RAPID COMMUNICATION
LXA4, aspirin-triggered 15-epi-LXA4, and their analogs selectively downregulate PMN azurophilic degranulation

Andrew T. Gewirtz1, Valery V. Fokin2, Nicos A. Petasis2, Charles N. Serhan3, and James L. Madara1

1 Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia; 2 Department of Chemistry, University of Southern California, Los Angeles, California; and 3 Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The eicosanoid lipoxin A4 (LXA4) is biosynthesized in vivo by cells present at inflammatory sites and appears to be an endogenous anti-inflammatory mediator. Further, in the presence of aspirin, the 15-epimer of LXA4 (15-epi-LXA4) is biosynthesized and may mediate some of aspirin's desirable bioactions. LXA4, 15-epi-LXA4, and their stable analogs inhibit inflammation in established animal models, indicating that these compounds may be useful for treating inflammatory disease states. To investigate the cellular mechanisms by which these lipid mediators downregulate inflammation, we investigated whether these eicosanoids could influence receptor-mediated degranulation of human neutrophils, an event thought to play a major causative role in several inflammatory disease states. LXA4, 15-epi-LXA4, and their stable analogs potently (IC50 < 1 nM) and selectively downregulated neutrophil release of azurophilic granule contents but did not affect other neutrophil secretory functions. Thus the cellular basis of action of these natural off-switches to inflammation appears to involve downregulation of neutrophil azurophilic granule release.

anti-inflammatory mediators; elastase; eicosanoids; Fcgamma receptors; immune complexes; neutrophils


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE LIPOXYGENASE INTERACTION PRODUCTS, lipoxins (LX), appear to play an important role in downregulating inflammation (25, 30, 31). In humans, these eicosanoids are cooperatively (i.e., involves multiple cell types) biosynthesized from arachidonate by cells present at sites of inflammation (25). In contrast to other eicosanoids (e.g., leukotrienes, prostaglandins) that promote leukocyte infiltration (i.e., inflammation), LX have bioactivities that downregulate polymorphonuclear leukocytes (PMN or neutrophils) movement, thus making these eicosanoids "anti-inflammatory." Particularly potent anti-inflammatory activity is exhibited by LXA4 and its stable analogs (26). One compound that behaves as an LXA4 stable analog, in that it competes for the LXA4 receptor, is resistant to enzymatic degradation, and has anti-inflammatory bioactivity, is the 15-epimer of LXA4 (15-epi-LXA4) (3, 31). However, 15-epi-LXA4 is in fact biosynthesized in vivo by a pathway involving cyclooxygenase that has been acetylated by aspirin. 15-Epi-LXA4 biosynthesized in this manner may play a role in mediating some of aspirin's bioactions.

LXA4 (as well as 15-epi-LXA4 and LXA4 analogs) potently downregulate inflammation in vivo in well-defined animal models such as the hamster cheek pouch (21) and the mouse ear (31). LX also exhibit anti-inflammatory bioactivity in isolated model systems with isolated cells or cell systems, including inhibition of PMN chemotaxis (14) as well as migration across model endothelium (19) and epithelium (6). Additionally, LXA4 can attenuate PMN movement by downregulating (in epithelial cells) secretion of chemokines that direct PMN movement (9, 12). LX can also downregulate vascular endothelial P-selectin expression, resulting in reduced PMN adherence to endothelium (23) (an early step in PMN movement to inflammatory sites). Although these in vitro LX bioactions are likely partially responsible for the in vivo anti-inflammatory effects of LX, the profound downregulation of inflammation in animal models [e.g., mouse ear (31)] by LX suggests that, in addition to these known cellular mechanisms, LX likely have as yet still unrecognized bioactions that can account, in part, for their overall in vivo anti-inflammatory effect.

We reasoned that perhaps LX could downregulate PMN secretion, thus providing an additional means by which LX could attenuate inflammation. When appropriately activated, PMN secrete their antibacterial arsenal of superoxide anions and the contents of their two major granule populations. Although PMN-secreted products play an important role in host defense, in some inflammatory disease states PMN granule contents also appear to be responsible for damage to host tissue and subsequently for promoting the inflammatory state (7, 33, 34). This can occur if PMN are lysed by phagocytosing urate crystals that are present in gout patients (35). Alternatively, PMN granule contents are also released extracellularly during the receptor-mediated ingestion of immune complexes, which are abundant in rheumatoid arthritis, via a process that has been termed "regurgitation during feeding" (36). Some PMN azurophilic granule contents are directly chemotactic for PMN (2), whereas others induce chemokine release from other cells (17), perhaps long after any foreign entities have been eliminated. One particularly important secreted molecule thought to be involved in host tissue damage in chronic inflammatory disease states is the protease elastase, the source of which is PMN azurophilic granules.

