Proresolving Actions of Synthetic and Natural Protease Inhibitors Are Mediated by Annexin A1 Free

The inflammatory process triggered by infection or tissue damage is characterized by microscopic events that include increased vascular permeability and leukocyte accumulation. Leukocyte recruitment, mainly polymorphonuclear leukocytes (PMN), is triggered by a number of proinflammatory mediators generated at the site of inflammation acting as chemotactic agents (1). Once recruited, PMNs release several granules rich in proteases that are important against infection. However, neutrophil products can also be harmful to the host leading to intense tissue injury (2).

Proteases are enzymes produced by a variety of phagocytic inflammatory cells, including neutrophils (3, 4). Neutrophil elastase (NE) and proteinase 3 (PR3) are destructive serine proteases with a range of substrates causing impact on cell and tissue function through diverse mechanisms, from degradation of ingested pathogens to favoring cell motility through the extracellular matrix (2). Therefore, it is not surprising that protease activity is tempered by anti-protease molecules, which are secreted to neutralize any excess of these enzymes. Antiproteases are classified as both systemic (produced by hepatocytes and distributed through the circulating) and alarm (synthesized and secreted by local cells to the site of inflammation) (5, 6). Alarm antiproteases such as secretory leukocyte protease inhibitor (SLPI) and Elafin are secreted predominantly by the mucosal epithelium, and their levels are modulated during multiple pathological conditions (3, 4, 6). Recent investigations indicate that SLPI and Elafin are inducible in human alveolar macrophages and neutrophils (7).

Despite the release of antiproteases as counterregulatory mechanism for excessive inflammation, the inflammatory response is also coupled to the release of local anti-inflammatory and proresolving factors preventing future or excessive recruitment of neutrophils and tissue damage and allowing the resolution of inflammation (8). Among these mediators, there are anti-inflammatory and proresolving lipids, such as lipoxins and resolvins (9), and proresolving proteins, such as annexin A1 (AnxA1) (10, 11).

AnxA1 is a glucocorticoid (GC)-regulated protein known as a mediator of several GC functions. The N-terminal region contains the main pharmacophore for the anti-inflammatory properties of AnxA1 (12); therefore, the intact protein of 37 kDa can stimulate multiple activities to help resolve acute inflammation. The regulation is quite unusual, with large amounts of the protein within innate immune cells. However, after cell activation, AnxA1 is externalized on the cell surface, and the N-terminal region is exposed and can interact in a paracrine/autocrine fashion with its receptor named formyl peptide receptor 2/lipoxin A4 receptor (FPR2/ALX). However, within this microenvironment AnxA1 is vulnerable to be cleaved at the N-terminal region by proteases including NE and PR3, generating the 33-kDa isoform of poorly known properties (13, 14). Studies have shown that the 33-kDa isoform of AnxA1 may be associated with proinflammatory effects (15, 16). Congruently, cleavage-resistant (CR) AnxA1 exhibited greater anti-inflammatory effect compared with the parent protein, in different animal models of inflammation (17, 18). In addition, an AnxA1 peptide with mutation on a distinct cleavage site was potently active in promoting resolution, inducing neutrophil apoptosis and efferocytosis (19) and exerting protection in the complex settings of sepsis (20).

The calculation of resolution indices was introduced for the first time by Bannenberg et al. (21) and allows assessment of the proresolving properties of agents by the temporal regulation of leukocyte recruitment at inflammatory sites. These indices chart and take into account 1) magnitude ψ_max (maximal of neutrophil numbers that are present in the exudates) and T_max (time when ψ_max is maximal [i.e., time when neutrophil numbers reach maximum]), 2) duration of the resolution interval (R_i) from T_max (i.e., the time that it takes for the number of neutrophils to reach half of ψ_max [T₅₀]). This is an important proresolving parameter that quantifies how efficient a new agent is.

In this study, we investigated the effects of synthetic (sivelestat [SIV]; Eglin) and natural (SLPI; Elafin) specific neutrophil protease inhibitors on resolution of LPS-induced pleural inflammation and queried if and how these effects could be associated with preservation of AnxA1 integrity. Collectively, our data show that strategies aiming at protecting and/or incrementing endogenous AnxA1 levels may be harnessed for the treatment of unresolved inflammation.

