Abstract
Elevated plasma levels of the acute-phase reactant serum amyloid A (SAA) have been used as a marker and predictor of inflammatory diseases. SAA regulates leukocyte activation; however, it is not known whether it also modulates neutrophil apoptosis, which is critical to the optimal expression and resolution of inflammation. Culture of human neutrophils with SAA (0.1–20 μg/ml) markedly prolonged neutrophil longevity by delaying constitutive apoptosis. SAA evoked concurrent activation of the ERK and PI3K/Akt signaling pathways, leading to phosphorylation of BAD at Ser112 and Ser136, respectively, and to prevention of collapse of mitochondrial transmembrane potential, cytochrome c release, and caspase-3 activation. These actions were abrogated by pharmacological inhibition of the formyl peptide receptor, ERK or PI3K. Furthermore, aspirin-triggered 15-epi-lipoxin A4 (15-epi-LXA4) and its stable analog 15-epi-16-p-fluorophenoxy-LXA4, which binds to the same receptor as SAA, effectively overrode the antiapoptosis signal from SAA even when neutrophils were treated with 15-epi-LXA4 at either 1 or 4 h postculture with SAA. 15-Epi-LXA4 itself did not affect neutrophil survival and apoptosis. Our results indicate that SAA at clinically relevant concentrations promotes neutrophil survival by suppressing the apoptotic machinery, an effect that can be opposed by 15-epi-LXA4. The opposing actions of SAA and aspirin-triggered 15-epi-LXA4 may contribute to the local regulation of exacerbation and resolution of inflammation, respectively.
Serum amyloid A (SAA)4 is a major acute-phase protein with a unique profile of biological activities (1, 2). In healthy subjects, serum concentration of SAA is <1 μg/ml or 80 nM; it can increase as much as 1000-fold within 24 h in response to infection or tissue damage (2). Elevated plasma levels of SAA have been noted in a variety of pathological conditions (2, 3) and portend a worse prognosis in rheumatoid arthritis (4) and severe unstable angina (5), conditions that are also associated with extensive neutrophil activation (6, 7). SAA was also detected in inflamed tissues (8, 9), suggesting a role in local inflammation.
The precise role of SAA as modulator of inflammation remains elusive, as it possesses both beneficial and harmful actions. For instance, SAA facilitates reverse transport of cholesterol (10) and opsonizes bacteria (11), but it is also precursor of amyloid A, the deposit of which causes amyloidosis (3). SAA is chemotactic to polymorphonuclear leukocytes (PMN) and monocytes (12, 13), regulates L-selectin and CD11b expression on leukocytes (13), promotes PMN adhesion to endothelial cells (13), and stimulates cytokine release from PMN (14, 15). PMN activation is intimately linked to prolonged survival. Mature PMN undergo constitutive programmed cell death that renders them unresponsive to chemoattractants and allows their recognition and removal by scavenger macrophages (16), leading to the resolution of inflammation (17). PMN survival and apoptosis are, however, profoundly influenced by the inflammatory environment and suppression of PMN apoptosis ensue chronic inflammation (18, 19). Indeed, markedly suppressed PMN apoptosis was detected in patients with inflammatory diseases (20, 21). The regulation of PMN apoptosis during the acute phase of inflammation is less well defined, yet it is critical to the optimal expression and resolution of inflammation.
The in vitro actions of SAA are mediated through the G protein-coupled formyl peptide receptor-like 1/lipoxin A4 (LXA4) receptor (ALX) (12, 15, 22). ALX is a pleiotropic receptor that binds a variety of ligands including the glucocorticoid-inducible protein annexin-1 and the anti-inflammatory lipids LXA4 and aspirin-triggered 15-epi-LXA4 (22, 23). Lipoxins are typically generated by transcellular biosynthesis. In particular, acetylation at Ser530 by aspirin (22) or S-nitrosylation at Cys526 by atorvastatin (24) redirect the catalytic activity of cyclooxygenase 2 to catalyze the conversion of arachidonate to 15R-HETE that can be converted by neutrophils and other cells to 15-epi-LXA4. Unlike SAA, LXA4 and 15-epi-LXA4 inhibit PMN chemotaxis, adhesion, and transmigration across the vascular endothelium (see review Ref. 22) and suppress IL-8 secretion (25). Annexin-1 accelerates PMN apoptosis (26), whereas LXA4/15-epi-LXA4 does not affect PMN apoptosis (25, 27). Thus, activation of ALX may result in potent proinflammatory or anti-inflammatory responses in a ligand-specific fashion.
