Lipoxin A4 Receptor

Lipoxin A4 Receptor Nan Chiang, Karsten Gronert, Fei-Hua Qiu and Charles N. Serhan* Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, USA * corresponding author tel: 617-732-8822, fax: 617-278-6957, e-mail: [email protected] DOI: 10.1006/rwcy.2000.23006.

SUMMARY Lipoxin A4 (LXA4) elicits biological actions via at least two classes of receptors known to date: (1) ALXR on leukocytes and enterocytes; and (2) a shared CysLT1 subtype on endothelial and mesangial cells. ALXR belongs to the group of classical G protein-coupled receptors and was identified in both human and mouse and characterized using direct evidence including specific [3H]LXA4 binding and activation of functional responses with LXA4. In several tissues and cell types other than leukocytes, results of pharmacological experiments indicate that LXA4 acts via a subclass of peptido-leukotriene receptors (CysLT1) as a partial agonist. In addition, endothelial cells (HUVECs) exhibit specific [3H]LXA4 binding which can be inhibited by LTD4 and SKF104353 (CysLT1 antagonist). The molecular origin of CysLT1 is currently under investigation. ALXR is the first cloned lipoxygenase-derived eicosanoid receptor and, together with BLT, they are more akin to chemokine receptors than prostanoid receptors. The cytoplasmic signaling pathways and bioactions of ALXR are cell type specific. In human PMNs, LXA4 stimulates rapid lipid remodeling with release of arachidonic acid in a pertussis toxin-sensitive fashion, and does not trigger significant increases in intracellular Ca2‡ to serve as a second messenger. LXA4 inhibits PMN adhesion, chemotaxis, transmigration as well as degranulation and was implicated as endogenous `stop signals' acting on PMNs. In human monocytes and THP-1 cells, LXA4 initiates intracellular Ca2‡ release via ALXR but neither Ca2‡ nor cAMP proved to be the required second messengers of lipoxin actions in these cell types, indicating different intracellular signaling pathways despite identical receptor cDNA sequences. LXA4 stimulates chemotaxis and adherence in monocytes but no other downstream responses of

these cells, which may relate to the recruitment of monocytes to sites of wound healing and clearance. In agreement with in vitro results, ALXR agonists, namely LXA4, 15-epi-LXA4 (an aspirin-triggered LX) and their stable analogs, are topically active in inhibiting PMN infiltration as well as vascular permeability in murine skin inflammation. The development of these stable analogs will provide valuable tools to evaluate biological and pharmacological roles of ALXR as well as a novel means to develop selective therapies for inflammatory diseases. Since another eicosanoid, PGE2, couples to a variety of signal transduction pathways (i.e. generation of IP3 and [Ca2‡]i as well as decrease or increase of cAMP) via distinct receptor subtypes and/or isoforms that are cell type and tissue specific (Negishi et al., 1995), it is likely that given the range of LXA4 actions in vivo and its impact in isolated cell types, additional receptor systems will be identified.

BACKGROUND Lipid-derived mediators play critical roles in inflammation and other multicellular processes (HaeggstroÈm and Serhan, 1998). Among them, lipoxins (LX) and aspirin-triggered lipoxins (ATLs) evoke actions of interest in a range of physiologic and pathophysiologic processes. These unique components possess a trihydroxytetraene structure and are both structurally and functionally distinct among lipid-derived bioactive mediators. LXA4 and 15-epiLXA4 (a member of the ATL series) display leukocyte-selective actions that enable them to serve as endogenous `stop signals' in multicellular events since they modulate adherence, transmigration, and chemotaxis. LXA4 and 15-epi-LXA4 elicit these cellular responses via a G protein-coupled receptor

2220 Nan Chiang et al. (GPCR) called ALXR, which has been identified in both human and mouse tissues. The characterization of ALXR and development of synthetic lipoxin and ATL stable analog mimetics have rapidly advanced our appreciation of the mechanism of lipoxin's actions and the potential utility of these counter-regulatory biocircuits in the control of local inflammatory events. In this review, ALXR is discussed with respect to its pharmacology, molecular biology, signal transduction, and bioactions in several cell types and animal models studied to date.

Discovery Lipoxins are trihydroxytetraene-containing eicosanoids first found to be generated as lipoxygenase (LO) interaction products in human systems during cell± cell interactions and interactions between specific eicosanoid-generating enzymes. They are both immunoregulatory and vasoactive and their bioactions are sharply distinct from those evoked by other eicosanoids such as leukotrienes (LT), prostaglandins (PG), or thromboxanes (TX). Most members of these eicosanoid classes are well-characterized proinflammatory mediators of inflammation and prothrombolic stimuli. The biosynthesis and actions of lipoxins and ATL are reviewed in the chapter on lipoxin. Here, we provide an update and overview of current knowledge of the actions of LXA4 and the characterization of one of its seven transmembrane receptors, namely LXA4R, recently termed ALXR, that is involved in regulating PMN, monocyte, and epithelial responses. As a class, lipoxins possess physiologic, pathophysiologic, and pharmacologic actions in several target tissues. All the actions of lipoxin are stereoselective in that changes in potencies accompany double bond isomerization, change in alcohol chirality (R or S ) at key positions (Figure 1), as well as selective dehydrogenation of alcohols and reduction of double bonds. The self-limited, local microenvironment impact of lipoxin suggest that they contribute to resolution of injury sites and/or resolve inflammatory loci by regulating further recruitment of PMN and stimulating monocyte migration to promote healing and remodeling. In the vasculature, lipoxins act on both leukocytes and vessels (Serhan, 1997). In this regard, LXA4 is the best studied because of the availability of synthetic compounds that match the physical properties of endogenous LXA4 and are clearly defined in physiology and bioactions. For example, LXA4 is active in several microvascular preparations and in vivo systems (DahleÂn et al., 1988). LXA4 is also active in the guinea pig lung strips and ileum, where its impact proved

