Evidence for an anti-inflammatory loop centered on polymorphonuclear leukocyte formyl peptide receptor 2/lipoxin A4 receptor and operative in the inflamed microvasculature

The importance of proresolving mediators in the overall context of the resolution of acute inflammation is well recognized, although little is known about whether these anti-inflammatory and proresolving molecules act in concert. In this article, we focused on lipoxin A(4) (LXA(4)) and annexin A1 (AnxA1) because these two very different mediators converge on a single receptor, formyl peptide receptor type 2 (FPR2/ALX). Addition of LXA(4) to human polymorphonuclear leukocytes (PMNs) provoked a concentration- and time-dependent mobilization of AnxA1 onto the plasma membrane, as determined by Western blotting and flow cytometry analyses. This property was shared by another FPR2/ALX agonist, antiflammin-2, and partly by fMLF or peptide Ac2-26 (an AnxA1 derivative that can activate all three members of the human FPR family). An FPR2/ALX antagonist blocked AnxA1 mobilization activated by LXA(4) and antiflammin-2. Analysis of PMN degranulation patterns and phospho-AnxA1 status suggested a model in which the two FPR2/ALX agonists mobilize the cytosolic (and not the granular) pool of AnxA1 through an intermediate phosphorylation step. Intravital microscopy investigations of the inflamed mesenteric microvasculature of wild-type and AnxA1(-/-) mice revealed that LXA(4) provoked leukocyte detachment from the postcapillary venule endothelium in the former (>50% within 10 min; p < 0.05), but not the latter genotype (～15%; NS). Furthermore, recruitment of Gr1(+) cells into dorsal air-pouches, inflamed with IL-1β, was significantly attenuated by LXA(4) in wild-type, but not AnxA1(-/-), mice. Collectively, these data prompt us to propose the existence of an endogenous network in anti-inflammation centered on PMN AnxA1 and activated by selective FPR2/ALX agonists.     

Lipoxin A4 and aspirin-triggered 15-epi-lipoxin A4 inhibit human neutrophil migration: comparisons between synthetic 15 epimers in chemotaxis and transmigration with microvessel endothelial cells and epithelial cells

Lipoxins (LX) are bioactive eicosanoids that can be formed during cell to cell interactions in human tissues to self limit key responses in host defense and promote resolution. Aspirin treatment initiates biosynthesis of carbon 15 epimeric LXs, and both series of epimers (LX and aspirin-triggered 15-epi-LX) display counter-regulatory actions with neutrophils. In this study, we report that synthetic lipoxin A(4) (LXA(4)) and 15-epi-LXA(4) (i.e., 15(R)-LXA(4) or aspirin-triggered LXA(4)) are essentially equipotent in inhibiting human polymorphonuclear leukocytes (PMN) in vitro chemotaxis in response to leukotriene B(4), with the maximum inhibition ( approximately 50% reduction) obtained at 1 nM LXA(4). At higher concentrations, 15-epi-LXA(4) proved more potent than LXA(4) as its corresponding carboxyl methyl ester. Also, exposure of PMN to LXA(4) and 15-epi-LXA(4) markedly decreased PMN transmigration across both human microvessel endothelial and epithelial cells, where 15-epi-LXA(4) was more active than LXA(4) at "stopping" migration across epithelial cells. Differences in potency existed between LXA(4) and 15-epi-LXA(4) as their carboxyl methyl esters appear to arise from cell type-specific conversion of their respective carboxyl methyl esters to their corresponding carboxylates as monitored by liquid chromatography tandem mass spectrometry. Both synthetic LXA(4) and 15-epi-LXA(4) as free acids activate recombinant human LXA(4) receptor (ALXR) to regulate gene expression, whereas the corresponding methyl ester of LXA(4) proved to be a partial ALXR antagonist and did not effectively regulate gene expression. These results demonstrate the potent stereospecific actions shared by LXA(4) and 15-epi-LXA(4) for activating human ALXR-regulated gene expression and their ability to inhibit human PMN migration during PMN vascular as well as mucosal cell to cell interactions.     