PMN secretion of azurophilic granule contents can be mediated via Fcgamma receptors. These receptors are activated by IgG-coated entities (bacteria or other antigens) or high-valency immune complexes (HIC) (1, 15, 28). Thus we investigated whether LXA4 and its stable analogs could regulate PMN secretion induced by HIC. We report that LXA4 attenuated PMN Fcgamma receptor-mediated release of azurophilic granule contents, indicating that LX downregulation of inflammation in vivo may involve attenuation of PMN degranulation. This effect was selective for release of azurophilic granules and did not extend to specific granules or superoxide release evoked by Fcgamma receptor-mediated activation. This selective inhibition of specific PMN activation responses by an endogenously biosynthesized compound is unique for LX.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

LX. LXA4 and LXB4 were obtained from Cayman (Ann Arbor, MI). LXA4 analogs (15[R/S]-methyl-LXA4, 16-phenoxy-LXA4, and 15-deoxy-LXA4) as well as 15-epi-LXA4 and its stable analog [15-epi-16-(para-fluoro)-phenoxy-LXA4] were prepared as methyl esters by total organic synthesis as previously described (26, 30).

LX exposure to PMN. PMN [50 × 106/ml Hanks' balanced salt solution (not supplemented with Ca2+)], isolated from healthy volunteers as previously described (20), were exposed to indicated LX or vehicle (always 0.1% ethanol) for 2.5 h unless otherwise indicated. PMN were kept at 37°C and mixed every 15 min by gentle pipetting. PMN were then used within 20 min or placed on ice and used within 1 h.

Secretion assays. Elastase release was measured in real time by spectrofluorometry as modified from Sklar et al. (27) as previously described (24). Unless otherwise noted, HIC [prepared as previously described (11, 28, 29)] was added at a final concentration of 100 µg/ml. The assay was calibrated with purified elastase generously supplied by Dr. Mustapha Si-Tahar (Pasteur Institute, Paris, France). Lactoferrin was measured by ELISA as previously described (10), except that standardization was performed with purified lactoferrin (Sigma) instead of PMN lysates. Myeloperoxidase activity was measured in supernatants of stimulated PMN, which had been centrifuged for 2 min after activation by HIC [using a peroxidase substrate as previously described (20)]. Superoxide release was measured by following the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome C at 550 nm (5). An endpoint version of this assay was utilized by measuring the total amount of SOD-inhibitable reduction of ferricytochrome C that occurred within 2 min following addition of HIC. Chelation of intracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), as well as measurement of intracellular Ca2+, were performed as previously described (24).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To investigate whether LX could regulate PMN secretion, PMN were treated with LXA4 before being stimulated with HIC, an agonist that, via an Fcgamma receptor-mediated mechanism, induces secretion in intact PMN (1). LXA4 attenuated PMN release of azurophilic granule contents (Fig. 1), as measured by release of elastase (utilizing an assay that continuously measures release of this protease). LXA4 did not by itself induce release of elastase (indicated by the fluorescence before addition of HIC being comparable with control) or affect the time at which maximal release in response to HIC occurred (~1 min, the time at which the slope becomes maximal). Inhibition of HIC-induced elastase release by LXA4 was concentration dependent, with IC50 approx  1.0 nM and maximal inhibition occurring at 100 nM (Fig. 1B). LXA4 attenuation of azurophilic degranulation was also time dependent, requiring that PMN be exposed to LXA4 for 2 h or more before activation with HIC for significant inhibition to occur (Fig. 2).


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Fig. 1.   Lipoxin A4 (LXA4) attenuates polymorphonuclear leukocyte (PMN) Fcgamma receptor-mediated elastase release. PMN were treated for 2.5 h with vehicle (0.1% ethanol) or LXA4 and elastase release was measured as described in MATERIALS AND METHODS. A: representative experiment showing effect of 100 nM LXA4 on high-valency immune complex (HIC)-induced elastase release. HIC were added to control and LXA4-treated PMN at time indicated by arrow. Note that immediate increase in fluorescence is due to light scatter by this particulate agonist, whereas increase in fluorescence that begins ~50 s later is due to substrate cleavage by elastase. B: concentration dependence of LXA4 attenuation of PMN Fcgamma receptor-mediated elastase. Values shown are those observed 2 min after addition of HIC. Data are means ± SE of an experiment performed in triplicate.


