIV-1 envelope protein domains (gp 120) ( 32 ); major histocompatibilty complex (MHC) binding peptide ( 17 ); an urokinase type plasminogen activator receptor (uPAR) fragment ( 91 ); a mitochondria peptide fragment MYFINILTL derived from NADH dehydrogenase subunit ( 17 ); annexin 1 ( 87 ); and neurotoxic prion peptide fragments (PrP 106-126 ) ( 65 ) ( Fig. 4 ). It should be noted that many of these exogenous or endogenous ligands do not share significant sequence homology with one another or with fMLP. Whereas multiple ligands have been shown to activate the ALXR in in vitro systems, this may reflect artifactual responses due to altered stoichiometry of signaling compounds. Presumably, exploitation of the ALXR/FPRL-1 null mouse model will address these issues in the future.

Fig. 4. Schematic represention of the potential lipid and nonlipid ligands that bind the ALXR. The peptide ligands include synthetic peptides, several N -formylated hexapeptides, serum amyloid A (SAA) protein, amyloid- (A ), HIV-1 envelope protein domains (gp 120), major histocompatibilty complex (MHC) binding peptide, a urokinase-type plasminogen activator receptor (uPAR) fragment, a mitochondria peptide fragment MYFINILTL derived from NADH dehydrogenase subunit, and neurotoxic prion peptide fragments (PrP 106-126 ).

Responses evoked through ALXR have been observed in response to either lipid or peptide ligands ( 17, 21, 22, 42, 55, 119 ). Many of the above molecules are chemotactic and elicit proinflammatory responses in human leukocytes (annexin 1 being an exception). Chemotactic responses of CHO cells overexpressing ALXR to MHC binding peptide and a previously identified surrogate FPRL-1 agonist were blocked by ATLa ( 17 ). Intriguingly, N -glycosylation of the ALXR is a key component for peptide but not LXA 4 recognition ( 17 ). Stimulation of ALXR by SAA induces IL-8 and, to a lesser extent, TNF- secretion in human neutrophils ( 55 ). LXA 4 activation of the same receptor does not induce IL-8 secretion but decreases SAA-induced IL-8 secretion ( 55 ). It is likely that SAA and LXA 4 may compete for a common binding site on the ALXR as demonstrated by cross-densitization studies ( 119 ). The functional significance of multiple lipid and peptide ligand binding at the ALXR remains uninvestigated. That a GPCR can switch recognition as well as function with certain chemotactic and other peptides from stimulatory to inhibitory with ATL and LX raises the possibility that ALXR activation by LX or ATL can protect the host from deleterious PMN-induced responses associated with innate immune responses. Although the recognition of both lipid and peptide ligands by a single receptor was originally first reported for ALXR, it is now being reported for other receptors such as peroxisome proliferator-activated receptor and CRTH2.

Chimerae constructed from receptors with opposing functions, namely, ALXR and LTB 4, have revealed that the third extracellular loop, seventh transmembrane segment, and COOH terminus of ALXR are essential for LXA 4 recognition and that additional regions of ALXR are required for high-affinity binding of the peptide ligands ( 17 ). It appears that the G protein interactions evoked by ligand-receptor binding and their intracellular amplification mechanisms are different for peptide vs. lipid ligands of ALXR. This has been proposed as a mechanism whereby they can dictate distinct functional responses.

SAA, A 42, and PrP 106-126 are endogenous proteins that when aggregated tend to precipitate and result in amyloid deposition in pathological states such as systemic amyloidosis (SAA), Alzheimer's disease (A 42 ), and prion disease (PrP 106-126 ), respectively. Amyloid, in particular, has been highlighted to be neurotoxic by a process that involves the activation of mononuclear phagocytes (microglia) that gather in and around senile plaques ( 64 ). ALXR transcripts are detectable at high levels by leukocytes infiltrating senile plaques in brain tissues from Alzheimer's disease patients ( 64 ). This is of interest given the role of LXs in promoting the non-phlogistic phagocytosis of apoptotic PMN in vitro and in vivo ( 48, 81 ). In this setting, the potential role of LXs in preventing amyloid -mediated PMN accumulation in senile plaques merits investigation. In addition, there are numerous reports describing the beneficial effects of nonsteroidal anti-inflammatory drugs in retarding the progression of Alzheimer's disease ( 118 ). Thus the benefits of aspirin therapy in Alzheimer's disease may include mechanisms that not only inhibit prostaglandin biosynthesis in senile plaques but may also include the generation of the ATLs that act locally to downregulate leukocyte activity and/or clear the the accumulation of apoptotic cells.