highly stereospecific: the 5S,6R-orientation of the two vicinal hydroxyls (Figure 1), positioned immediately adjacent to the carboxylic end of the conjugated tetraene, is essential for activity, and this suggested the presence of specific LXA4 recognition sites. In addition to vasoregulation and modulation of contractile responses in several tissues, LXA4 was also shown to display human leukocyte-selective actions which implicate them as endogenous `stop signals' since LXA4 modulates adherence, transmigration, and chemotaxis (Serhan, 1997). The human neutrophil (PMN) and monocyte responses with LXA4 have been examined in further detail. Early findings showed that LXA4 stimulates rapid lipid remodeling within seconds and releases arachidonic acid in PMN without oxygenation, which is sensitive to pertussis toxin (PTX) treatment (Grandordy et al., 1990; Nigam et al., 1990), pointing to the involvement of a GPCR. The synthesis of the radiolabeled [11,12-3H]LXA4 (Brezinski and Serhan, 1991) enabled the first direct characterization of specific LXA4-binding sites present on PMN that are likely to mediate many of its selective actions on these cells (Fiore et al., 1992). Intact PMN demonstrate specific and reversible [11,12-3H]LXA4 binding (Kd  0.5 nM and Bmax  1830 sites/PMN) that are modulated by guanosine stable analogs. The subcellular distribution of LXA4binding sites is listed in Table 1. These LXA4-binding sites are inducible in promyelocytic lineage (HL-60) cells exposed to differentiating agents (e.g. retinoic acid, DMSO, and PMA) and confer LXA4-stimulated phospholipase activation (Fiore et al., 1993). Together, these findings provided further evidence that LXA4 interacts with specific membrane-associated receptors on human leukocytes which belong to the classical GPCR since, in the absence of LXA4-specific binding, lipid-signaling events are not initiated by LXA4. Based on the finding that functional LXA4 receptors are inducible in HL-60 cells, several putative receptor cDNAs that are also induced within this temporal frame, cloned earlier from myeloid lineages and designated orphans (Perez et al., 1992; Nomura et al., 1993), were systematically examined for their ability specifically to bind and signal with LXA4. Chinese hamster ovary (CHO) cells transfected with one of the orphans (previously denoted pINF114, also known as FPRL1 and FPR2) displayed both specific [3H]LXA4 binding with high affinity (Kd=1.7 nM) and demonstrated selectivity when compared with LXB4, LTB4, LTD4, and PGE2 (Figure 1 and Table 2) (Fiore et al., 1994). These transfected CHO cells transmitted signal with LXA4, activating both GTPase and the release of arachidonic acid (C20:4) from membrane phospholipid,

Lipoxin A4 Receptor 2221 Figure 1 Ligand specificity and structure±activity relationship of ALXR. LXA4 interaction with ALXR is highly stereospecific, that is the 5S,6R-orientation of the two hydroxyl groups as well as 11-cis double bond conformation are essential for bioactions. 15-epi-LXA4 (an aspirin-triggered lipoxin, ATL) carries a C-15 alcohol at the R configuration, opposite to the S configuration in native LXA4 and was shown to have higher potency than native LXA4 in certain bioassays. In 15(R/S )-methyl-LXA4, hydrogen at C-15 was replaced by a methyl group at a racemate at C-15. 16-phenoxy-LXA4 has a phenoxyl group at C-16. These compounds which are more resistant to rapid dehydrogenation by 15-hydroxyprostaglandin dehydrogenase (15-PGDH) than native LXA4, compete with [3H]LXA4-specific binding on PMNs and are potent inhibitors for PMN functions in vitro and in vivo.

cis

trans

S trans

RS

Table 1 Subcellular distribution of LXA4- and LTB4-specific binding in human Component

Distribution of specific binding (%) [3H]LXA4

[3H]LTB4

Plasma membrane

 42%

 16%

Granule fraction

 34.5%

 75.7%

Nuclear fraction

 23.3%

 8.3%

indicating that this cDNA encodes a functional receptor for LXA4 in myeloid cells. It is essential to use GTPase and C20:4 release to test the relationship between specific binding and function because, with human PMNs, LXA4 stimulates only a modest  10% of the Ca2‡ response observed with fMLP. In human monocytes and THP1 cells, a Ca2‡ transit is initiated by LXA4, but Ca2‡ mobilization proved not to be a second messenger of lipoxin actions since LXA4-triggered adherence to laminin is insensitive to BAPTA-AM (Figure 2), an intracellular Ca2‡ chelator (Romano et al., 1996; Maddox et al., 1997). The