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Fig. 2.   Time dependence of LXA4 attenuation of Fcgamma receptor-mediated elastase release. PMN were treated for indicated time with vehicle (0.1% ethanol) or 100 nM LXA4 before measuring elastase release in response to HIC. Data are normalized means ± SE of 3 separate experiments utilizing different PMN donors.

We next ascertained whether LXA4 treatment led to inhibition of all Fcgamma receptor-mediated secretion or selectively attenuated azurophilic degranulation. We measured PMN release of superoxide anions as well as the specific granule component, lactoferrin, in response to HIC. LXA4 treatment, which inhibited HIC-induced elastase release by >50%, did not significantly affect HIC-induced secretion of lactoferrin or superoxide anions (Fig. 3). Next, we measured whether elastase release induced by another agonist was also inhibited by LXA4. If PMN are pretreated with cytochalasins (fungal metabolites that prevent actin polymerization), stimulation with chemoattractants such as N-formyl-methionyl-leucyl-phenylalanine (FMLP) results in elastase release (37). In these experiments, cytochalasin-treated PMN that were stimulated with a subsaturating dose of FMLP (2.0 × 10-8 M) exhibited elastase release, which was attenuated by pretreatment with LXA4 (Fig. 4). PMN (vehicle or LXA4 treated) stimulated with 1.0 × 10-8 M FMLP did not secrete detectable amounts of elastase, whereas stimulation with higher concentrations of FMLP gave a maximal response that was not affected by LXA4 (Fig. 4). In contrast, Fcgamma receptor-mediated elastase release was attenuated by LXA4 in response to all tested concentrations of HIC (Fig. 4). These results indicate that LXA4 downregulated azurophilic degranulation in response to a physiological agonist as well as an unrelated receptor-mediated agonist but blocked neither other Fcgamma receptor-mediated superoxide generation nor azurophilic degranulation induced by saturating stimulation. To verify that the inhibition of elastase release that we measured truly reflected inhibition of azurophilic degranulation rather than specific inhibition of only elastase release, we also measured HIC-induced myeloperoxidase release (another azurophilic granule component) in the absence and presence of LXA4. This response was similarly inhibited by LXA4 (inhibition ranged from 40 to 65% among 3 different PMN donors, P < 0.01), indicating that, indeed, LXA4 attenuation of elastase release represented an attenuation of azurophilic degranulation.


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Fig. 3.   LXA4 does not affect Fcgamma receptor-mediated specific degranulation or superoxide generation. PMN were exposed to 100 nM LXA4 (solid bars) or vehicle (0.1% ethanol; open bars) for 2.5 h, at which time elastase release (A), lactoferrin release (B), and superoxide generation (C) were measured in response to HIC. Data are means ± SE of a representative experiment performed in triplicate.


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Fig. 4.   LXA4 attenuates azurophilic degranulation in response to subsaturating doses of N-formyl-methionyl-leucyl-phenylalanine (FMLP) and a wide range of HIC concentrations. PMN were exposed to 100 nM LXA4 or vehicle (0.1% ethanol; control) for 2.5 h, at which time elastase release was measured in response to FMLP (A; in presence of 5 µg/ml cytochalasin B) or HIC (B) at indicated concentrations.

We next began to explore potential mechanisms by which LXA4 downregulated PMN Fcgamma receptor-mediated elastase release. The previously characterized bioactions of LXA4 appear to be mediated via the LXA4 receptor (8, 9, 12, 31). To find out whether LX attenuation of degranulation was also mediated via the LXA4 receptor, we investigated which members of a panel of LXA4 stable analogs, which are or are not specific ligands for the LXA4 receptor, could mimic the ability of LXA4 to downregulate Fcgamma receptor-mediated azurophilic degranulation. The LXA4 stable analogs that are specific ligands for the LXA4 receptor (15-[R/S]-methyl-LXA4, 16-phenoxy-LXA4; Ref. 26) were able to mimic the ability of LXA4 to downregulate Fcgamma receptor-mediated azurophilic degranulation, whereas two compounds that are not specific ligands for the LXA4 receptor (15-deoxy-LXA4, an inactive LXA4 stable analog, and lipoxin B4, an eicosanoid whose bioactivity differs in some cases from LXA4) were without significant effect (Fig. 5A). Aspirin-triggered 15-epi-LXA4, which is also a specific ligand for the LXA4 receptor (3), and its stable analog [15-epi-16-(para-fluoro)-phenoxy-LXA4 (30)] also downregulated PMN elastase release to an extent similar or perhaps slightly greater than LXA4. 15-Epi-LXA4 exhibited this bioactivity at subnanomolar concentrations with an IC50 of ~0.2 nM. An analog of this compound, 15-epi-16-(para-fluoro)-phenoxy-LXA4, exhibited similar bioactivity at saturating concentrations but had an IC50 approximately one log unit less potent (Fig. 5B).


