15-Epi-lipoxin A₄ Inhibits Myeloperoxidase Signaling and Enhances Resolution of Acute Lung Injury

MPO and Lipoxins

MPO purified from human leukocytes was obtained from Athens Research and Technology (Athens, GA) (purity > 97% on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, RZ value of 0.82, no detectable eosinophil peroxidase contamination). 15-Epi-LXA₄ was from Calbiochem (La Jolla, CA). 15-Epi-16-p-fluorophenoxy-LXA₄ (ATLa), a metabolically stable analog of 15-epi-LXA_4, and the biologically inactive analog 15-deoxy-LXA₄ were prepared by total organic synthesis (27). Structures were confirmed by reversed-phase high performance liquid chromatography (HPLC), nuclear magnetic resonance, and mass spectral analysis.

Neutrophil Isolation and Culture

Neutrophils were isolated (28) from the venous blood of healthy volunteers who had denied taking any medication for at least 2 weeks. Neutrophils (5 × 10⁶ cells per ml, purity >96%, viability >98%, apoptotic <3%) were cultured in Hanks' balanced salt solution supplemented with 10% autologous serum on a rotator. The Clinical Research Committee at the Maisonneuve-Rosemont Hospital approved the experimental protocols. Neutrophils were either pretreated for 20 minutes with 15-epi-LXA₄, ATLa, or 15-deoxy-LXA₄ (0.03–2 μM) and then challenged with MPO (10–160 nM) at 37°C or were first challenged with MPO and then treated with 15-epi-LXA₄ at 60 minutes and 4 hours post MPO. In some experiments, MPO was pretreated with 4-aminobenzoic acid hydrazide (20 μM) to inhibit its enzymatic activity (17) before addition to neutrophils. At the designated time points, cells were processed as described below.

Assessment of Apoptosis

Apoptosis was assessed with flow cytometry using fluorescein isothiocyanate–conjugated annexin-V (BD Biosciences, Mountain View, CA) in combination with propidium iodide (Molecular Probes, Eugene, OR) and the percentage of cells with hypoploid DNA (29). DNA cleavage was assayed by detection of cytoplasmic histone-associated DNA fragments (Cell Death ELISA; Roche, Laval, QC, Canada) and gel electrophoresis (17).

Caspase-3 Activation

Activated caspase-3 in neutrophils was detected with flow cytometry using fluorescein isothiocyanate–labeled DEVD-fmk (Calbiochem) (17).

Detection of Mitochondrial Transmembrane Potential (ΔΨ_m)

Neutrophils (5 × 10⁵ cells) were incubated for 15 minutes with the lipophilic fluorochrome chloromethyl-X-rosamine (CMXRos, 200 nM, Molecular Probes) and the fluorescence was analyzed in a FACScan flow cytometer (29).

Western Blot Analysis

Proteins from 10⁷ neutrophils were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to polyvinylidene flouride (PVDF) membranes, blocked with 5% nonfat milk, and probed with antibodies to phosphorylated extracellular signal–regulated kinase (ERK) 1/2, Akt, p38 mitogen-activated protein kinase (MAPK), Bad(Ser¹¹²), and Bad(Ser¹³⁶) (all from Cell Signaling Technologies, Danvers, MA), Mcl-1 (Santa Cruz Biotechnology, Santa Cruz, CA), or β-actin (Sigma, Oakville, ON, Canada) (29).

Mac-1 Expression

Surface expression of Mac-1 on blood or isolated neutrophils was assessed using R-phycoerythrin–conjugated anti-CD11b (Becton Dickinson) antibody with a FACScan flow cytometer (BD Biosciences, Mountain View, CA) (25).

MPO Release

Conditioned media were analyzed for MPO by a specific ELISA (Assay Design, Ann Arbor, MI). The intraassay and interassay coefficients of variation were less than 8%.

In Vivo ALI

Female BALB/c mice (aged 8–12 wk, Charles River Laboratories, St-Constant, QC, Canada) were housed in pathogen-free conditions. The Animal Care Committee of the Maisonneuve-Rosemont Hospital approved the protocols. Mice were injected intratracheally with 0.1 ml of 0.25% λ-carrageenan (Fluka-Sigma-Aldrich, Buchs, Switzerland) plus 10 μl of 16 μM MPO (17), followed 24 hours later by intravenous injection of 200 μg/kg 15-epi-LXA₄ in 100 μl saline or appropriately diluted ethanol as a vehicle control. At the same time, some mice were also injected intraperitoneally with the pan-caspase inhibitor z-Val-Ala-DL-Asp-fluoromethylketone (zVAD-fmk, Calbiochem, 10 μg/kg in 0.2 ml), followed by two additional doses of zVAD-fmk 4 and 8 hours later (30). Additional groups of mice were challenged intraperitoneally with 2 × 10⁸ live E. coli (American Type Culture Collection, ATCC 25922) or 1 × 10⁹ live bacteria for survival studies (19). At 1 hour postinfection, mice were treated with 15-epi-LXA₄ or vehicle as just described.