2222 Nan Chiang et al. Table 2 Competitive binding of [3H]LXA4 with structure-related eicosanoids

Competition

PMNs

HL-60 cells

CHO-ALXR

HUVECs

LXA4 (IC50  1.5 nM)

LXA4

LXA4 (Ki  5.6 nM)

LXA4

LXA4-methyl ester

LTD4

15(R/S )-methyl-LXA4

SKF104353

16-phenoxy-LXA4

(CysLT1 antagonist)

15-epi-LXA4 Partial competition

LTC4 (IC50  62 nM)

LTC4

LTD4 (Ki  80 nM)

LTD4 (IC50  56 nM)

fMLP

fMLP (IC50  1000-fold higher than LXA4) No competition

LTB4

LTB4

LXB4 6S-LXA4 11-trans-LXA4

SKF104353

LTB4

ONO-4057

ONO-4057

LXB4

(LTB4 and antagonist)

LXB4

PGE2

SKF104353

Figure 2 LXA4 and ALXR evoke differential signalings and responses with human PMNs versus monocytes. ALXR inhibits PMN and stimulates monocyte functions via pertussis toxin (PTX)-sensitive G proteins (G) upon activation by LXA4, 15-epi-LXA4, as well as LX analogs. In PMNs, neither intracellular calcium ([Ca2‡]i) nor cAMP were increased in response to lipoxins. In monocytes, LXA4 induced an increase of [Ca2‡]i which is not the second messenger for LXA4stimulated adherence or chemotaxis since these responses were unaffected by BAPTA-AM (a Ca2‡ chelator). See text for details.

Surrogate peptides

mouse LXA4 receptor cDNA was cloned from a spleen cDNA library (Figure 3) and displays specific [3H]LXA4 binding and LXA4-initiated GTPase activity when transfected into CHO cells (Takano et al., 1997).

The human and mouse LXA4 receptors represented the first cloned LO-derived eicosanoid receptors. Several prostaglandin receptors were cloned earlier and, more recently, using a similar approach as for

Lipoxin A4 Receptor 2223 Figure 3 Human and mouse ALXR: homology and tissue distribution. ALXR mRNAs  1.4 kb in both human and mouse tissues are indicated by arrows. e1±e3 represent the putative extracellular loops, T1±T7, transmembrane segments and i1±i3, intracellular loops for ALXR, respectively. Percentage homology between individual domains of human and mouse ALXR in deduced amino acid sequences are indicated by numbers. High homology is observed in second intracellular loop (100%) and sixth transmembrane segment (97%).

ALXR, enabled identification of the long-sought human LTB4 receptor (BLT) (Yokomizo et al., 1997) and its murine homolog (Huang et al., 1998). It is of interest that the BLT, which displays sequence homology with ALXR, was originally cloned as a purinergic receptor (Akbar et al., 1996), and that both ALXR and BLT are more akin to chemokine receptor structure than prostaglandin receptors (Figure 4), which probably reflects the fact that lymphotoxins and lipoxins are eicosanoids that contain thermally sensitive conjugated double bond systems and prostaglandins contain a cyclopentane structure. BLT was recently identified as a novel coreceptor mediating HIV-1 entry into CD4‡ cells (Owman et al., 1998). Along these lines, ALXR was also recently shown to interact with surrogate peptide ligands and evoke Ca2‡ mobilization when expressed in HEK 293 cells carrying G16 (Klein et al., 1998). These observations suggest that receptors of this class interact with both small endogenous lipophilic ligands of the host and larger exogenous proteinaceous structures: this is an interesting aspect of these receptors that might relate to their potential multifunctional roles as sensing receptors in host defense.

Alternative names ALXR cDNA was initially cloned as an orphan receptor and reported independently by several different

Figure 4 Phylogenetic tree of human eicosanoid and chemokine receptors. Structure similarity of deduced amino acid sequences of human eicosanoid and chemokine receptors is determined by the average linkage cluster analysis. Abbreviations: TP (thromboxane A2 receptor), EP1, EP2, EP3, EP4 (subtypes of prostaglandin E2 receptor), FP (prostaglandin F2 receptor), IP (prostacyclin receptor), ALXR (lipoxin A4 receptor) and BLT (leukotriene B4 receptor). BLT (Owman et al., 1998), CXCR4 (Feng et al., 1996), and CCR5 (Alkhatib et al., 1996) were identified as coreceptors for HIV-1 entry.

groups using fMLP receptor (FPR) cDNA as a probe with low-stringency hybridization conditions. The cDNA has high sequence homology ( 70%) to FPR and hence, on the basis of sequence homology alone, was named FPRL1 (FPR-like 1) (Murphy et al., 1992), FPRH1 (Bao et al., 1992), and also FPR2

2224 Nan Chiang et al. (Ye et al., 1992) or RFP (receptor related to FPR; Perez et al., 1992). Convincing functional responses were not presented for these clones. It was also cloned by Nomura et al. (1993) from a human monocyte cDNA library and renamed as an orphan receptor, denoted by these investigators as HM63. None of the reported cloning efforts attached functions to these clones. However, since the bacterial peptide surrogate fMLP interacts with FPR on phagocytic cells, the assumption of a phagocytic or chemotactic response in host defense for the sequence-related receptors such as FPRL1 remained a plausible hypothesis. These cDNA sequences are available from GenBank (see Accession numbers). Note that ALXR and BLT are the recently proposed renomenclature for LXA4 receptor and LTB4 receptor, respectively (DahleÂn et al., International Union of Pharmacologic Sciences Nomenclature Committee, Stockholm, Nobel Forum, November, 1998). In addition, the terms CysLT1 and CysLT2 were introduced for peptido-LT (LTC4, LTD4, and LTE4) receptors which are classified on the basis of sensitivity to antagonism rather than agonist properties. CysLT1 and CysLT2 are sensitive and resistant, respectively to the class of drugs currently being introduced in the clinic, including SKF104353.