Macrophage sub-populations and the lipoxin A4 receptor implicate active inflammation during equine tendon repair

Macrophages (Mφ) orchestrate inflammatory and reparatory processes in injured connective tissues but their role during different phases of tendon healing is not known. We investigated the contribution of different Mφ subsets in an equine model of naturally occurring tendon injury. Post mortem tissues were harvested from normal (uninjured), sub-acute (3-6 weeks post injury) and chronically injured (>3 months post injury) superficial digital flexor tendons. To determine if inflammation was present in injured tendons, Mφ sub-populations were quantified based on surface antigen expression of CD172a (pan Mφ), CD14(high)CD206(low) (pro-inflammatory M1Mφ), and CD206(high) (anti-inflammatory M2Mφ) to assess potential polarised phenotypes. In addition, the Lipoxin A(4) receptor (FPR2/ALX) was used as marker for resolving inflammation. Normal tendons were negative for both Mφ and FPR2/ALX. In contrast, M1Mφ predominated in sub-acute injury, whereas a potential phenotype-switch to M2Mφ polarity was seen in chronic injury. Furthermore, FPR2/ALX expression by tenocytes was significantly upregulated in sub-acute but not chronic injury. Expression of the FPR2/ALX ligand Annexin A1 was also significantly increased in sub-acute and chronic injuries in contrast to low level expression in normal tendons. The combination of reduced FPR2/ALX expression and persistence of the M2Mφ phenotype in chronic injury suggests a potential mechanism for incomplete resolution of inflammation after tendon injury. To investigate the effect of pro-inflammatory mediators on lipoxin A(4) (LXA(4)) production and FPR2/ALX expression in vitro, normal tendon explants were stimulated with interleukin-1 beta and prostaglandin E(2). Stimulation with either mediator induced LXA(4) release and maximal upregulation of FPR2/ALX expression after 72 hours. Taken together, our data suggests that although tenocytes are capable of mounting a protective mechanism to counteract inflammatory stimuli, this appears to be of insufficient duration and magnitude in natural tendon injury, which may potentiate chronic inflammation and fibrotic repair, as indicated by the presence of M2Mφ.     

Multi-pronged inhibition of airway hyper-responsiveness and inflammation by lipoxin A(4)

The prevalence of asthma continues to increase and its optimal treatment remains a challenge. Here, we investigated the actions of lipoxin A(4) (LXA(4)) and its leukocyte receptor in pulmonary inflammation using a murine model of asthma. Allergen challenge initiated airway biosynthesis of LXA(4) and increased expression of its receptor. Administration of a stable analog of LXA(4) blocked both airway hyper-responsiveness and pulmonary inflammation, as shown by decreased leukocytes and mediators, including interleukin-5, interleukin-13, eotaxin, prostanoids and cysteinyl leukotrienes. Moreover, transgenic expression of human LXA(4) receptors in murine leukocytes led to significant inhibition of pulmonary inflammation and eicosanoid-initiated eosinophil tissue infiltration. Inhibition of airway hyper-responsiveness and allergic airway inflammation with a stable LXA(4) analog highlights a unique counter-regulatory profile for the LXA(4) system and its leukocyte receptor in airway responses. Moreover, our findings suggest that lipoxin and related pathways offer novel multi-pronged therapeutic approaches for human asthma.     

Cutting edge: lipoxin (LX) A4 and aspirin-triggered 15-epi-LXA4 block allergen-induced eosinophil trafficking

Tissue eosinophilia prevention represents one of the primary targets to new anti-allergic therapies. As lipoxin A4 (LXA4) and aspirin-triggered 15-epi-LXA4 (ATL) are emerging as endogenous "stop signals" produced in distinct pathologies including some eosinophil-related pulmonary disorders, we evaluated the impact of in situ LXA4/ATL metabolically stable analogues on allergen-induced eosinophilic pleurisy in sensitized rats. LXA4/ATL analogues dramatically blocked allergic pleural eosinophil influx, while concurrently increasing circulating eosinophilia, inhibiting the earlier edema and neutrophilia associated with allergic reaction. The mechanisms underlying this LXA4/ATL-driven allergic eosinophilia blockade was independent of mast cell degranulation and involved LXA4/ATL inhibition of both IL-5 and eotaxin generation, as well as platelet activating factor action. These findings reveal LXA4/ATL as a novel class of endogenous anti-allergic mediators, capable of preventing local eosinophilia.     