Assessment of Inflammation

At the indicated times, mice were killed and the lungs were lavaged and bronchoalveolar lavage (BAL) fluid protein, IL-6 levels, total and differential leukocyte counts, and neutrophil viability and apoptosis were determined (17). BAL fluid cells, cytospinned into poly-l-lysine-coated slides, were stained with hematoxylin and eosin and assayed for macrophages containing apoptotic bodies. In separate groups of mice, the lungs were removed without lavage, fixed in 4% formaldehyde, and processed for standard histological evaluation (17). Lung dry-to-wet weight ratio was determined after placement of tissues in a drying oven at 56°C for 3 days. Tissue MPO activity was measured using o-dianisidine as a substrate and human MPO (Sigma) as a standard (31). BAL fluid IL-6 levels were measured by using a mouse IL-6 ELISA kit (BD Biosciences). The intra-assay and inter-assay coefficients of variation were less than 6%.

Statistical Analysis

Results are expressed as mean ± SEM. Statistical comparisons were made by analysis of variance using ranks (Kruskal-Wallis test) followed by Dunn multiple contrast hypothesis tests to identify differences between various treatments or by the Mann-Whitney U test (two-tailed). Correlations were analyzed by the Spearman test. P values less than 0.05 were considered statistically significant. Kaplan-Meyer survival curves were compared using the log-rank test.

Aspirin-triggered 15-epi-LXA₄ Overcomes MPO Delay of Apoptosis in Human Neutrophils

Confirming our previous results (17), MPO prolonged neutrophil life span by delaying intrinsic apoptosis (Figure 1A). MPO partially prevented disruption of mitochondrial transmembrane potential (ΔΨ_m) that precedes development of apoptotic morphology in neutrophils undergoing constitutive programmed cell death (29, 32). Because the apoptosis-delaying action of MPO is mediated through the β₂ integrin Mac-1 (17) and 15-epi-LXA₄ down-regulates Mac-1 expression on human neutrophils (23–25), we compared 15-epi-LXA₄ and its metabolically stable analog ATLa with MPO for their effects on apoptosis and cellular signaling. Although neither 15-epi-LXA₄ nor ATLa, up to a concentration of 2 μM, affected neutrophil survival (data not shown), they markedly attenuated the MPO actions on ΔΨ_m and apoptosis (Figures 1B and 1C). Apparent maximum inhibition was achieved at approximately 2 μM with 15-epi-LXA₄ and ATLa being virtually equally potent inhibitors (Figure 1C). The biologically inactive analog 15-deoxy-LXA₄ had no detectable effects. Electrophoretic analysis confirmed the ability of 15-epi-LXA₄ to prevent MPO suppression of DNA fragmentation (Figure 1D). 15-Epi-LXA₄ did not inhibit the catalytic activity of MPO (Figure 1E). Pretreatment of MPO with 4-aminobenzoic acid hydrazide resulted in greater than 95% loss of peroxidase activity (17). Like MPO, catalytically inactive MPO suppressed apoptosis and this was also reversed by 15-epi-LXA₄ (Figure 1C). Binding of MPO to Mac-1 results in rapid phosphorylation of ERK 1/2 and Akt (17) that generate survival signals for neutrophils. We further probed the effects of 15-epi-LXA₄ on these signaling pathways. 15-Epi-LXA₄ alone slightly increased phosphorylation of p38 MAPK, but not Akt and ERK 1/2 (Figure 1F). By contrast, 15-epi-LXA₄ reduced MPO-induced phosphorylation of ERK 1/2 and Akt (Figure 1F) and their downstream target Bad (Figure 1G). Expression of Mcl-1, a key regulator of apoptosis in neutrophils (33, 34), decreased rapidly within 2 hours of culture, an effect that was prevented by MPO (Figure 1H). 15-epi-LXA₄ inhibited MPO-mediated preservation of Mcl-1 (Figure 1H) and produced a small inhibition of p38 MAPK phosphorylation by MPO (Figure 1F).