Structure Deduced amino acid sequence places ALXR within the GPCR superfamily, characterized by seven putative transmembrane (TM) segments (Figure 5) with the N-terminus on the extracellular side of the membrane and the C-terminus on the intracellular side (Baldwin, 1993). The overall homology between human and mouse ALXRs is 76% in nucleotide sequence and 73% in deduced amino acid (Takano et al., 1997). An especially high homology is evident for their second intracellular loop (100%) and between their sixth TM segment (97%), followed by the second, third, and seventh TM segment as well as the first extracellular loop (87±89%), suggesting essential roles for these regions in ligand recognition and G protein coupling (Figure 3). Molecular evolution analysis suggests that ALXR is only distantly related to prostanoid receptors and belongs to the cluster of chemoattractic peptide receptors, exemplified by fMLP, C5a, and IL-8 receptors (Toh et al., 1995), which is now known to include BLT. The recently cloned BLT was obtained from human HL-60 cells (Yokomizo et al., 1997) and mouse eosinophils (Huang et al., 1998) and found to share an overall  30% homology with ALXR in deduced amino acid sequences (Figure 5). A highly

homologous region ( 46%) is present within the second TM segment in both ALXR and BLT with the amino acid sequence LNLALAD. Prostanoids interact with their receptors via COOÿ interacting with an arginine residue within the seventh TM segment (Ushikubi et al., 1995). Neither ALXR nor BLT share this Arg (in seventh TM segment) requirement (Fiore et al., 1994; Yokomizo et al., 1997), yet both ligands contain COOH that at physiological pH could present as a counteranion. Together, these findings provide further evidence that the origin of receptors for leukotriene and lipoxin is distinct from that for prostanoids.

Main activities and pathophysiological roles As mentioned above, LXA4 evokes vasodilatory and counter-regulatory roles in both in vivo and in vitro models. These counter-regulatory actions are initiated via unique cell surface receptors on leukocytes and enterocytes (Figure 6). With other cell types, such as endothelium and mesangial cells (Figure 7), LXA4 evokes bioactions and interacts with a subclass of peptido-LT receptors (CysLT1), reviewed by Serhan (1997). The leukocyte receptors are physiologically and pharmacologically distinct and evoke selective actions on each type of leukocyte tested to date. With human peripheral blood leukocytes, LXA4 inhibits both isolated PMN and eosinophil chemotaxis in vitro in the nanomolar range (Lee et al., 1989; Soyombo et al., 1994) and blocks human natural killer (NK) cell cytotoxicity in a stereoselective fashion (Serhan, 1997). In cell±cell interaction systems, LXA4 inhibits PMN transmigration across both endothelial and epithelial monolayers (Colgan et al., 1993; Papayianni et al., 1996) via actions on both cell types (i.e. PMNs and endothelial cells, PMNs and epithelial cells). These responses are also evident in vivo with murine receptors (Takano et al., 1998). These immunoregulatory actions implicate them as endogenous `stop signals' acting on human PMNs defining novel antiinflammatory receptors and signaling pathways (Serhan, 1997). Recently, it was reported that NK cells possess cell surface receptors that recognize major histocompatibility complex (MHC) class I peptides and inhibit NK cell-mediated cytotoxicity (Lanier, 1997), further supporting the existence of the inhibitory receptors within the immune system. In human monocytes, LXA4 stimulates chemotaxis and adherence via ALXR (Figure 2), which may be related to the recruitment of monocytes to sites of wound healing and a protective role for LXA4 (Maddox et al., 1997). The main in vitro and in vivo

Lipoxin A4 Receptor 2225 biological actions of LXA4 are summarized in Figure 6 and Figure 7. Also see Table 3 and Table 5 in the lipoxin chapter where the pathophysiological roles of LXA4 are discussed in detail.

GENE

Accession numbers The cDNAs for both human ALXR from THP1 cells (Maddox et al., 1997) and enterocytes (Gronert et al., 1998) are available in GenBank: the accession numbers are U81501 and AF054013, respectively. It was also previously cloned by several individual groups as an FPR homolog without functional data or ligands and deposited as orphan receptors and submitted with accession numbers X63819 (Perez et al., 1992), M84562 (Murphy et al., 1992), D10922 (Nomura et al., 1993), M88107 (Ye et al., 1992), and M76672 (Bao et al., 1992). Mouse ALXR cDNA is also available from GenBank: U78299 (Takano et al., 1997).

Sequence Both human (Fiore et al., 1994) and mouse (Takano et al., 1997) ALXR cDNA contain an open reading frame of 1051 nucleotides which encode a protein of 351 amino acids. Northern blot analysis (Figure 3) demonstrated that ALXR mRNA is  1.4 kb in both human and mouse (Takano et al., 1997).