Lipoxin A4 inhibits immune cell binding to salivary epithelium and vascular endothelium

Lipoxins are formed by leukocytes during cell-cell interactions with epithelial or endothelial cells. Native lipoxin A(4) (LXA(4)) binds to the G protein-coupled lipoxin receptors formyl peptide receptor 2 (FPR2)/ALX and CysLT1. Furthermore, LXA(4) inhibits recruitment of neutrophils, by attenuating chemotaxis, adhesion, and transmigration across vascular endothelial cells. LXA(4) thus appears to serve as an endogenous "stop signal" for immune cell-mediated tissue injury (Serhan CN; Annu Rev Immunol 25: 101-137, 2007). The role of LXA(4) has not been addressed in salivary epithelium, and little is known about its effects on vascular endothelium. Here, we determined that interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) receptor activation in vascular endothelium and salivary epithelium upregulated the expression of adhesion molecules that facilitates the binding of immune cells. We hypothesize that the activation of the ALX/FPR2 and/or CysLT1 receptors by LXA(4) decreases this cytokine-mediated upregulation of cell adhesion molecules that enhance lymphocyte binding to both the vascular endothelium and salivary epithelium. In agreement with this hypothesis, we observed that nanomolar concentrations of LXA(4) blocked IL-1β- and TNF-α-mediated upregulation of E-selectin and intercellular cell adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells (HUVECs). Binding of Jurkat cells to stimulated HUVECs was abrogated by LXA(4). Furthermore, LXA(4) preincubation with human submandibular gland cell line (HSG) also blocked TNF-α-mediated upregulation of vascular cell adhesion molecule-1 (VCAM-1) in these cells, and it reduced lymphocyte adhesion. These findings suggest that ALX/FPR2 and/or CysLT1 receptor activation in endothelial and epithelial cells blocks cytokine-induced adhesion molecule expression and consequent binding of lymphocytes, a critical event in the pathogenesis of Sj?gren's syndrome (SS).     

Inflamm-Aging and Arachadonic Acid Metabolite Differences with Stage of Tendon Disease

The contribution of inflammation to the pathogenesis of tendinopathy and high prevalence of re-injury is not well established, although recent evidence suggests involvement of prostaglandins. We investigated the roles of prostaglandins and inflammation-resolving mediators in naturally occurring equine tendon injury with disease stage and age. Levels of prostaglandins E₂ (PGE₂), F_2α (PGF_2α), lipoxin A₄ (LXA₄) and its receptor FPR2/ALX were analysed in extracts of normal, sub-acute and chronic injured tendons. To assess whether potential changes were associated with altered PGE₂ metabolism, microsomal prostaglandin E synthase-1 (mPGES-1), prostaglandin dehydrogenase (PGDH), COX-2 and EP₄ receptor expression were investigated. The ability of tendons to resolve inflammation was determined by assessing FPR2/ALX expression in natural injury and IL-1β stimulated tendon explants.Alterations in the profile of lipid mediators during sub-acute injury included low PGE₂ and elevated LXA₄ levels compared to normal and chronic injuries. In contrast, PGF_2α levels remained unchanged and were three-fold lower than PGE₂. The synthetic capacity of PGE₂ as measured by the ratio of mPGES-1:PGDH was elevated in sub-acute injury, suggesting aberrations in tendon prostaglandin metabolism, whilst COX-2 and EP₄ receptor were unchanged. Paradoxically low tendon PGE₂ levels in early injury may be attributed to increased local clearance via PGDH or the class switching of lipid mediators from the prostaglandin to the lipoxin axis. PGE₂ is therefore implicated in the development of tendon inflammation and its ensuing resolution. Whilst there was no relationship between age and tendon LXA₄ levels, there was an age-associated decline in FPR2/ALX receptor expression with concurrent increased PGE₂ levels in injury. Furthermore, uninjured tendon explants from younger (<10 years) but not older horses (≥10 years) treated with IL-1β responded by increasing FPR2/ALX suggesting aged individuals exhibit a reduced capacity to resolve inflammation via FPR2/ALX, which may present a potential mechanism for development of chronic tendinopathy and re-injury.     