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Fig. 5.   Stable analogs of LXA4 and aspirin-triggered 15-epimer of LXA4 (15-epi-LXA4), which are specific ligands for the LXA4 receptor, also attenuated PMN azurophilic degranulation. A: PMN were exposed to 100 nM indicated compound or vehicle (0.1% ethanol) for 2.5 h and then elastase release was measured in response to HIC. B: PMN were exposed to indicated concentrations of 15-epi-LXA4 (), 15-epi-16-(para-fluoro)-phenoxy-LXA4 (open circle ), or vehicle (control values normalized to 100%). Data are normalized means ± SE of 3 separate experiments utilizing different PMN donors.

One intracellular signaling event that occurs in PMN in response to LXA4 is a small increase in cytosolic Ca2+ concentration ([Ca2+]) (18). Our bioactive LXA4 stable analogs also induce a small increase in cytosolic [Ca2+] in PMN (increase was ~25 nM above baseline, reaching maximal levels ~1 min after addition of LXA4 analog). To investigate whether this small Ca2+ mobilization was required for LX attenuation of PMN Fcgamma receptor degranulation, the Ca2+ increase was inhibited by the intracellular Ca2+ chelator BAPTA. First, we verified the effectiveness of our BAPTA loading procedure by checking that, as previously shown (24), this BAPTA treatment prevented any detectable increase of [Ca2+] in response to FMLP. We then measured intracellular [Ca2+] in control (DMSO-treated) and BAPTA-loaded cells in response to our most potent LXA4 stable analog. As expected, the small increase in intracellular [Ca2+] observed in response to 15-[R/S]-methyl-LXA4 was completely prevented by BAPTA. Preventing the LXA4 analog-induced increase in intracellular [Ca2+] in this manner did not affect LXA4 analog attenuation of Fcgamma receptor-mediated elastase release (100 nM 15-[R/S]-methyl LXA4 attenuated HIC-induced elastase release by 46 ± 7.5% in DMSO-treated PMN and by 54 ± 5.0% in BAPTA-loaded PMN). Thus the small increase in intracellular [Ca2+] observed in response to LXA4 and its stable analogs does not appear to mediate this LXA4 bioaction.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Inflammation appears to be downregulated via the biosynthesis of LX by cells present at inflammatory sites. These LX can then act to counter the bioactions of proinflammatory mediators (e.g., leukotrienes, platelet-activating factor, tumor necrosis factor) (12, 31). In part, LX attenuate inflammation by downregulating PMN motility to chemoattractants as well as by downregulating secretion of the chemokines that direct PMN movement (9, 12). Here we demonstrate that LXA4 and its stable analogs can also downregulate Fcgamma receptor-mediated release of azurophilic granules. This previously unappreciated bioaction of LXA4 may be an important means by which LX regulates inflammation in vivo. Because the proteases released from PMN azurophilic granules are thought to play an important role in the pathologies of a number of chronic inflammatory disease states, these results suggest that LXA4, aspirin-triggered LX, and their stable analogs are not merely tools for studying LX action but may have therapeutic potential for treating these disease states. This newly described bioaction of LX likely adds to the ability of LX to attenuate PMN movement in reducing inflammation in vivo.