Male BALB/c mice (8–10 wk) were housed under standard conditions and had free access to commercial chow and water. Mice were obtained from the Bioscience Unit of Instituto de Ciências Biológicas (Brazil). All described procedures had prior approval from the Animal Ethics Committee of Universidade Federal de Minas Gerais (protocol number 15/2011).

SIV (number S7198; Sigma-Aldrich, St. Louis, MO), Eglin c (number SP3133b; Cambridge Bioscience), Elafin (number 61641; AnaSpec), and recombinant human SLPI (number 1274-PI-100; R&D Systems) were dissolved in DMSO and diluted further in PBS. Endotoxin level in recombinant human SLPI was <1.0 EU/1 μg of the protein by the Limulus amebocyte lysate method (as presented in R&D Systems catalog number1274-PI-100). Rabbit anti–P-ERK1/2 (number 4377), anti–caspase-3 (number 9665), anti–Mcl-1 (number 5453), anti-GAPDH (number 3683), and mouse anti–P-IκB-α (number 9246) were purchased from Cell Signaling Technology (Beverly MA). Anti-SLPI, anti-elastase, and secondary anti-rabbit and anti-mouse peroxidase conjugate Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Elafin was from Bioss. Rabbit anti-AnxA1 was from Invitrogen (Carlsbad, CA). Anti–β-actin and LPS (from Escherichia coli serotype O:111:B4) were from Sigma-Aldrich. BOC-1 (N-t-Boc-Met-leu-Phe) was from MP Biomedicals (Santa Ana, CA). ZVAD-fmk was from Tocris (Ellisville, MO). The peptides AnxA1_2–50 and CR-AnxA1_2–50 were purchased as described (19). Anti-AnxA1 antiserum (D3428) was a donation from National Institute for Biological Standards and Control (B. Lane, Potters Bar Hertfordshire, U.K.).

Mice received an intrapleural (i.pl.) injection of LPS (250 ng/cavity) or PBS as described previously (22, 23). The cells present in the pleural cavity were harvested by washing the cavity with 2 ml PBS at different time points after injection of LPS. The monosodium urate (MSU) crystals were prepared as described previously (24). Mice were placed under anesthesia (150:10 mg/kg ketamine:xylazine; i.p.; Syntec, Sao Paulo, Brazil) and were injected with MSU crystals (100 μg) into the tibiofemoral joint. Knee washes were performed at 18 h after MSU injection. The total cell counts were performed in a Neubauer chamber using Turk’s stain. Differential cell counts were performed on cyto-centrifuge preparations (Shandon III) stained with May-Grunwald-Giemsa using standard morphological criteria to identify cell types. The results are presented as the number of cells per cavity.

To evaluate the effect of protease inhibitors agents on LPS-induced pleurisy, mice were treated with specific neutrophil elastase inhibitors SIV (1, 5, and 25 mg/kg, i.p.), Elafin peptide (10 μg/mouse, i.p.), and a recombinant human secretory leukocyte protease inhibitor, SLPI (10 μg/mouse, i.p.); an inhibitor of elastase and cathepsin G, Eglin c peptide (100 μg/mouse, i.p.) 4 or 8 h after LPS challenge. We used also a recently described AnxA1 peptide CR-AnxA1_2–50 and its control peptide AnxA1_2-50 (19). To prevent the action of AnxA1 induced by SIV, mice were treated with anti-AnxA1 antiserum (0.1 ml hyperimmune serum diluted in 100 μl PBS/mice, i.p.) and with BOC-1 (5 mg/kg, i.p.), a nonselective FPR antagonist that blocks the FPR and ALXR receptors (25). Nonimmune goat serum was used as control (data not showed). ZVAD-fmk (1 mg/kg), and a broad-spectrum-caspase inhibitor was given systemically (i.p.) 15 min before SIV injection, as described previously (23). Drugs were dissolved in DMSO and diluted further in PBS. Control mice received only vehicle.