Lipoxin A(4) analogues inhibit leukocyte recruitment to Porphyromonas gingivalis: a role for cyclooxygenase-2 and lipoxins in periodontal disease

The potential involvement of the inducible cyclooxygenase isoform (COX-2) and the role of novel lipid mediators were investigated in the pathogenesis of periodontal disease. Crevicular fluids from localized juvenile periodontitis (LJP) patients contained prostaglandin (PG)E(2) and 5-lipoxygenase-derived products, leukotriene B(4), and the biosynthesis interaction product, lipoxin (LX)A(4). Neutrophils from peripheral blood of LJP patients, but not from asymptomatic donors, also generated LXA(4), suggesting a role for this immunomodulatory molecule in periodontal disease. To characterize host responses of interest to periodontal pathogens, Porphyromonas gingivalis was introduced within murine dorsal air pouches. In the air pouch cavity, P. gingivalis elicited leukocyte infiltration, concomitant with elevated PGE(2) levels in the cellular exudates, and upregulated COX-2 expression in infiltrated leukocytes. In addition, human neutrophils exposed to P. gingivalis also upregulated COX-2 expression. Blood borne P. gingivalis gave significant increases in the murine tissue levels of COX-2 mRNA associated with both heart and lungs, supporting a potential role for this oral pathogen in the evolution of systemic events. The administration of metabolically stable analogues of LX and of aspirin-triggered LX potently blocked neutrophil traffic into the dorsal pouch cavity and lowered PGE(2) levels within exudates. Together, these results identify PMN as an additional and potentially important source of PGE(2) in periodontal tissues. Moreover, they provide evidence for a novel protective role for LX in periodontitis, limiting further PMN recruitment and PMN-mediated tissue injury that can lead to loss of inflammatory barriers that prevent systemic tissue invasion of oral microbial pathogens.     

Cutting edge: Humanized nano-proresolving medicines mimic inflammation-resolution and enhance wound healing

Endogenous microparticles (MPs) were systematically profiled during the time course of self-limited inflammation. Precursors for specialized proresolving lipid mediators were identified in MPs from inflammatory exudates using liquid chromatography tandem mass spectrometry-based metabolomics. Hence, we postulated that formation of anti-inflammatory and proresolving lipid mediators could underlie beneficial effects attributed to MPs and that this process could serve as a basis for biomimicry. Using human neutrophil-derived MPs, we constructed novel nanoparticles (NPs) containing aspirin-triggered resolvin D1 or a lipoxin A(4) analog. Enriched NPs dramatically reduced polymorphonuclear cell influx in murine peritonitis, shortened resolution intervals, and exhibited proresolving actions accelerating keratinocyte healing. The enriched NPs protected against inflammation in the temporomandibular joint. These findings indicate that humanized NPs, termed nano-proresolving medicines, are mimetics of endogenous resolving mechanisms, possess potent beneficial bioactions, can reduce nanotoxicity, and offer new therapeutic approaches.     