In this study, we investigated whether SAA modulates constitutive PMN apoptosis, what signaling events are associated with this event, and whether such actions can be reversed by aspirin-triggered 15-epi-LXA4. Our results indicate that SAA through ALX prolongs the lifespan of PMN by suppressing the apoptotic machinery. SAA stimulates phosphorylation of the proapoptotic protein BAD through activation of ERK and Akt, leading to prevention of mitochondrial dysfunction and activation of caspase-3. Finally, we demonstrate that aspirin-triggered 15-epi-LXA4 overrides the antiapoptosis signal from SAA through suppression of ERK and Akt signaling.
Materials and Methods
Materials
Recombinant human SAA (purity >98%) was obtained from PeproTech and BioVision. The endotoxin content was <0.1 ng/μg protein by the Limulus assay. 15-Epi-LXA4 was purchased from EMD Biosciences, the formyl peptide receptor antagonist Boc2 (N-t-Boc-Phe-Leu-Phe-Leu-Phe) (28) was from MP Biomedicals. Aspirin-triggered 15-epi-16-p-fluorophenoxy-LXA4 (ATLa), a metabolically stable analog of 15-epi-LXA4 and 15-deoxy-LXA4, was prepared by total organic synthesis as described (29). Structures were confirmed by reversed-phase HPLC, nuclear magnetic resonance, and mass spectral analysis.
Isolation and culture of neutrophils
Venous blood (anticoagulated with sodium heparin, 50 U/ml) was obtained from healthy volunteers who had denied taking any medication for at least 2 wk. The Clinical Research Committee approved the experimental protocols. PMN were isolated as described (30). Neutrophils (5 × 106 cells/ml, purity >96%, viability >98%) resuspended in HBSS supplemented with 10% autologous plasma were placed on a rotator, preincubated for 20 min with 15-epi-LXA4 or ATLa (0.03–2 μM), PD98059 (50 μM), wortmannin (100 nM), SB203580 (1 μM), Z-VAD-FMK (20 μM), or Z-FA-FMK (20 μM) and then challenged with SAA (0.1−20 μg/ml) at 37°C in 5% CO2 atmosphere. In some experiments, PMN were first challenged with SAA (10 μg/ml) and then treated with 15-epi-LXA4 (2 μM) at 15 and 60 min after addition of SAA. At the designated time points, cells were processed as described below.
Assessment of cell viability and apoptosis
PMN viability was assessed by flow cytometry after staining with propidium iodide (0.5 μg/ml). Specific binding of PE-labeled annexin V and the percentage of cells with hypodiploid DNA (30) were used as markers of apoptosis. For DNA detection, PMN (∼106) were suspended in 0.5 ml hypotonic fluorochrome solution (50 μg/ml propidium iodide in 0.1% sodium citrate plus 0.1% Triton X-100). Propidium iodide fluorescence of 10,000 individual nuclei per sample was acquired and analyzed with a FACScan flow cytometer (BD Biosciences).
Caspase-3 activity
Cell lysates, prepared from 107 PMN, were incubated for 60 min at 37°C in a buffer containing 28 μM N-acetyl-Asp-Glu-Val-Asp-AMC (BD Biosciences). Release of AMC from N-acetyl-Asp-Glu-Val-Asp-AMC was measured using a CytoFluor microplate reader (PE Biosystems) with excitation and emission wavelengths of 340 and 460 nm, respectively (30).
Analysis of mitochondrial transmembrane potential (Δψm)
At the indicated times, aliquots of neutrophils (5 × 105 cells) were incubated for 15 min at 37°C with the lipophilic fluorochrome chloromethyl-X-rosamine (CMXRos, 200 nM; Molecular Probes) (31). Fluorescence was analyzed in a FACScan flow cytometer. In control experiments, freshly isolated PMN were incubated with CMXRos in the presence of the uncoupling agent carbonyl cyanide m-chloro-phenylhydrazone (5 μM, 15 min at 37°C) that abolishes mitochondrial transmembrane potential.