Although both azurophilic degranulation and superoxide secretion are important components of the PMN arsenal, LXA4 treatment downregulated the former but had no detectable effect on the latter in response to an Fcgamma receptor-mediated agonist. A possible rationale for this can be envisaged by considering the physiological role of Fcgamma receptor-mediated secretion as well as some of the fundamental differences between superoxide anions and azurophilic granule components. Fcgamma receptors mediate the phagocytosis of microorganisms, various other foreign entities, and host-generated immune complexes (4). Accompanying phagocytosis are PMN generation of superoxide anions and secretion of granule contents into the phagovacuole and extracellular space, which act to kill microorganisms (32) and possibly to digest immune complexes. Although both superoxide anions and azurophilic granule proteases are also capable of damaging host tissue and subsequently promoting inflammation, azurophilic granule enzymes (especially proteases like elastase and cathepsin G) have characteristics that may make them much more proinflammatory than superoxide anions. First, superoxide is rapidly reduced by superoxide dismutase, whereas azurophilic granule proteases like elastase can subsist for many hours (especially perhaps in nonserosal environments such as the lumens of the lung and gastrointestinal tract). Second, the azurophilic granule proteases (elastase and cathepsin G) are directly chemotactic for immune cells (2). Third, granule proteases (and azurophilic glycosidases) can digest matrix tissue, thus providing access routes for immune cells to the inflammatory site and perhaps also for locally generated proinflammatory molecules back to the vasculature. This latter event results in the recruitment of yet more immune cells. Considering that LX biosynthesized at inflammatory sites in vivo are likely generated from products of PMN 5-lipoxygenase (and/or 15-lipoxygenase interactions), LX will not likely be present until many immune cells have already arrived at the infected/inflamed site. Thus, perhaps by selectively downregulating extracellular release of their azurophilic granule contents, but not Fcgamma receptor-mediated superoxide generation, PMN can maintain their ability to kill microorganisms while minimizing tissue damage. Coherent with this view, release of specific granules was also not affected by LX, and the content of these granules is antibacterial but does not seem to pose a threat to host tissue or to promote the inflammatory state.

The intracellular mechanisms that mediate downregulation of azurophilic granule secretion by LXA4, aspirin-triggered LX, and their stable analogs are not yet clear, although some insights can be drawn. Because, of the compounds tested, only those that are specific ligands for the LXA4 receptor (26, 30) were able to downregulate azurophilic degranulation, it is likely that ligand binding of this receptor is a prerequisite for such attenuation of this secretory response. One postreceptor signal induced by LXA4 is a small increase in cytosolic [Ca2+]. However, since preventing the generation of this signal had no effect on the ability of LXA4 to attenuate azurophilic degranulation, this Ca2+ mobilization appears not to be required for this anti-inflammatory action of this eicosanoid. Analogously, LXA4-induced Ca2+ mobilization does not play a role in mediating LXA4 effects on monocyte adherence, indicating that intracellular Ca2+ is not a second messenger of LX receptor activation (16, 22). Identification of the signaling pathway by which LXA4 acts to downregulate PMN azurophilic granule release will be important for developing rational therapeutic strategies for disease states mediated by PMN tissue destruction.

This report is the first description of an agent that selectively attenuates azurophilic degranulation but not oxidative burst in response to a receptor-mediated agonist. This downregulation was exhibited not by a pharmacological compound but by a biomolecule that is naturally biosynthesized in vivo at inflammatory sites. Furthermore, this downregulation of azurophilic degranulation was observed in response to HIC analogous to those that are characteristically found in inflammation in the lung and joints (in rheumatoid arthritis) (13). Because azurophilic granule components (especially the protease elastase) are thought to damage host tissue in these and other inflammatory disease states, downregulating release of these granules via LXA4 and LXA4 stable analogs may offer therapeutic potential for these conditions. Because LXA4 stable analogs are much more resistant to degradation than the native eicosanoid, these compounds might be especially useful in treating inflammatory disease states. Further, it is possible that some of the therapeutic benefits of currently used nonsteroidal anti-inflammatory agents, specifically aspirin, are in fact due to the endogenous biosynthesis of 15-epi-LX that is induced by this drug. If this notion (supported in part by our observations that this aspirin-triggered compound downregulated elastase release at subnanomolar concentrations) is correct, then direct administration of 15-epi-LX (or the analogs studied here) may offer potent anti-inflammatory activity while avoiding some of the potential undesirable side effects caused by drugs that interfere with eicosanoid production via inhibition of cyclooxygenases and lipoxygenases.


    ACKNOWLEDGEMENTS

These studies were supported by National Institutes of Health Grants DK-47662 and DK-35932 (to J. L. Madara) and DK-50305 and GM-38765 (to C. N. Serhan) and by a discovery grant from Schering AG (to J. L. Madara, C. N. Serhan, and N. A. Petasis). A. Gewirtz was supported by an individual National Research Service Award.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: A. Gewirtz, Dept. of Pathology and Laboratory Medicine, Emory Univ. Hospital, Atlanta, GA 30322 (E-mail: agewirt{at}emory.edu).

Received 11 November 1998; accepted in final form 8 January 1999.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Cell Physiol 276(4):C988-C994
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