Perretti et al. ( 87 ) have identified novel ligands, annexin 1-derived peptides, which bind ALXR to inhibit PMN diapedesis. Annexin 1 is induced by glucocorticoid and, like LXA 4, is generated at the inflammatory milieu. Both ATL and annexin 1-derived peptides limited PMN infiltration and reduced production of inflammatory mediators. These findings were later demonstrated in vivo using an ischemia-reperfusion model to promote leukocyte-endothelium interactions in the mouse mesenteric microcirculation ( 44 ). In naive mice, the annexin 1-mimetic peptide Ac2-26 abolished ischemia-reperfusion-induced cell adhesion and emigration, but not rolling. In vitro, PMNs taken from these animals could be activated at high concentrations of fMLP, and this effect was blocked by cell incubation with peptide Ac2-26. Both ALXR agonists LXA 4 and peptide Ac2-26 provoked detachment of adherent leukocytes equally in naive and FPR-deficient mice, whereas the CXC chemokine KC or fMLP was inactive. These findings demonstrate that LXA 4 and annexin 1-derived peptides interact directly with human ALXR/FPRL1 to limit PMN trafficking. Therefore, the mechanism of anti-inflammatory bioactions of glucocorticoids may be explained, in part, as the result of the generation of lipoximimetic peptide ligands, such as annexin 1, that bind and activate ALXR ( 44, 87, 88 ). In the context of LX-mediated non-phlogistic phagocytosis of apoptotic cells, it is noteworthy that this activity is also shared by glucocorticoids, albeit after a much longer exposure ( 73 ). Furthermore, annexin 1 exposure in the plasma membrane of apoptotic cells facilitates clearance by phagocytes ( 63, 73 ). These data may suggest that during the acute inflammatory state functional redundancies in endogenous anti-inflammation circuits may exist.

LXS AND THEIR IMPACT IN VIVO

LX generation during inflammatory disease is being increasingly documented ( 79, 102 ). Table 2 illustrates the current data on the effects of LX and 15-epi-LXs in both human clinical investigations and animal disease models. The precise role of LX generation in the pathophysiology of inflammatory diseases is uncertain. Diminished LX production is demonstrated in chronic asthma ( 8 ), chronic liver disease ( 24 ), and chronic myelogenous leukemia ( 116 ). On the contrary, LX generation may be upregulated in other disease scenarios, such as juvenile periodontitis ( 89 ), artherosclerotic plaque rupture ( 11 ), and nasal polyps ( 35 ). In general, it is proposed that LX and the 15-epi LXs are generated in vivo at the inflammatory milieu, and impaired LX biosynthesis may correlate with an inability to resolve the acute inflammatory reaction contributing to a more chronic inflammatory phenotype.

Table 2. Effect of lipoxins and their analogs in vivo

LXs Convey Protection in Acute Inflammatory Renal Diseases

Current pharmacological therapies such as cytotoxic agents and corticosteroids are used to treat inflammatory renal diseases and are often unsatisfactory, nonselective, and highly toxic ( 10 ). Given the extensive display of complex pathophysiological events that may occur in many renal diseases, there is an urgent need for the development of therapeutic interventions that simultaneously target two or more of these detrimental pathological events.