Lipoxins and aspirin-triggered lipoxins in neutrophil adhesion and signal transduction

Lipoxin A4 (LXA4) and aspirin-triggered 15-epi-LXA4 (ATL) are emerging as endogenous braking signals for neutrophil-mediated tissue injury. LXA4 and ATL and their metabolically stable analogues display potent inhibitory actions in human isolated cells and blood, including attenuation of expression of adhesion molecules on leukocytes and endothelial cells, neutrophil adhesion to endothelial cells and platelets under shear, and IL-8 production, key events of the acute inflammatory response. The underlying molecular mechanisms include interference with MAPK signaling pathways, modulation of the oxidative chemistry of superoxide, NO and ONOO-, inhibition of activation of NF-kappaB and AP-1, and consequently the expression of interleukin-8 and likely other pro-inflammatory genes. Collectively, these results add to the profile of LXA4/ATL rapid actions that contribute to "stop signaling" involved in regulating neutrophil functions during acute inflammation and suggest that aspirin inhibits neutrophil accumulation through triggering the synthesis of 15-epi-LXA4.     

Lipoxin biosynthesis in inflammatory bowel disease

BACKGROUND AND AIMS: Lipoxins are anti-inflammatory lipid mediators that are produced in gut mucosa, which serve to limit and resolve persistent inflammation. The purpose of this study was to evaluate colonic lipoxin biosynthesis in patients with ulcerative colitis (UC) to establish a possible biochemical basis for persistent inflammation in UC. METHODS: Colonic mucosa from patients with UC or organ donors (controls) was placed into tissue culture for 90 min. The conditioned media was assayed (ELISA) for lipoxin A4 (LXA) and the biologically active isomer 15-epi-LXA4 (aspirin triggered lipoxin, ATL). Mucosal tissue 15-lipoxygenase protein was determined by Western blot. RESULTS: Patient colonic mucosa produced significantly lower (12-fold) amounts of LXA, relative to organ donors. This occurred irregardless of patient steroid treatment. However, patient tissue responded to in vitro aspirin by synthesizing biologically active ATL. For the first time, human colonic mucosa was found to synthesize 15-lipoxygenase-2, an epithelial-derived isoenzyme used for lipoxin synthesis. These levels were significantly lower in UC patients compared to the control tissue. Finally, mice chronically treated with a putative selective 15-lipoxygenase inhibitor (PD 146176) experienced significantly worse intestinal function during experimental colitis, relative to untreated mice. CONCLUSION: Colonic mucosa from UC patients demonstrated defective lipoxin biosynthesis, which may contribute to the inability of these patients to resolve persistent colonic inflammation.     

Molecular circuits of resolution: formation and actions of resolvins and protectins

The cellular events underlying the resolution of acute inflammation are not known in molecular terms. To identify anti-inflammatory and proresolving circuits, we investigated the temporal and differential changes in self-resolving murine exudates using mass spectrometry-based proteomics and lipidomics. Key resolution components were defined as resolution indices including Psi(max), the maximal neutrophil numbers that are present during the inflammatory response; T(max), the time when Psi(max) occurs; and the resolution interval (R(i)) from T(max) to T(50) when neutrophil numbers reach half Psi(max). The onset of resolution was at approximately 12 h with proteomic analysis showing both haptoglobin and S100A9 levels were maximal and other exudate proteins were dynamically regulated. Eicosanoids and polyunsaturated fatty acids first appeared within 4 h. Interestingly, the docosahexaenoic acid-derived anti-inflammatory lipid mediator 10,17S-docosatriene was generated during the R(i). Administration of aspirin-triggered lipoxin A(4) analog, resolvin E1, or 10,17S-docosatriene each either activated and/or accelerated resolution. For example, aspirin-triggered lipoxin A(4) analog reduced Psi(max), resolvin E1 decreased both Psi(max) and T(max), whereas 10,17S-docosatriene reduced Psi(max), T(max), and shortened R(i). Also, aspirin-triggered lipoxin A(4) analog markedly inhibited proinflammatory cytokines and chemokines at 4 h (20-50% inhibition), whereas resolvin E1 and 10,17S-docosatriene's inhibitory actions were maximal at 12 h (30-80% inhibition). Moreover, aspirin-triggered lipoxin A(4) analog evoked release of the antiphlogistic cytokine TGF-beta. These results characterize the first molecular resolution circuits and their major components activated by specific novel lipid mediators (i.e., resolvin E1 and 10,17S-docosatriene) to promote resolution.     