Chromosome location and linkages Chromosome mapping revealing that the gene encoding ALXR (Fiore et al., 1994) is located on chromosome 19q (Bao et al., 1992), denoted in this early report of the orphan receptor as FPRH1.

PROTEIN

Accession numbers Not available.

Description of protein Hydrophobicity analysis of the deduced amino acid sequence of ALXR revealed seven repeated hydrophobic clusters of 20±25 amino acids interspersed

with varying lengths of hydrophilic sequences, which are the common features of GPCR (Figure 5). The seven hydrophobic clusters were proposed to form membrane spanning  helices, whereas the hydrophilic segments form loops that project alternately into the extracellular space and the cytoplasm (Baldwin, 1993). Several potential posttranslational modification sites were observed in the deduced amino acid sequences of both human and mouse ALXR (Figure 5): disulfide linkage, N-glycosylation, and phosphorylation. Disulfide Linkage Conserved cysteine residues at the first (Cys99) and second (Cys176) extracellular loops are found in both human and mouse ALXR. These are proposed to form a disulfide bond to stabilize the tertiary integrity of most GPCR. N-glycosylation Human ALXR contains two putative N-glycosylation sites located at the N-terminus (Asn4) and second extracellular loop (Asn179). In mouse ALXR, both putative N-glycosylation sites are located at the Nterminus (Asn4 and Asn10). Phosphorylation The C-terminus of mouse ALXR contains nine potential phosphorylation sites (e.g. serine and threonine residues), among which six are conserved within the human ALXR.

Relevant homologies and species differences The overall homology between human and mouse ALXRs is 76% in the nucleotide sequence and 73% in deduced amino acid with high homology within their second intracellular loops (100%) (Takano et al., 1997). Information for other species is not yet available. For details, see Structure.

Affinity for ligand(s) Table 2 and Figure 1 summarize the knowledge of ligand affinity and specificity for ALXR. Intact human PMNs and retinoic acid-differentiated HL-60 cells demonstrate specific and reversible [3H]LXA4 binding with Kd  0.5 and  0.6 nM, respectively (Fiore et al., 1992, 1993). Several isomers of LXA4

2226 Nan Chiang et al. Figure 5 The predicted membrane topology model of ALXR: homology between ALXR and BLT. Deduced amino acid sequence of ALXR demonstrates seven putative transmembrane segments with Nterminus on the extracellular side of the membrane and C-terminus on the intracellular side. ALXR possesses potential N-glycosylation sites (±CHO), phosphorylation sites (±P) and disulfide linkage (C±C). Blue circles indicate the residues conserved between human ALXR (anti-inflammatory) and BLT (proinflammatory). Unlike prostanoid receptors that use Arg in the seventh TM segment as a counterion for ligand binding, neither ALXR nor BLT possess arginine residues in the seventh TM segment as a structural feature. (Full colour figure may be viewed online.)

tested, namely 11-trans-LXA4, 6S-LXA4, and LXB4, did not compete for this recognition site, consistent with their functional responses in these systems. Results from Scatchard analyses indicate that [3H]LXA4 binds PMN granule membrane-enriched fractions with comparable Kd values (0.8 nM), but with a larger Bmax (4.1  10ÿ11 M) than plasma membrane (Kd=0.7 nM, Bmax=2.1  10ÿ11 M) fractions (Fiore et al., 1994). Hence, it appears that additional receptors can be mobilized by granule fusion to the plasma membranes of PMNs (Table 1). [3H]LXA4-specific binding is stereoselective since neither LTB4, LXB4, 6S-LXA4, 11-trans-LXA4, nor SKF104353 (a CysLT1 antagonist) compete for [3H]LXA4 in human PMNs (Table 2 and Figure 1) (Fiore et al., 1992). Among the related eicosanoid heteroligands tested in the HL-60 cell system, only LTC4 at  3-log molar excess competes for

[3H]LXA4-specific binding (Fiore et al., 1993). The peptido-LT receptors remain to be cloned. The crosscompetition of LTC4 and LTD4 observed with LXA4 in several systems suggests that the `true' peptido-LT receptors may also be of this class of receptors. In several tissues and cell types other than leukocytes, results from pharmacological experiments indicate that LXA4 interacts with a subclass of peptido-LT receptors (CysLT1) as a partial agonist to mediate its actions (Badr et al., 1989; Fiore et al., 1992). Along these lines, both LTC4 and LXA4, albeit at high concentrations (> 1 mM), induce contractions of guinea pig lung parenchyma and release of thromboxane A2, which is sensitive to CysLT1 receptor antagonists (Wikstrosm Jonsson, 1998). This is not likely to be a physiologic action of LXA4. In certain cell types, LXA4 (in the nanomolar range) blocks LTD4 actions (Figure 7) and in this regard it

Lipoxin A4 Receptor 2227 Figure 6 LXA4 actions via ALXR in leukocytes and epithelial cells. Illustration of the regulatory actions of LXA4 in leukocytes (reviewed in Serhan, 1997) and human epithelial cells (Gronert et al., 1998 and Gewirtz et al., 1998). Upper left panel: Ear biopsies: Inhibition of LTB4-induced PMN infiltration into mouse ear by topical application of LXA4 analogs in acute skin inflammation (Tokano et al., 1997). PMN is indicated by an arrow. Upper right panel: Photomicrograph: Internalization of Salmonella typhimurium (shown in green) by intestinal epithelium (indicated by an arrow). In response to this gastrointestinal pathogen, intestinal epithelium secretes chemokines which promote neutrophil infiltration. This chemokine (IL-8) secretion can be downregulated by LXA4 analogs (Gewirtz et al., 1998).