When neutrophils were first challenged with MPO and then treated with 15-epi-LXA₄ at 60 minutes post MPO, the proapoptotic action of 15-epi-LXA₄ was dominant over MPO-mediated effects (Figure 2A). Treatment with 15-epi-LXA₄ attenuated MPO-induced phosphorylation of Akt and ERK 1/2 (Figure 2B). Such an inhibitory action was still detectable when 15-epi-LXA₄ was added 4 hours post MPO (data not shown), further highlighting the therapeutical potential of this antiinflammatory lipid.

MPO up-regulates neutrophil CD11b expression and induces MPO release (15). We found that 15-epi-LXA₄ inhibited MPO-stimulated CD11b expression on isolated neutrophils as well as on whole blood neutrophils (Figure 3A) and attenuated MPO release (Figure 3B) in a concentration-dependent fashion. We observed a negative correlation between neutrophil CD11b expression levels assayed at 30 minutes post MPO and development of apoptosis assessed at 24-hour culture (Figure 3C), further corroborating the importance of CD11b-mediated “outside-in” signaling in determining the fate of neutrophils.

15-Epi-LXA₄ Promotes Neutrophil Apoptosis and Enhances Inflammatory Resolution

Having shown the ability of 15-epi-LXA₄ to override the apoptosis-delaying action of MPO in vitro, we investigated the effects of 15-epi-LXA₄ on the resolution of inflammation in mice. In the carrageenan model, low concentration of intratracheal carrageenan instillation evokes neutrophil-mediated ALI that resolves within 5 days without treatment (35). Coadministration of MPO prolongs carrageenan-induced inflammation parallel with suppression of neutrophil apoptosis (17). Treatment with 15-epi-LXA₄ accelerated the resolution of established inflammation when administered intravenously at near the peak of inflammation (24 h) evoked by carrageenan plus MPO (Figure 4). We observed, over a period of 4 days of administering 15-epi-LXA₄, decreases in BAL fluid total leukocyte and neutrophil numbers with increases in the number of monocytes/macrophages (Figure 4A). 15-Epi-LXA₄ reduced tissue MPO content, an index of neutrophil accumulation (Figure 4B). Antiinflammatory actions of 15-epi-LXA₄ were also evident, as 15-epi-LXA₄ attenuated edema formation (Figures 4C and 4D) and the release of IL-6 into the bronchoalveolar space (Figure 4E). Furthermore, 15-epi-LXA₄ augmented the amount of cytoplasmic histone-associated DNA fragments in BAL cells (Figure 4F) and caspase-3 activity in BAL neutrophils (Figure 4G). Lungs of mice treated with 15-epi-LXA₄ exhibited markedly reduced inflammatory infiltrate and lung injury (Figure 5A). Decreases in BAL fluid neutrophil number (Figure 6A) occurred parallel with increases in the percentage of annexin-V–positive cells (Figure 6B) and the percentage of macrophages containing apoptotic bodies (Figures 5B and 5C).

Next, we investigated whether the enhanced resolution was mediated by 15-epi-LXA₄ induction of caspase-3–dependent neutrophil apoptosis in vivo. Intraperitoneal injection of the pan-caspase inhibitor zVAD-fmk prevented the 15-epi-LXA₄–induced reduction of neutrophil accumulation (Figure 6A), increase in neutrophil apoptosis (Figure 6B), and suppression of edema formation (Figure 6C) in carrageenan plus MPO-induced lung inflammation. Of note, zVAD-fmk enhanced inflammatory cell accumulation (Figure 6A) and edema formation (Figure 6C) in the airways.

The proresolution action of 15-epi-LXA₄ was further confirmed in the E. coli septicemia-associated lung injury model, which was shown to involve endogenous MPO (19). In this model, treatment with 15-epi-LXA₄ reduced tissue MPO content (Figure 7A) and BAL fluid neutrophil numbers (Figure 7B) with concomitant increases in the number of apoptotic neutrophils (Figure 7C) and caspase-3 activity in BAL neutrophils (Figure 7D). 15-Epi-LXA₄ also reduced E. coli–induced edema formation (Figure 7E) and tissue injury (Figure 7F). We observed greater survival in 15-epi-LXA₄–treated animals. Approximately 60% of mice died within 7 hours after E. coli (10⁹) challenge, whereas all 15-epi-LXA₄–treated mice were alive at this time (Figure 7G).