Aspirin-triggered 15-Epi-Lipoxin A4 (LXA4) and LXA4 Stable Analogues Are Potent Inhibitors of Acute Inflammation: Evidence for Anti-inflammatory Receptors
Lipoxins are bioactive eicosanoids that are immunomodulators. In human myeloid cells, lipoxin (LX) A4 actions are mediated by interaction with a G protein–coupled receptor. To explore functions of LXA4 and aspirin-triggered 5(S),6(R),15(R)-trihydroxy-7,9,13-trans-11-cis–eicosatetraenoic acid (15-epi-LXA4) in vivo, we cloned and characterized a mouse LXA4 receptor (LXA4R). When expressed in Chinese hamster ovary cells, the mouse LXA4R showed specific binding to [3H]LXA4 (Kd ≈ 1.5 nM), and with LXA4 activated GTP hydrolysis. Mouse LXA4R mRNA was most abundant in neutrophils. In addition to LXA4 and 15-epi-LXA4, bioactive LX stable analogues competed with both [3H]LXA4 and [3H]leukotriene D4 (LTD4)– specific binding in vitro to neutrophils and endothelial cells, respectively. Topical application of LXA4 analogues and novel aspirin-triggered 15-epi-LXA4 stable analogues to mouse ears markedly inhibited neutrophil infiltration in vivo as assessed by both light microscopy and reduced myeloperoxidase activity in skin biopsies. The 15(R)-16-phenoxy-17,18, 19,20-tetranorLXA4 methyl ester (15-epi-16-phenoxy-LXA4), an analogue of aspirin triggered 15-epi-LXA4, and 15(S)-16-phenoxy-17,18,19,20-tetranor-LXA4 methyl ester (16-phenoxy-LXA4) were each as potent as equimolar applications of the anti-inflammatory, dexamethasone. Thus, we identified murine LXA4R, which is highly expressed on murine neutrophils, and showed that both LXA4 and 15-epi-LXA4 stable analogues inhibit neutrophil infiltration in the mouse ear model of inflammation. These findings provide direct in vivo evidence for an anti-inflammatory action for both aspirin-triggered LXA4 and LXA4 stable analogues and their site of action in vivo.
Lipoxins are trihydroxytetraene-containing eicosanoids that are generated within vascular lumen by plateletleukocyte interactions and transcellular biosynthetic pathways during multicellular responses such as inflammation, atherosclerosis, and thrombosis (as reviewed in reference 1). This branch of the eicosanoid cascade generates specific tetraenecontaining products that appear to function as stop signals. In this regard, lipoxins display selective actions on human leukocytes in vitro that include inhibition of (a) FMLP and leukotriene B4 (LTB4)1-induced neutrophil chemotaxis (2), (b) FMLP-induced neutrophil transmigration through epithelial cells (3), and (c) neutrophil adhesion and transmigration with endothelial cells (4). We have recently shown that these actions of lipoxin (LX) A4; 5(S),6(R),15(S)-trihydroxy7,9,13-trans-11-cis-eicosatetraenoic acid are mediated via signal transduction events initiated by engagement of highaffinity G protein–coupled receptors in human cells (4–6). This includes LXA4-induced downregulation of CD11b/ CD18 in human neutrophils (5), an adhesion molecule that plays an important role in endothelial–leukocyte interactions (7). Although lipoxins do not directly inhibit the generation of reactive oxygen species by activated neutrophils (reviewed in reference 8), the ability of LX to block endothelial cell–leukocyte interactions (4) can also prevent injury initiated by leukocyte-derived reactive oxidants (9, 10). Taken together, these results suggest that lipoxins play important regulatory roles in leukocyte trafficking and inflammation.
The biosynthesis of lipoxins is initiated through cell–cell and lipoxygenase (LO) interactions that are regulated by specific cytokines (1). One major pathway is mounted during PMN–platelet interaction and involves both the 5-LO and 12-LO, and the other involves interactions between the 5-LO and 15-LO (recently reviewed in reference 8) that are controlled by the cytokines IL-4 and IL-13 (11). Given the wide use of aspirin, the mechanism of aspirin's beneficial actions in inflammation remains a topic of intense interest. Aspirin has no direct impact on the lipoxygenases (8). In this regard, a third major pathway for lipoxin biosynthesis was recently uncovered, which involves prostaglandin H synthase-II (PGHS-II) in endothelial cells and 5-LO in leukocytes that generate novel 15-epi-lipoxins when PGHS-II is acetylated after treatment with aspirin (12). The aspirin-triggered lipoxins, for example, 5(S),6(R),15(R)- trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid (15-epiLXA4), carries its C-15 alcohol in the R configuration, instead of S as in native LXA4, and has potent inhibitory actions in neutrophil adhesion, and 15-epi-LXB4 blocks cell proliferation in vitro (12, 13). This pathway that leads to 15-epiLXA4 may mediate, in part, some of the beneficial actions of aspirin.
Lipoxins are also generated in vivo in humans and in experimental animals (reviewed in reference 8). LXA4 and LXB4 are both formed in ischemic rat brain (14), and LXA4 is generated in mouse kidneys with glomerulonephritis in a P-selectin–dependent fashion predominantly via interactions between platelets and neutrophils (15). In rats, the infiltration of neutrophils to glomerulonephritic kidneys is markedly inhibited by prior exposure of neutrophils to LXA4 (16). Also, LXA4 has recently been found to regulate LTB4mediated delayed hypersensitive reactions in guinea pig (17). The actions of LXA4 are not mediated by competition at the LTB4 receptor (18), but LXA4 is reported to antagonize the formation of intracellular signals such as IP3 (19). In addition to its selective actions with leukocytes, LXA4 also modulates the vasoconstrictor actions of leukotriene D4 (LTD4) in renal hemodynamics and is vasodilatory (20). These actions of LXA4 are mediated by a receptor distinct from that of the myeloid LXA4R and are consistent with LXA4 acting on a subtype of the peptido-leukotriene receptors, competing for LTC4 and LTD4 high-affinity sites that are present on both mesangial (20) and endothelial cells (21). Interest in the actions of LXA4 is also heightened by findings with human subjects that indicate that LXA4 administration via inhalation significantly blocks airway constriction in asthmatic subjects (22).
To explore biological functions of both lipoxins and the recently identified aspirin-triggered lipoxins in vivo, it is essential to identify the molecular basis of their response in experimental animals. To this end, we report here isolation of the mouse lipoxin A4 receptor (LXA4R) and that stable analogues of LXA4 and the aspirin-triggered 15-epi-LXA4 that specifically compete at this site are potent inhibitors of acute neutrophil infiltration in vivo.