blocks specific [3H]LTD4 binding to mesangial cells (Badr et al., 1989) and human umbilical vein endothelial cells (HUVECs) (Fiore et al., 1993; Takano et al., 1997). HUVECs specifically bind [3H]LXA4 at a Kd of 11 nM, which can be inhibited by LTD4 and SKF104353 (Fiore et al., 1993). Therefore, it appears that LXA4 interacts with at least two classes of cell surface receptors: one specific for LXA4 that is present on leukocytes and enterocytes (ALXR) (Figure 6), the other shared by LTD4 and present on HUVECs and mesangial cells (CysLT1) (Figure 7). The molecular origins of these LXA4/LTD4-binding sites (pharmacologically defined as CysLT1) are currently of considerable interest. Several synthetic analogs of both LXA4 and 15-epiLXA4 have been designed to resist rapid metabolic inactivation and have been tested for their ability to compete with [3H]LXA4-specific binding to ALXR. For detailed information on the formation of 15-epiLXA4, the design of stable LX analogs and their

pharmacological activities, see the lipoxin chapter. Both 15(R/S )-methyl-LXA4 and 16-phenoxy-LXA4 compete with [3H]LXA4 as well as [3H]LTD4 for specific binding on human PMNs (Table 2) and HUVECs, respectively (Takano et al., 1997). Hence, each of the bioactive lipoxin mimetics acts at sites that compete with [3H]LXA4. Moreover, each LXA4 analog that competes with [3H]LXA4 is also topically active in inhibiting PMN migration in acute dermal inflammation. Human and mouse ALXR cDNA transfected into CHO cells display specific binding with [3H]LXA4; the Kd is 1.7 nM for human (Fiore et al., 1994) and Kd 1.5 nM for mouse ALXR (Takano et al., 1997). Human ALXR-transfected CHO cells were also tested for binding with other eicosanoids, including LXB4, LTD4, LTB4, and PGE2 (Figure 1). Only LTD4 shows competition with [3H]LXA4 binding, giving a Ki of 80 nM (Table 2) (Fiore et al., 1994). It is of interest that, although ALXR shares  70%

2228 Nan Chiang et al. Figure 7 LXA4 interaction with CysLT1 in several cell types and tissues. Illustration of the regulatory actions of LXA4 in vascular endothelial cells (reviewed in Serhan, 1997), smooth muscle contraction (DahleÂn et al., 1988; Christie et al., 1992), rat glomerular mesangial cells (Badr et al., 1989) as well as in bone marrow (Stenke et al., 1991) via a subclass of peptido-LT receptors (CysLT1).

in vivo

homology with FPR, ALXR only binds [3H]fMLP with low affinity (Kd  5 mM) and proves to be selective for LXA4 by three log orders of magnitude (Fiore and Serhan, 1995). Recently, it was reported that some surrogate peptides can also interact with ALXR (also known as FPRL-1) in model systems (Klein et al., 1998), but the functional role of these peptides in human biology is not clear. The apparent EC50 value for receptor activation (determined by mobilization of [Ca2‡]i) by the best synthetic rogue peptide of this synthetic series is approximately 2 nM (Klein et al., 1998), whereas LXA4 and its analogs stimulate monocyte adherence via ALXR at concentrations less than 1 nM (EC50 for analogs  8  10ÿ11 M, EC50 for LXA4  8  10ÿ10 M) (Maddox et al., 1997) or inhibit PMN transmigration and adhesion at 10ÿ10 M (Serhan et al., 1995). These new findings suggest that small peptides as well as bioactive lipids can both function as ligands for the same receptor, with different affinity and/or distinct interaction sites within the receptor and separate intracellular signaling, depending on the cell types. It appears likely that the protein interactions following ligand±receptor binding are different for peptide in comparison with lipid ligands of this receptor.

Cell types and tissues expressing the receptor To date, ALXR has been identified by its functions and direct actions, and cloned in both human and mouse PMNs (Fiore et al., 1994; Takano et al., 1997), human monocytes (Maddox et al., 1997), as well as human enterocytes (Gronert et al., 1998). In human PMNs, the results of subcellular fractionation experiments revealed that [3H]LXA4-binding sites are associated with plasma membrane and endoplasmic reticulum (42.1%), granule (34.5%) as well as nuclear-enriched fractions (23.3%), a distribution distinct from that of [3H]LTB4-binding sites (Table 1) (Fiore et al., 1992). The finding that LXA4 blocks both PAF- and fMLP-stimulated eosinophil chemotaxis (Soyombo et al., 1994) suggests that functional ALXR is also present on eosinophils. In human enterocytes, ALXR is present in crypt and brush border colonic epithelial cells, as demonstrated by Gronert et al. (1998). Functional blockage of LTD4 binding by LXA4 and vice versa on HUVECs indicates the presence of specific LXA4/LTD4-binding sites (CysLT1) on both endothelial and mesangial cells (Figure 7).