Mitochondrial cytochrome c release
Mitochondrial and cytosolic fractions were prepared from 2 × 107 PMN using the Mitochondrial Fractionation kit (Active Motif). Cytochrome c levels in mitochondrial and cytosolic extracts were measured by an ELISA (Active Motif) (32). The intraassay and interassay coefficients of variation were <10%.
Western blot analysis
Protein extracts from 2 × 107 neutrophils prepared as previously described (30) were analyzed for phosphorylation of ERK1/2, Akt, and p38 MAPK by Western blotting using polyclonal Abs (Cell Signaling Technology). ERK- and Akt-mediated phosphorylation of BAD was assessed with an anti-phospho-BAD(Ser112) Ab (BioSource International) and an anti-phospho-BAD(Ser136) Ab (Upstate Biotechnology), respectively.
Statistical analysis
Results are expressed as mean ± SEM. Statistical comparisons were made by ANOVA using ranks (Kruskal-Wallis test) followed by Dunn’s multiple contrast hypothesis test to identify differences between various treatments or by Mann-Whitney U test. Values for p < 0.05 were considered significant for all tests.
Results
SAA prolongs neutrophil survival by delaying apoptosis through ALX
As expected, isolated untreated PMN developed prominent features of apoptosis, including loss of membrane asymmetry (assessed by annexin V binding) and internucleosomal cleavage of DNA, resulting in hypoploid nuclei within 24 h of culture. SAA effectively suppressed the development of apoptotic morphology, and increased the percentage of viable PMN (Fig. 1⇓A). The apoptosis inhibitory action was concentration-dependent with an EC50 value of ∼3.8 μg/ml (Fig. 1⇓B). The apparent maximum suppression of apoptosis that can be achieved with SAA was similar to that of LPS (1 μg/ml) (percentage of annexin V-positive cells at 24 h culture: untreated, 46.6 ± 2.2%; SAA, 16.9 ± 1.6%; LPS, 19.7 ± 1.1%, n = 7 experiments). Although considerable proportions of SAA-treated PMN retained a nonapoptotic phenotype after 48 h in culture, cell viability decreased below 32%, and ∼85% of cells stained positive for annexin V by 72 h. By 96 h, <3% of cells were viable. LPS at 2 ng/ml (the maximum level of contamination detected in SAA preparations) was without detectable effect (data not shown).
SAA delays apoptosis of human neutrophils. Kinetic analysis (A) and concentration dependence (B) of the effects of SAA on PMN viability and development of apoptotic morphology assessed at 24 h of culture. PMN were stained with propidium iodide (to assess viability) or with annexin V, or processed for nuclear DNA content analysis. Results are mean ± SEM for five to seven experiments with different blood donors. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with untreated PMN.
SAA induces IL-8 secretion from human PMN through ALX (15). To assess the functional receptor that mediates the apoptosis suppressing action of SAA, we used Boc2 (27). Preincubation of PMN with Boc2 attenuated the effects of SAA on PMN viability and annexin V staining by 80–97% (Fig. 2⇓).
Boc2 prevents neutrophil responses to SAA. PMN were cultured for 24 h at 37°C with or without Boc2 (50 μM) and SAA (10 μg/ml). Cell viability and staining with annexin V were assessed by cytofluorometry. Results are mean ± SEM of five independent experiments. **, p < 0.01 compared with untreated PMN.
SAA induces phosphorylation of BAD via activation of ERK and Akt
To assess the downstream intracellular signaling pathways that mediate the apoptosis-delaying action of SAA, we studied the activation of several MAPK known to regulate PMN survival. SAA induced a rapid, transient phosphorylation of ERK1/2 and Akt relative to unstimulated controls, reaching a peak within 15 min (Fig. 3⇓A). Culture of PMN resulted in time-dependent phosphorylation of p38 MAPK that was further enhanced in the presence of SAA (Fig. 3⇓B).