Identification,cloning, and functional characterization of a murine lipoxin A4 receptor homologue gene

To identify additional members of the murine N-formyl-Met-Leu-Phe peptide receptor family (fMLF-R), a mouse macrophage cDNA library was screened using the open reading frame of murine N-formyl peptide receptor. Four individual hybridizing cDNA clones were maintained through tertiary screening. One cDNA clone was a truncated, polyadenylated version of the previously described murine-fMLF-R. The other three cDNA clones varied in length, but contained identical open reading frame sequences. One clone, 8C10, was selected for further study and shared 70% sequence identity with murine-fMLF-R and 89% sequence identity with murine lipoxin A4 receptor cDNA. When placed into the pcDNA-3 expression vector and cotransfected with Galpha16 cDNA into COS-1 cells, 8C10 cDNA induced the production of inositol-1,4,5-triphosphate when concentrations of 1-1600 nM lipoxin A4 (LXA4) were tested as ligands. Northern blot analysis of murine organs indicated that the 8C10 message is present in lung, spleen, and adipose tissue. Moreover, mice treated with LPS demonstrated increased expression of 8C10 message in spleen and adipose tissue, while showing a slight reduction in lung. We have also characterized the 8C10 structural gene from a 129Sv/J genomic library and have determined its size to be >6.1 kb in length and comprised of two exons separated by a 4.8-kb intron. Collectively, these data indicate that this homologue receptor is closely related to the murine LXA4 receptor and functionally responds to LXA4 as a ligand.     

Role of lipoxin A4 in the cell-to-cell interaction between all-trans retinoic acid-treated acute promyelocytic leukemic cells and alveolar macrophages

Differentiation therapy with all-trans retinoic acid (ATRA) has been used successfully to treat acute promyelocytic leukemia (APL), but such treatment also causes differentiation syndrome (DS) by inducing APL cell infiltration into alveolar spaces. The mechanism underlying the clearance of infiltrated APL cells has not been investigated in detail. Lipoxin A(4) (LXA(4)) is an important anti-inflammatory mediator during the resolution of inflammation. In this study, the role of LXA(4) in the cell-cell interaction between alveolar macrophages (AMφ; NR8383 cells) and APL NB4 cells was investigated and found that conditioned medium (CM) harvested from ATRA-treated NR8383 (ATRA-NR8383) cells was able to induce the transmigration of ATRA-NB4 cells. However, the pro-migratory activity of CM was attenuated progressively when ATRA-NR8383 cells were co-cultured with increased cell dosages of apoptotic NB4 cells. A significantly higher amount of LXA(4) was released into the CM by ATRA-NR8383 cells when they were co-cultured with apoptotic ATRA-NB4 cells. Expression of a receptor for LXA(4) (ALX/FPR2) was enhanced in both ATRA-NB4 cells and ATRA-NR8383 cells. Exogenous LXA(4) treatment was able to inhibit the transmigration of ATRA-NB4 cells and induce the phagocytic clearance of apoptotic cells by ATRA-NR8383 cells. The anti-migratory activity of exogenous LXA(4) was attenuated by pre-treating ATRA-NB4 cells with an ALX/FPR2 inhibitor. We conclude that AMφ-derived LXA(4) plays an important role in the interaction between AMφ and APL cells and that this contributes to clearance of apoptotic APL cells.     