Lipoxin A4 Receptor 2229 Northern blot analysis (Figure 3) of multiple murine tissues demonstrated that ALXR mRNA is most abundant in PMNs, spleen, and lung, with lesser amounts in heart and liver (Takano et al., 1997). In the absence of disease, the pattern is similar in human tissues. In humans, ALXR mRNA is also abundant in PMNs, followed by spleen, lung, placenta, and liver (Fiore et al., 1994; Takano et al., 1997).

mRNA (Gronert et al., 1998). In view of the cytokine regulation of ALXR, it is likely that the expression of these receptors will change dramatically in disease states which, in turn, might downregulate mucosal inflammatory and allergic responses.

SIGNAL TRANSDUCTION

Regulation of receptor expression

Associated or intrinsic kinases

Retinoic acid, PMA, and DMSO, which lead to granulocytic phenotypes in HL-60 cells, each induce a 3- to 5-fold increase in the expression of ALXR as monitored by specific [3H]LXA4-binding (Fiore et al., 1993). Transcription of ALXR was dramatically upregulated by cytokines in human enterocytes, with lymphocyte-derived IL-13 and IFN being most potent (Figure 8), followed by IL-4 and IL-6. IL-1 and LPS also showed moderate induction of ALXR

In retinoic acid-differentiated HL-60 cells, LXA4 stimulated phospholipase D (PLD) activation that is staurosporine-sensitive, suggesting the involvement of PKC in signal transduction in these cells (Fiore et al., 1993). It was also demonstrated that LXA4 blocks LTB4- or fMLP-stimulated PMN transmigration or adhesion by regulation of 2 integrin-dependent PMN adhesion (Fiore and Serhan, 1995). This modulatory action is partially reversed by prior exposure to

Figure 8 Regulatory role of LX and ALXR during PMN±epithelial cell interaction. Illustration of the anti-inflammatory actions of LXA4, 15-epi-LXA4, and LXA4 analogs that are mediated via the epithelial and PMN ALXR. Multistep recruitment of PMNs to a site of mucosal inflammation and intestinal epithelial ALXR gene regulation by lymphocyte-derived cytokines is depicted. Activation of ALXR present on both PMNs and epithelial cells inhibits PMN migration to sites of inflammation. Bioactions of epithelial and PMN ALXR activation are indicated by asterisks. These anti-inflammatory actions include inhibition of PMN adhesion to the transmigration across both endothelia and epithelia, as well as inhibition of the generation of a gradient of IL-8 at both the protein and gene level. The biosynthesis of aspirin-triggered 15-epi-LXA4 is illustrated during PMN and epithelial communication (see the lipoxin chapter for details).

2230 Nan Chiang et al. genistein, a tyrosine kinase inhibitor (Papayianni et al., 1996).

Cytoplasmic signaling cascades The cytoplasmic signaling cascade of ALXR appears to be highly cell type-specific. For example, in human PMNs, LXA4 stimulates rapid lipid remodeling (within seconds) with release of arachidonic acids that are evoked via PTX-sensitive G proteins (Nigam et al., 1990) without formation of either LT or PG. Only a modest Ca2‡ mobilization was observed. Also, LXA4 was reported to block intracellular generation of IP3 (Grandordy et al., 1990) as well as Ca2‡ mobilization in response to other stimuli (Lee et al., 1989). In human peripheral blood monocytes and cultured THP1 cells, LXA4 triggers intracellular Ca2‡ release and adherence to laminin (Romano et al., 1996; Maddox et al., 1997). Thus, different intracellular signaling pathways are present in PMN versus monocytes despite identical receptor sequences. It is of interest that Ca2‡ is not the second messenger for lipoxin actions in monocytes, since LXA4-stimulated monocyte adherence to laminin is not dependent on an lipoxin-stimulated increase in [Ca2‡]i. The EC50 value for LXA4-stimulated increase in [Ca2‡]i is > 100 nM in monocytes, which is more than two log orders of magnitude higher than that required for LXA4-stimulated adherence (EC50 < 1 nM). In view of G protein-coupling events in monocytes, both Ca2‡ mobilization and adherence are PTX-sensitive. This indicates that the receptor coupling in monocytes and PMNs is similar to this point, although there could be different PTX-sensitive G protein subtypes coupled to the intracellular domains of the receptors, which then diverge downstream in the signal transduction pathways, leading to chemotaxis of monocytes and inhibition of PMNs. Distinct signaling in monocytes and PMNs is further evidenced by different responses to LXA4 in these cell types (Figure 2). Recently, results from this laboratory indicate that activation of ALXR leads to polyisoprenyl phosphate (PIPP) remodeling and accumulation of presqualene diphosphate (PSDP) (Levy et al., 1999), a key component in the PIPP-signaling pathway and a potent negative intracellular signal in PMNs (Levy et al., 1997), indicating that ALXR stimulates intracellular lipids that appear directly to mediate some of its actions. The characteristics of ALXRs in various cell types are briefly summarized in Table 3. Also, LXA4 modulates MAP kinase activities on mesangial cells in a PTX-insensitive manner (McMahon et al., 1998),

suggesting the presence of additional novel LXA4 receptor subtypes and/or signaling pathways in these cells.