SAA suppression of neutrophil apoptosis is mediated via ERK and Akt signaling. Western blot analysis of phosphorylated Akt and ERK1/2 (A) and p38 MAPK (B). Freshly isolated PMN were challenged with SAA (10 μg/ml) for the indicated times. Proteins were then isolated and probed sequentially with phospho-specific Abs, and β-actin was used as control for equal protein loading. The results are representative of four independent experiments. C, SAA-induced phosphorylation of BAD. PMN were challenged with SAA for 30 min. The experiments were repeated three times. D, Pharmacological blockade of MAPK kinase/ERK and PI3K attenuates the apoptosis delaying action of SAA. PMN were cultured for 24 h at 37°C in the presence of PD98059 (50 μM), wortmannin (100 nM), or SB203580 (1 μM) with or without SAA (10 μg/ml). Cell viability and staining with annexin V were assessed by cytofluorometry. Results are mean ± SEM for five to seven experiments with different donor cell preparations. *, p < 0.05; **, p < 0.01 vs untreated PMN; ††, p < 0.01 compared with SAA-treated PMN.
To confirm the role of Akt and ERK activation in mediating the actions of SAA, we used selective pharmacological inhibitors. Both the MAPK kinase/ERK inhibitor PD98059 and the PI3K (the upstream regulator of Akt activation) inhibitor wortmannin effectively blocked the responses to SAA, albeit complete inhibition was never detected, not even with a combination of PD98059 and wortmannin (Fig. 3⇑D). Neither PD98059 nor wortmannin alone affected PMN viability or the percentage of annexin V-positive cells. By contrast, the p38 MAPK inhibitor SB203580 significantly increased the percentage of viable cells and reduced the percentage of annexin V-positive PMN. Cotreatment of PMN with SAA and SB203580 resulted in similar changes as those observed with either SAA or SB203580 alone (Fig. 3⇑B). SAA induced concentration-dependent phosphorylation of BAD at both Ser112 and Ser136, a downstream target for ERK and Akt, respectively (Fig. 3⇑C).
SAA inhibits disruption of mitochondrial transmembrane potential, cytochrome c release and activation of caspase-3
Reductions in mitochondrial transmembrane potential precede development of apoptotic morphology in PMN undergoing constitutive programmed cell death (33, 34). Thus, following 24 h culture, ∼67% of PMN exhibited reduced mitochondrial transmembrane potential (decreased CMXRos staining) (Fig. 4⇓) compared with ∼46% and ∼37% of cells that stained positive with annexin V and had apoptotic nuclei, respectively (Fig. 1⇑). Consistent with suppression of development of apoptotic morphology, SAA partially prevented disruption of mitochondrial transmembrane potential in a concentration-dependent fashion (Fig. 4⇓). Similar results were obtained using another fluorochrome JC-1 (data not shown).
SAA attenuates disruption of mitochondrial transmembrane potential. PMN were cultured for 24 h at 37°C with or without SAA (10 μg/ml), then aliquots of 105 cells were incubated for 15 min with CMXRos (200 nM) and analyzed by cytofluorometry. A, Staining with CMXRos (indicating mitochondrial transmembrane potential) in freshly isolated PMN (0 h) and in PMN treated with carbonyl cyanide m-chloro-phenylhydrazone (mClCCP, 5 μM) for 15 min or cultured for 24 h with SAA or LPS (1 μg/ml). The percentages indicate cells with reduced mitochondrial transmembrane potential. B, Concentration-dependent effect of SAA. Results are mean ± SEM (n = 6 experiments). *, p < 0.05; **, p < 0.01 vs untreated PMN.
To link changes in mitochondrial transmembrane potential to cytochrome c release, we measured cytochrome c levels in the mitochondrial and cytosolic fractions with an ELISA. Cytochrome c was predominantly localized in the mitochondria in freshly isolated PMN (Fig. 5⇓A). A 24-h culture resulted in release of cytochrome c into the cytosol that was markedly attenuated in the presence of SAA (Fig. 5⇓A). Caspase-3 activity was barely detectable in freshly isolated PMN. Culture of PMN for 24 h resulted in marked increases in caspase-3 activity that were reduced by SAA in a concentration-dependent fashion (Fig. 5⇓B). Both PD98059 and wortmannin attenuated the caspase-3 inhibitory action of SAA, though complete reversal was not achieved, not even with the coadministration of PD98059 and wortmannin (Fig. 5⇓C).
