Lipoxiini LXA4 ja aspiriini

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2196289/

J Exp Med. 1997 May 5; 185(9): 1693–1704.

PMCID: PMC2196289

Articles

Aspirin-triggered 15-Epi-Lipoxin A₄ (LXA₄) and LXA₄ Stable Analogues Are Potent Inhibitors of Acute Inflammation: Evidence for Anti-inflammatory Receptors

Tomoko Takano,^*‡ Stefano Fiore,^* Jane F. Maddox,^* Hugh R. Brady,^‡ Nicos A. Petasis,^§ and Charles N. Serhan^*

Abstract

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 (K_d ≈ 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.