DOWNSTREAM GENE ACTIVATION

Genes induced In human enterocytes (T84), ALXR activation by LXA4 and LX analogs diminishes Salmonella typhimurium-induced IL-8 transcription (Gewirtz et al., 1998). The reduction of IL-8 mRNA levels parallels decreases in IL-8 secretion, indicating that in these cells ALXR's mechanism of action for blocking this chemokine is at the gene transcriptional level.

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

Unique biological effects of activating the receptors ALXR activation on human PMNs evoke leads to inhibition of both LTB4- and fMLP-induced PMN adhesion (by downregulating CD11/CD18), chemotaxis, transmigration (Serhan, 1997), as well as degranulation (Gewirtz et al., 1999). Recently, it was demonstrated that both LXA4 and ATL analogs applied to mouse ears dramatically reduced both LTB4- and PMA-initiated PMN infiltration as well as LTB4-triggered vascular permeability (Takano et al., 1998) (Figure 6). In addition, LXA4 analogs inhibit leukocyte rolling and adherence by attenuating Pselectin expression in rat mesenteric microvasculature (Scalia et al., 1997). Native LXA4 also inhibits PMN recruitment to inflamed glomeruli in vivo (Papayianni et al., 1995), further supporting the antiinflammatory actions of LX in vivo. In cell types other than leukocytes, LX analogs inhibit TNF-induced IL-8 release (Gronert et al., 1998) as well as pathogeninduced IL-8 secretion at the mRNA level in human enterocytes (Figure 8) (Gewirtz et al., 1998). In rabbit trachea, LXA4 stimulates nitric oxide generation, reducing airway smooth muscle contraction (Tamaoki et al., 1995). For details, see Figure 6 and Figure 7 and the lipoxin chapter.

Lipoxin A4 Receptor 2231 Table 3 Characterization of human and mouse ALXR Cell type

Kd (nM)

Signal transduction

Kinase associated

Upregulated by

Human HL-60 (differentiated)

0.6

PLD activation (lipid remodeling)

Protein kinase C (staurosporin sensitive)

Retinoic acid, DMSO, PMA

Human PMNs

0.5

PLD activation

Tyrosine kinase (genistein sensitive)

GTPase activity C20:4 release PIPP signal (raises PSDP accumulation) (with second signal) No increase of cAMP, proton efflux and very weak [Ca2‡]i Human PMNs (expressed in CHO)

1.7

GTPase activity Arachidonic acid release (PTX sensitive) No increase of cAMP and [Ca2‡]i Increase of [Ca2‡]i (PTX sensitive)

Human monocytes

No increase of cAMP and proton efflux Human enterocytes Human endothelium

No proton efflux 11

IL-13, IL-4, IFN

Prostacyclin generation

Protein kinase C

Nitric oxide generation No increase of [Ca2‡]i and proton efflux Mouse leukocyte (expressed in CHO)

1.5

GTPase activity

Phenotypes of receptor knockouts and receptor overexpression mice ALXR knockout and transgenic mice are not currently available. However, an in vitro receptor knockout was carried out using an antisense oligonucleotide selected from a region of ALXR cDNA sequence which gives low homology with the formyl peptide receptor. Retinoic acid-differentiated HL-60 cells exposed to this antisense oligonucleotide selectively lost [3H]LXA4 binding as well as LXA4initiated lipid remodeling paralleling the loss of ALXR mRNA (Fiore and Serhan, 1995).

Human abnormalities The formation of native LXA4 as well as 15-epi-LXA4 is documented in vivo. During chronic myelocytic

leukemia, platelets from these individuals lose 12lipoxygenase and their ability to generate lipoxin. Of particular interest, the loss of lipoxin generation correlates with the onset of blast crisis observed in chronic myelocytic leukemia (Stenke et al., 1994).

THERAPEUTIC UTILITY

Effect of treatment with soluble receptor domain ALXR agonists, namely LXA4 and ATL analogs, are topically active in inhibiting PMN infiltration and vascular permeability in murine skin inflammation (Serhan, 1997). Inhalation of LXA4 by human asthmatic patients blocks LTC4-induced contraction in vivo (Christie et al., 1992). In renal hemodynamics,

2232 Nan Chiang et al. LXA4 blocks LTD4 induced vasoconstriction (Badr et al., 1989) and prevents PMN entry into the kidney in glomerulonephritis (Papayianni et al., 1995). For details, see the section on Unique biological effects of activating the receptors and the lipoxin chapter.

Effects of inhibitors (antibodies) to receptors A specific antipeptide antibody was prepared against the second extracellular loop of ALXR. This antibody blocks specific [3H]LXA4-binding as well as LXA4-inhibitory actions on fMLP-stimulated CD11b expression and aggregation in human PMNs (Fiore and Serhan, 1995) and inhibits LXA4-triggered intracellular Ca2‡ mobilization in human peripheral blood monocytes (Maddox et al., 1997).

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LICENSED PRODUCTS Licensed products for lipoxin investigation are listed in the chapter on lipoxin.

ACKNOWLEDGEMENTS For the purpose of brevity, review articles are preferentially cited. We apologize to our colleagues whose names do not appear within the reference list, yet who have contributed substantially to this area of research. This work was supported in part by National Institutes of Health grants GM-38765 and DK-50305 to CNS and National Arthritis Foundation fellowships to NC and KG.