SAA prevents cytochrome c release and activation of caspase-3. A, Mitochondrial and cytosolic cytochrome c levels were determined in freshly isolated PMN (0 h) and in PMN cultured for 24 h with or without SAA (10 μg/ml). The results are mean ± SEM (n = 6). *, p < 0.05; **, p < 0.01. B, Concentration-dependent inhibition of caspase-3 activity. Caspase-3 activity was determined using N-acetyl-Asp-Glu-Val-Asp-AMC as a substrate and expressed as fluorescence units (FU). No fluorescence was detected in the presence of N-acetyl-Asp-Glu-Val-Asp-aldehyde, an inhibitor of caspase-3 activity. C, Reversal of the SAA effects by PD98059 (50 μM) and wortmannin (100 nM). D, Effects of pan-caspase inhibition. PMN were cultured for 24 h at 37°C with or without Z-VAD-FMK (20 μM) and SAA (10 μg/ml). Cells were stained with propidium iodide (to assess viability) or with annexin V, or processed for nuclear DNA content analysis. Results are mean ± SEM for five experiments with different blood donors. *, p < 0.05; **, p < 0.01 vs untreated PMN; †, p < 0.05 vs SAA-treated PMN.
Pretreatment of PMN with the pan-caspase inhibitor Z-VAD-FMK effectively suppressed apoptosis, resulting in significant increases in the number of viable cells as assessed after 24 h of culture (Fig. 5⇑D). Cotreatment with Z-VAD-FMK and SAA produced similar changes than SAA or Z-VAD-FMK alone (Fig. 5⇑D). Neither Z-FA-FMK (a negative control) nor the vehicle (0.1% DMSO) elicited any detectable effects (data not shown).
Aspirin-triggered 15-epi-LXA4 reverses SAA suppression of neutrophil apoptosis
Because 15-epi-LXA4 is also a ligand for ALX (22, 23), we compared 15-epi-LXA4 and a metabolically stable analog ATLa with SAA for their effects on apoptosis and intracellular signaling. Although neither 15-epi-LXA4 nor ATLa, at nanomolar to low micromolar concentrations, produced detectable effects on mitochondrial transmembrane potential, apoptosis, and cell viability following 24 h culture, pretreatment of PMN with these lipids resulted in a marked attenuation of SAA prevention of mitochondrial transmembrane potential collapse and suppression of apoptosis (Fig. 6⇓). Apparent maximum inhibition was achieved at ∼2 μM with 15-epi-LXA4 and ATLa being virtually equally potent inhibitors. For instance, EC50 values for ATLa and 15-epi-LXA4 to attenuate the SAA effect on annexin V staining were 0.42 and 0.48 μM, respectively. The biologically inactive LXA4 analog 15-deoxy-LXA4 did not share the 15-epi-LXA4 or ATLa actions on the PMN responses to SAA (Fig. 6⇓D). 15-Epi-LXA4 alone evoked slight increases in phosphorylation of p38 MAPK, but did not induce Akt and ERK1/2 phosphorylation (Fig. 7⇓A). Furthermore, 15-epi-LXA4 markedly reduced SAA-induced ERK1/2 and Akt phosphorylation and consequently phosphorylation of BAD at Ser112 and Ser136 (Fig. 7⇓B). 15-Epi-LXA4 also produced a small inhibition of p38 MAPK phosphorylation by SAA (Fig. 7⇓A).
Reversal of SAA suppression of neutrophil apoptosis by pretreatment with 15-epi-LXA4. PMN were pretreated with various concentrations of 15-epi-LXA4 for 20 min and then cultured for 24 h at 37°C with or without SAA (10 μg/ml). Cells were stained with propidium iodide (to assess viability) (A) or with annexin V (B), or processed for analysis of mitochondrial transmembrane potential (Δψm) or nuclear DNA content. C, Untreated PMN (control) are shown for comparison. Lack of effects of 15-epi-LXA4 and ATLa on PMN survival and apoptosis. PMN were cultured with 15-epi-LXA4 and ATLa (both at 2 μM) for 24 h and then stained with propidium iodide and annexin V. D, 15-Deoxy-LXA4 does not affect PMN responses to SAA. PMN were pretreated with 15-deoxy-LXA4 (2 μM) for 20 min and then cultured for 24 h with SAA (10 μg/ml). Results are mean ± SEM (n = 5 experiments). *, p < 0.05; **, p < 0.01 vs SAA-treated PMN.