Lipoxin A4 stimulates calcium-activated chloride currents and increases airway surface liquid height in normal and cystic fibrosis airway epithelia

Cystic Fibrosis (CF) is a genetic disease characterised by a deficit in epithelial Cl(-) secretion which in the lung leads to airway dehydration and a reduced Airway Surface Liquid (ASL) height. The endogenous lipoxin LXA(4) is a member of the newly identified eicosanoids playing a key role in ending the inflammatory process. Levels of LXA(4) are reported to be decreased in the airways of patients with CF. We have previously shown that in normal human bronchial epithelial cells, LXA(4) produced a rapid and transient increase in intracellular Ca(2+). We have investigated, the effect of LXA(4) on Cl(-) secretion and the functional consequences on ASL generation in bronchial epithelial cells obtained from CF and non-CF patient biopsies and in bronchial epithelial cell lines. We found that LXA(4) stimulated a rapid intracellular Ca(2+) increase in all of the different CF bronchial epithelial cells tested. In non-CF and CF bronchial epithelia, LXA(4) stimulated whole-cell Cl(-) currents which were inhibited by NPPB (calcium-activated Cl(-) channel inhibitor), BAPTA-AM (chelator of intracellular Ca(2+)) but not by CFTRinh-172 (CFTR inhibitor). We found, using confocal imaging, that LXA(4) increased the ASL height in non-CF and in CF airway bronchial epithelia. The LXA(4) effect on ASL height was sensitive to bumetanide, an inhibitor of transepithelial Cl(-) secretion. The LXA(4) stimulation of intracellular Ca(2+), whole-cell Cl(-) currents, conductances and ASL height were inhibited by Boc-2, a specific antagonist of the ALX/FPR2 receptor. Our results provide, for the first time, evidence for a novel role of LXA(4) in the stimulation of intracellular Ca(2+) signalling leading to Ca(2+)-activated Cl(-) secretion and enhanced ASL height in non-CF and CF bronchial epithelia.     

Annexin A1 interaction with the FPR2/ALX receptor: identification of distinct domains and downstream associated signaling

Understanding how proresolving agonists selectively activate FPR2/ALX is a crucial step in the clarification of proresolution molecular networks that can be harnessed for the design of novel therapeutics for inflammatory disease. FPR2/ALX, a G protein-coupled receptor belonging to the formyl peptide receptor (FPR) family, conveys the biological functions of a variety of ligands, including the proresolution mediators annexin A1 (AnxA1) and lipoxin A(4), as well as the activating and proinflammatory protein serum amyloid A. FPR2/ALX is the focus of intense screening for novel anti-inflammatory therapeutics, and the small molecule compound 43 was identified as a receptor ligand. Here, we used chimeric FPR1 and FPR2/ALX clones (stably transfected in HEK293 cells) to identify the N-terminal region and extracellular loop II as the FPR2/ALX domain required for AnxA1-mediated signaling. Genomic responses were also assessed with domain-specific effects emerging, so the N-terminal region is required for AnxA1 induction of JAG1 and JAM3, whereas it is dispensable for modulation of SGPP2. By comparison, serum amyloid A non-genomic responses were reliant on extracellular loops I and II, whereas the small molecule compound 43 activated extracellular loop I with downstream signaling dependent on transmembrane region II. In desensitization experiments, the N-terminal region was dispensable for AnxA1-induced FPR2/ALX down-regulation in both the homologous and heterologous desensitization modes.     

Anti-inflammatory circuitry: lipoxin, aspirin-triggered lipoxins and their receptor ALX

Endogenous chemical mediators or autacoids play key roles in controlling inflammation and its programmed resolution. Among them, it is known that lipoxins (LX) and aspirin-triggered LX (ATL) evoke bioactions in a range of physiologic and pathophysiologic processes and serve as endogenous lipid/chemical mediators that stop neutrophilic infiltration and initiate resolution. LXA4, ATL and their metabolic stable analogs elicit cellular responses and regulate PMN in vivo via interacting with their specific receptor, namely ALX. ALX is the first cloned and identified lipoxygenase-derived eicosanoid receptor with cell type-specific signaling pathways. Also, ALX could regulate PMN by interacting with each class of ligands (lipid vs. peptide) within specific phases of an inflammatory response. Together LX, ATL and ALX may provide new opportunities to design "resolution-targeted" therapies with high degree of precision in controlling inflammation. In this chapter, we give an overview and update of the current actions for LX and ATL, the identification of ALX and their novel anti-inflammatory and pro-resolving signals.     