The spectrum of bioactivities reported for LX in vitro and in vivo suggests that these trihydroxytetraenes may also be protective in various human renal diseases. LXs are potent intrarenal vasodilators; inhibitors of PMN chemotaxis, adhesion, and migration across glomerular endothelial cells ( 85 ); promotors of apoptotic PMN clearance from inflammed glomerui ( 48 ); inhibitors of mesangial cell proliferation in response to mitogens LTD 4 and PDGF ( 78, 80 ); and modulators of cytokine production ( 61 ). The therapeutic potential of LXs has been demonstrated in animal models of renal disease. Exposure of PMNs to LXA 4 ex vivo attenuates their recruitment to inflamed renal glomeruli ( 86 ). Overexpression of 15-LO in rat kidney has been shown to be protective in immune-mediated glomerulonephritis and is paralleled with enhanced LX formation ( 82 ). Of particular interest is the murine model of renal ischemia-reperfusion injury (IRI), in which the lipoxin analog 15-epi-16-(FPhO)-LXA 4 -Me was associated with reduced PMN infiltration ( 69 ). In this study, tubule epithelial integrity was preserved and serum creatinine levels were normalized to control levels in 15-epi-16-(FPhO)-LXA 4 -Me-treated IRI mice. Furthermore, the finding of reduced mRNA levels for IL-1, IL-6, and GRO-1 in LXA 4 -treated animals in association with increased expression of suppressor of cytokine signaling (SOCS)-1 and SOCS-2 is intriguing, given the putative role for SOCS as endogenous inhibitors of cytokine bioactivities ( 69 ). Subsequent to these findings was the oligonucleotide microarray-based comparison of mRNA expression profiles in the IRI kidneys in the presence and absence of 15-epi-16-(FPhO)-LXA 4 -Me ( 61 ). Pretreatment with LX analog resulted in a blunting of IRI-associated gene expression and was observed in all of the functional classes of genes induced by IRI, including inflammatory mediators such as chemoattractants, cytokines (e.g., IL-6), chemokines, and chemokine receptors (e.g., IL-1 receptor); growth factors and their receptors (e.g. thrombospondin-1, TGF-1 receptor); adhesion molecules (e.g. ICAM-1, VCAM-1); and other genes (claudin-1, claudin-7) ( 61 ). Interestingly, the data generated in this study indicate that the effects of LX were not restricted to modulating PMN infiltration, as effects on gene expression within the renal parenchyma were observed.

In general, the effects of LXs in conveying protection from acute inflammatory renal disease are promising; however, a role for these eicosanoids in renal fibrosis, the common final pathway in end-stage renal disease, remains the focal point of current research. In this context, the actions of LX to modulate mesangial cell receptor tyrosine kinases (RTKs) are intriguing, given the pivotal role of RTKs in renal fibrosis ( Fig. 5 ) ( 1, 29, 54, 58, 100 ). However, the role of LXA 4 in its ability to downregulate activity of RTKs in primary cultures of human mesangial cells in vitro is suggestive of a protective role for LXA 4 in the detrimental chronic inflammatory response ( 78, 80 ).

Fig. 5. Proposed involvement of LXA 4 and ATL analogs in the prevention of progressive renal disease. ECM, extracellular matrix; ESRD, end-stage renal disease.

LXs in Inflammatory Lung Disease

LXs were first identified in humans in broncheal alveolar lavage fluid from patients with respiratory diseases ( 66 ). Asthmatic patients possess the capacity to generate both LX and the 15-epi-LXs, with higher LX levels detected in the sputum of mildly asthmatic patients compared with lower levels detected in chronically asthmatic patients ( 8 ). In addition, aspirin-intolerant asthmatic patients display a lower biosynthetic capacity than do aspirin-tolerant asthmatic patients, suggesting a better prognostic outcome is associated with increased LX-generating capacity ( 8, 96 ). In murine models of asthma, it is noteworthy that LX blocked airway hyperresponsiveness and pulmonary inflammation ( 71 ). In mice expressing the ALXR transgene, protection against the development of allergy and experimental asthma has been observed ( 71 ).

LXs in Vascular Disease

As mentioned previously, LX generation within the vasculature has been well documented. Vascular effects of LXs both in vivo and in vitro have been reported (Tables 1 and 2 ). These include effects on vascular tone, leukocyte trafficking, and vascular permeability and changes in vascular proliferation. In the kidney, LXA 4 elicits selective vasorelaxant responses in preglomerular arteriolar resistance vessels, thereby increasing renal blood flow and glomerular filtration rate ( 4 ). The vasoactive properties of LXs may be mediated, in part, by the antagonistic activity at peptidoleukotriene receptors ( 4 ) and/or the formation of vasodilatory prostaglandins and prostacyclin ( 12 ). Several studies have also demonstrated LX inhibitory effects on leukocyte-endothelial interactions ( 77, 98, 103 ) and on changes in vascular permeability in vivo ( 52, 56, 120 ). More recent evidence suggests ATL antagonize VEGF-induced angiogenic phenotype in vivo ( 38 ).