Impact of 15-epi-LXA4 on SAA-induced phosphorylation of MAPK (A) and BAD (B). Freshly isolated PMN were cultured with SAA (10 μg/ml) in the absence or presence of 15-epi-LXA4 (2 μM) for 30 min. Cell lysates were prepared and immunoblotted with phospho-specific Abs. The results are representative of three independent experiments.
To investigate whether 15-epi-LXA4 could also exert these effects on PMN exposed to SAA, PMN were first challenged with SAA and then treated with 15-epi-LXA4 at various time points. Treatment with 15-epi-LXA4 at 60 min postchallenge with SAA resulted in marked attenuation of the SAA effects as assayed at 24 h of culture (Fig. 8⇓). Attenuation of PMN responses to SAA was still detectable, albeit to a lesser degree, when 15-epi-LXA4 was added 4 h postculture with SAA (viable cells: SAA, 83.7 ± 3.6%; SAA plus 15-epi-LXA4, 73.9 ± 3.1%, n = 4 experiments, p < 0.05 and annexin V-positive cells: SAA, 15.8 ± 1.9%; SAA plus 15-epi-LXA4, 34.1 ± 3.7%, n = 4 experiments, p < 0.05). Furthermore, SAA-induced Akt and ERK1/2 phosphorylation was also markedly attenuated when 15-epi-LXA4 was added to PMN 15 or 60 min postculture with SAA (Fig. 9⇓).
Posttreatment with 15-epi-LXA4 reverses the antiapoptosis action of SAA. Freshly isolated PMN were challenged with SAA (10 μg/ml) and treated with 15-epi-LXA4 (2 μM) at 60 min postchallenge with SAA. Cell viability (A), annexin V staining (B), mitochondrial transmembrane potential (Δψm) (C), and nuclear DNA content (D) were analyzed at 24 h culture with SAA. Results are mean ± SEM (n = 6 experiments). *, p < 0.05; **, p < 0.01.
SAA-induced phosphorylation of MAPK is reduced with posttreatment with 15-epi-LXA4. Freshly isolated PMN were challenged with SAA (10 μg/ml) and treated with 15-epi-LXA4 (2 μM) at 15 min (A) or 60 min (B) postchallenge with SAA. Cell lysates were prepared and immunoblotted for phospho-Akt and phospho-ERK1/2 30 min after addition of 15-epi-LXA4. The results are representative of three independent experiments.
Discussion
The present study provides evidence for a mechanism by which SAA may contribute to inflammation by rescuing neutrophils from apoptosis. Because prolonged PMN survival is required for excessive leukocyte trafficking into injured tissues, our observations bear directly on the mechanism for SAA amplification of the inflammatory response. Conversely, pretreatment of naive PMN or treatment of SAA-stimulated PMN with 15-epi-LXA4 overrides the apoptosis delaying signals activated by SAA and thus redirecting PMN to apoptosis, consistent with facilitation of the resolution of inflammation.
Consistent with the commitment of PMN to apoptosis, SAA at clinically relevant concentrations delayed, rather than blocked, apoptosis, resulting in prolonged PMN survival as assessed in vitro. These actions were not due to endotoxin contamination because culture of PMN with 2 ng/ml LPS (the highest level detected in SAA preparations) did not result in detectable effects. The degree of the SAA inhibition of apoptosis is comparable to levels found with LPS (35) and with structurally modified C-reactive protein (30).
In the blood, SAA is associated with high density lipoproteins, but it dissociates from lipoproteins at higher concentrations (36, 37). Free SAA was also detected in cultured vascular cells (8) and rheumatoid arthritis synovial tissues (9). When s.c. injected into mice, SAA recruited PMN and monocytes at the injection site (13). Our data suggest that even modest increases in SAA level over baseline value are sufficient to generate an antiapoptosis signal for PMN.
The formyl peptide receptor antagonist Boc2 effectively inhibited the SAA actions. Boc2 is a potent antagonist of binding of formyl peptides to PMN, and it also blocks binding of annexin 1 to ALX (28). SAA and annexin 1 may share recognition sites on PMN (23). Because the formyl peptide receptor does not bind SAA (12, 15, 22), Boc2 most likely inhibited SAA binding to ALX, though we cannot categorically exclude SAA binding to other unrelated receptors.