Hydrogen sulfide upregulates cyclooxygenase-2 and prostaglandin E metabolite in sepsis-evoked acute lung injury via transient receptor potential vanilloid type 1 channel activation

Hydrogen sulfide (H(2)S) has been shown to promote transient receptor potential vanilloid type 1 (TRPV1)-mediated neurogenic inflammation in sepsis and its associated multiple organ failure, including acute lung injury (ALI). Accumulating evidence suggests that the cyclooxygenase-2 (COX-2)/PGE(2) pathway plays an important role in augmenting inflammatory immune response in sepsis and respiratory diseases. However, the interactions among H(2)S, COX-2, and PGE(2) in inciting sepsis-evoked ALI remain unknown. Therefore, the aim of this study was to investigate whether H(2)S would upregulate COX-2 and work in conjunction with it to instigate ALI in a murine model of polymicrobial sepsis. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in male Swiss mice. dl-propargylglycine, an inhibitor of H(2)S formation, was administrated 1 h before or 1 h after CLP, whereas sodium hydrosulfide, an H(2)S donor, was given during CLP. Mice were treated with TRPV1 antagonist capsazepine 30 min before CLP, followed by assessment of lung COX-2 and PGE(2) metabolite (PGEM) levels. Additionally, septic mice were administrated with parecoxib, a selective COX-2 inhibitor, 20 min post-CLP and subjected to ALI and survival analysis. H(2)S augmented COX-2 and PGEM production in sepsis-evoked ALI by a TRPV1 channel-dependent mechanism. COX-2 inhibition with parecoxib attenuated H(2)S-augmented lung PGEM production, neutrophil infiltration, edema, proinflammatory cytokines, chemokines, and adhesion molecules levels, restored lung histoarchitecture, and protected against CLP-induced lethality. The strong anti-inflammatory and antiseptic actions of selective COX-2 inhibitor may provide a potential therapeutic approach for the management of sepsis and sepsis-associated ALI.     

Regulation of hepatocyte growth factor secretion by fibroblasts in patients with acute lung injury

The mechanisms of pulmonary repair in acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) are poorly known. Hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) are key factors involved in alveolar epithelial repair, present in the bronchoalveolar lavage fluid (BALF) from patients with ALI/ARDS. The role of BALF mediators in their production remains to be determined. We evaluated the overall effect of BALF from 52 patients (27 ventilated patients with ALI/ARDS, 10 ventilated patients without ALI, and 15 nonventilated control patients) on HGF and KGF synthesis by lung fibroblasts. Fibroblasts were cultured in the presence of BALF. HGF and KGF protein secretion was measured using ELISA, and mRNA expression was evaluated using quantitative real-time RT-PCR. Only BALF from ALI/ARDS patients upregulated both HGF and KGF mRNA expression and protein synthesis (+271 and +146% for HGF and KGF, respectively). BALF-induced HGF synthesis from ALI/ARDS patients was higher than that from ventilated patients without ALI (P < 0.05). HGF secretion was correlated with BALF IL-1beta levels (rho = 0.62, P < 0.001) and BALF IL-1beta/IL-1 receptor antagonist ratio (rho = 0.54, P < 0.007) in the ALI/ARDS group. An anti-IL-1beta antibody partially (>50%) inhibited the BALF-induced HGF and PGE(2) secretion, whereas NS-398, a specific cyclooxygenase-2 (COX-2) inhibitor, completely inhibited it. Anti-IL-1beta antibodies as well as NS-398 reversed the COX-2 upregulation induced by BALF. Therefore, IL-1beta is a main BALF mediator involved in HGF secretion, which is mediated through a PGE(2)/COX-2-dependent mechanism. BALF mediators may participate in vivo in the production of HGF and KGF by lung fibroblasts during ALI/ARDS.