A Role for LX in Gastrointestinal Inflammation

LX and LX analogs inhibit PMN adhesion to and transmigration across intestinal epithelia induced by TNF- and fMLP ( 49 ), IL-8 release from TNF- -primed colonic cell lines ( 51 ), human colon ex vivo ( 49 ), and from intestinal epithelia in response to challenge with S. typhimuium ( 45 ). The ALX R is preferentially expressed on the basolateral surface of intestinal epithelia; therefore, LX generation at the paracellular space via neutrophil-epithelial interactions can rapidly act on lateral membrane LXA 4 receptors to downregulate epithelial promotion of intestinal inflammation ( 62 ).

Further evidence of the anti-inflammatory effects of LX in intestinal epithelia was demonstrated by Gewirtz et al. ( 45 ). The effects of LXA 4 analog on gene expression in control and inflammed ( S. typhimuium -infected) model intestinal epithelia were examined by microarray analysis ( 45 ). LXA 4 analog by itself did not influence gene expression; however, it modulated expression of 57 of the 115 genes upregulated by S. typhimurium in in this in vitro model of gastroenteritis ( 45 ). In PMNs, NAB1, a transcriptional corepressor identified previously as a glucocorticoid response gene, was found to be upregulated by an LX stable analog ( 90 ). NAB1 can counterregulate or "switch off" proinflammatory programs, highlighting the protective transcriptional involvement of LX anti-inflammatory signaling. These transcriptomic changes induced by LX and LX analogs will help to underpin novel processes by which these agents offer protection in renal and inflammatory bowel diseases.

In the context of LX-mediated immunomodulation, it is noteworthy that a role for LX in defense against opportunistic infections has been proposed. Human GI epithelial cells treated with ATL stimulate production of a bactericidal/permeability-increasing protein (BPI), a protective protein that inhibits endotoxin signaling ( 15 ). Additional evidence that LXs promote mucosal host defense was demonstrated in a rat model of gastritis where aspirin elicited greater gastric 15( R )-epi-LXA 4 synthesis in the inflammed than in the normal stomach and this was correlated with further reduction in leukocyte-mediated gastric damage ( 114 ).

LXs in Cutaneous Inflammation

A role for LXA 4 in reducing cutaneous inflammation was addressed in a variety of skin inflammation models ( 101 ). These models exhibited pathological features of irritant contact dermatitis, psoriasis, allergic contact dermatitis, urticaria, and atopic dermatitis. Anti-inflammatory potency and efficacy were comparable to an LTB 4 -receptor antagonist and a mild glucocorticoid. ATL analog inhibited edema, leukocyte inflammation, and epidermal hyperproliferation ( 101 ). The anti-inflammatory potential of LX in cutaneous inflammation was further demonstrated further in transgenic mice overexpressing the human myeloid ALX receptor ( 33 ). When topically challenged via dermal ear skin, the mice showed attenuated neutrophil infiltration in response to LTB 4 and PGE 2 ( 33 ).

CONCLUSIONS AND THERAPEUTIC OPPORTUNITIES

Taken together, the data presented here suggest that LX, ATL, and their analogs influence leukocyte trafficking via modulation of chemotaxis, adhesion, transmigration, and phagocytic clearance of apoptotic cells as convincingly demonstrated in both in vivo and in vitro systems. With the gradual elucidation of the cellular and molecular events that underpin the inflammatory process, counterregulatory roles of LXs in conveying protection from leukocyte-mediated tissue injury are slowly being unravelled.

Future directions may involve investigating the potential role of LX and LX mimetics in preventing fibrogenesis, the chronic inflammatory process. The use of well-established experimental models of renal fibrosis will help to underpin the relative importance of these compounds in chronic renal failure ( Fig. 5 ), and a rationale for this work is suggested by evidence that LXs modulate RTK activity in renal cells ( 78 ), a key event in the development of fibrosis ( 1, 29, 54, 100 ).

The accumulating data concerning LXs and their stable analogs on modifying the pathogenesis of the inflammatory process suggest that these molecules may be suitable for pharmacological mimricy for therapeutic gain.

GRANTS

Work in the authors' lab is supported by The Health Research Board, Ireland, The Mater College, The Government of Ireland Programme for Research in Third Level Institutions and The Wellcome Trust.

ACKNOWLEDGMENTS

We thank H. R. Brady for helpful discussions.

Address for reprint requests and other correspondence: C. Godson, Dept. of Medicine and Therapeutics, The Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland (E-mail: catherine.godson{at}ucd.ie