Neutrophil apoptosis is controlled by a complex network of signaling pathways, including the ERK, Akt, and p38 MAPK pathways (30, 32, 35, 38). The present study provides evidence that SAA also uses these pathways. Culture of PMN with SAA resulted in a rapid phosphorylation of both ERK1/2 and Akt. In addition, inhibition of MAPK kinase/ERK with PD98059 or inhibition of PI3K with wortmannin markedly attenuated, though never fully reversed, the apoptosis-delaying action of SAA. However, the combination of PD98059 with wortmannin did not produce an additive inhibition, indicating that ERK1/2 and Akt work in concert to delay PMN apoptosis. Transient activation of PI3K without ERK activation may not be sufficient to delay apoptosis (38). The role of p38 MAPK in the regulation of PMN apoptosis has been a matter of controversy, for both proapoptosis (30, 32, 39) and antiapoptosis actions (40) have been reported. Constitutive PMN apoptosis appears to be associated with phosphorylation of p38 MAPK (30, 38) that was further enhanced by SAA during the first 6 h of culture. Conversely, the specific p38 MAPK inhibitor SB203580 partially rescued PMN from apoptosis (30, 38), and this result was confirmed in the present study. However, it is unlikely that p38 MAPK represents a survival signal under our experimental conditions because SB203580 by itself suppressed apoptosis, and the apoptosis delaying actions of SB203580 and SAA were not additive.
Our results indicate that the MAPK kinase/ERK and PI3K/Akt pathways converge on BAD protein, a member of the Bcl-2 family. Both Akt and ERK can phosphorylate BAD (41, 42). We observed SAA-induced phosphorylation of BAD at Ser112 and Ser136 consistent with activation of ERK and Akt, respectively. Phosphorylated BAD cannot associate with the antiapoptotic Bcl-2 homologs Mcl-1 or A1 expressed in PMN, thereby allowing expression of their antiapoptotic actions. Among other actions, these proteins could prevent mitochondrial membrane potential transition and, consequently, loss in potential that occurs in cells irreversibly committed to programmed cell death (43, 44). Contrary to general beliefs, mature PMN do contain an unexpectedly large number of mitochondria (45) that may have a role restricted to apoptosis (33). Our results demonstrate that SAA can partially prevent decreases in mitochondrial transmembrane potential and subsequent cytochrome c release occurring during spontaneous PMN apoptosis. Moreover, SAA preservation of mitochondrial transmembrane potential precedes reduction of PMN apoptosis. Prevention of collapse of mitochondrial transmembrane potential and attenuation of cytochrome c release by SAA are consistent with decreased caspase-3 activation, as detected in this study. The results from the experiments using the pan-caspase inhibitor Z-VAD-FMK and SAA confirm the importance of SAA reduction of caspase-3 activation. From these observations, we conclude that a major mechanism by which SAA rescues PMN from constitutive apoptosis is ERK- and PI3K-dependent prevention of mitochondrial dysfunction and repression of caspase-3 activity.
Because 15-epi-LXA4 also binds to ALX and exerts its anti-inflammatory functions through this receptor (22, 46, 47), we sought to investigate its impact on PMN apoptosis. Culture of PMN with 15-epi-LXA4 or a metabolically stable analog ATLa failed to produce detectable changes in development of PMN apoptosis. By contrast, LXA4 was reported to stimulate fibroblast apoptosis (48), suggesting cell-specific lipoxin actions. Previous studies reported that LXA4 and 15-epi-LXA4 facilitate phagocytosis of apoptotic PMN by macrophages rather than interfering directly with the PMN apoptotic machinery (27, 49). Our results show, for the first time, that an endogenous anti-inflammatory molecule aspirin-triggered 15-epi-LXA4 can override a powerful antiapoptosis signal from SAA, thus profoundly affecting the fate of human PMN. These actions may be stimulus-specific, as 15-epi-LXA4 did not attenuate the ability of LPS to prolong PMN survival and delay apoptosis (25). As expected, higher concentrations of 15-epi-LXA4 and ATLa were required in the presence of human plasma to block PMN responses to SAA than were required in other isolated cells. This might reflect interactions of lipoxins with serum components such as albumin. Nevertheless, it is impressive that these lipophilic compounds overcome interactions with plasma components to specifically regulate PMN. Modulation by 15-epi-LXA4 of the fate of PMN is similar to that reported with cyclin-dependent kinase inhibitors (50). Intriguingly, activation of ALX could generate intracellular signals that suppress or reverse the suppression of PMN apoptosis.
The molecular basis for how ALX differently responds to various ligands remains enigmatic. Although 15-epi-LXA4 stimulates limited phosphorylation of p38 MAPK, this response was insufficient to produce detectable changes in PMN apoptosis. The inability of 15-epi-LXA4 to evoke phosphorylation of ERK and Akt may explain the lack of effect on PMN apoptosis. It is unlikely that 15-epi-LXA4 attenuation of SAA-induced activation of ERK and Akt pathways was due to inhibition by 15-epi-LXA4 of SAA binding to ALX because these ligands may bind to distinct pockets on the receptor (51). Indeed, an Ab against the N-terminal domain of this receptor blocked SAA, but not LXA4 binding (15). Whether binding to such pockets leads to different conformational changes in the receptor remains to be investigated. It is possible that 15-epi-LXA4 may generate a yet unidentified negative signal that blocks ERK and Akt. LXA4/15-epi-LXA4 attenuates peroxynitrite signaling (25) via the regulation of presqualene diphosphate accumulation in human PMN (52). LXA4/15-epi-LXA4 through inhibition of PI3K antagonized proliferation of renal mesangial cells (53, 54) and human lung fibroblasts (55). In mesangial cells, LXA4 stimulated phosphorylation of ERK and p38 MAPK (53). However, these and the present results are not easily comparable because PMN are terminally differentiated cells that do not undergo cell division, and MAPK kinases play different roles in PMN than in proliferating cells.
In summary, our results demonstrate that SAA prolongs PMN survival by suppressing the apoptotic machinery. These actions are mediated, in part, through concurrent stimulation of the MAPK kinase/ERK and PI3K/Akt signaling pathways, leading to prevention of mitochondrial dysfunction and inhibition of caspase-3 activation. Combined with earlier observations that SAA is chemotactic for phagocytes (13) and evokes IL-8 release from PMN (14, 15), this acute-phase protein possesses powerful activities that may contribute to prolongation and amplification of the inflammatory response. Furthermore, the observation that 15-epi-LXA4 and its metabolically stable analog ATLa are potent inhibitors of SAA suppression of neutrophil apoptosis is an important addition to the anti-inflammatory proresolution profile of these compounds (22, 47) and demonstrates a hitherto unrecognized potential for treatment of inflammatory diseases associated with enhanced SAA formation.
Disclosures
Driss El Kebir, Levente József, Tarek Khreiss, Wanling Pan, and János G. Filep have no financial conflict of interest. Brigham and Women’s Hospital has licensed lipoxin stable analog patents for development by Schering AG-Berlex (now Bayer-Schering AG) for clinical development. Charles N. Serhan is the inventor of lipoxin stable analogs and Brigham and Women’s Hospital is the assignee. Charles N. Serhan and Nicos A. Petasis are consultants for Resolvyx Pharmaceuticals.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵1 This work was supported by Grant MOP-64283 (to J.G.F.) and Doctoral Research Awards (to L.J. and T.K.) from the Canadian Institutes of Health Research, and by Grant P50-DE016191 from the National Institutes of Health (to N.A.P. and C.N.S.).
↵2 D.E.K. and L.J. contributed equally to this work.
↵3 Address correspondence and reprint requests to Dr. János G. Filep, Research Center, Maisonneuve-Rosemont Hospital, 5415 Boulevard de l’Assomption, Montréal, Québec, Canada H1T 2M4. E-mail address: janos.g.filep{at}umontreal.ca
↵4 Abbreviations used in this paper: SAA, serum amyloid A; PMN, polymorphonuclear leukocyte; LXA4, lipoxin A4; ATLa, aspirin-triggered 15-epi-16-p-fluorophenoxy-LXA4; ALX, lipoxin receptor; CMXRos, chloromethyl-X-rosamine.
- Received March 16, 2007.
- Accepted April 20, 2007.
- Copyright © 2007 by The American Association of Immunologists
References
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