Apoptosis was assessed as previously reported (22, 23). Briefly, cells (5 × 10⁴) were collected after LPS challenge, cytocentrifuged, fixed, and stained with May-Grünwald-Giemsa. Posteriorly, to determine the proportion of cells with distinctive apoptotic morphology, the cells were counted using oil immersion microscopy (×100 objective). At least 500 cells/glass slide were counted, and results are expressed as the mean ± SEM of percentage of cells with apoptotic morphology. Of note, cells are considered apoptotic when presenting chromatin condensation, nuclear fragmentation, and formation of apoptotic bodies out or inside macrophages. Assessment of neutrophil (Ly6G⁺/F4/80⁻) apoptosis was also performed by flow cytometry using FITC-labeled annexin V and 7-aminoactinomycin D. Efferocytosis was assessed by flow cytometry as previously shown (19), considering the frequency of macrophages containing PMNs (F4/80⁺/Ly6G⁺ cells). Abs used were F4/80 (PEcy7; eBioscience, San Diego, CA) and Ly6G (BV421; BD Biosciences, San Jose, CA). Analysis of efferocytosis was also performed by preparing cytospin slides and determining the proportion of macrophages that ingested apoptotic bodies (500 cells/slides were counted).

Cells present in the pleural cavity were harvested 24 h after LPS injection (LPS 6 h + SIV 18 h). The leukocyte populations were analyzed by staining with fluorescent mAbs against F4/80 (PE; eBioscience), GR1 (BV421; BD Biosciences), and CD11b (FITC; BD Biosciences). After being stained for surface markers, cells were permeabilized with permeabilization buffer (eBioscience) for 30 min. Stained cells were acquired in BD FACSCanto II cell analyzer (BD Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, OR). Macrophage populations were defined according to F4/80, GR1, and CD11b expression, as shown previously (26, 27).

Neutrophils were isolated from human peripheral blood from healthy donors (Ethics Committee of the Universidade Federal de Minas Gerais, Brazil - Institutional Review Board Project number 0319.0.203.000-11) by using histopaque gradient (Histopaque 11191 and 10771; Sigma-Aldrich) as described previously (28). Neutrophils (1 × 10⁶ cells/well) were resuspended in RPMI 1640 medium, seeded in 24-well culture plates (BD Biosciences), and incubated at 37°C in a 5% CO₂ atmosphere. Cell viability was determined by trypan blue staining and the purity of preparations were >95%. To evaluate the effect of SIV on LPS-induced prosurvival/delayed apoptosis of neutrophils, isolated neutrophils were cultured in the presence of LPS (100 ng/ml) and 1 h after were treated with different concentrations of SIV (10, 30, and 100 μg/ml) for 2 or 5 h or with Elafin (100 ng/ml) or SLPI (100 ng/ml). In some experiments, neutrophils were pretreated by 1 h with zVAD (100 μM) before addiction of antiproteases. Apoptosis was evaluated as described above. Experiments were performed in triplicates.

Whole-cell extracts were quantified with the Bradford assay reagent from Bio-Rad (Hercules, CA). After quantification, 50 μg whole protein was separated by electrophoresis on a denaturing polyacrylamide-SDS gel (10–15%) and electrotransferred to nitrocellulose membranes, as described previously (22). Membranes were blocked with PBS containing 5% (w/v) nonfat dry milk and 0.1% Tween 20 (v/v) overnight at 4°C, washed with PBS–Tween-20 0.1% (v/v), and then incubated with specific primary Abs (Elastase, SLPI, Elafin, cleaved caspase-3, AnxA1, or anti–β-actin) using a dilution of 1:1000 in PBS-BSA 5% (w/v) and 0.1% Tween-20. After washing with PBS-Tween 20 0.1% (v/v), membranes were incubated with appropriated peroxidase-conjugated secondary Ab (1:3000). Immunoreactive bands were visualized by using ECL detection system, as described by the manufacturer (GE Healthcare, Piscataway, NJ). The values of intact and cleaved AnxA1 were quantified by using a densitometric analysis software (ImageJ, Image Processing and Analysis in Java; National Institutes of Health, Bethesda, MD). Changes in protein levels were estimated, and results are expressed as cleaved AnxA1, cleaved caspase-3, MCl-1, or P-IκB-α (in arbitrary units), and normalized to the values of β-actin in the same sample.

We quantified the resolution indices as described previously (21, 29). Murine pleural exudates were collected at 8, 24, 36, and 48 h after challenge with LPS. The treatment with SIV (5 mk/kg) was performed at the peak of inflammation, 8 h after the challenge. The number of PMN and mononuclear cells was determined by total and differential leukocyte counting. The resolution of acute inflammation were defined in quantitative terms by the following resolution indices: 1) magnitude (ψ_max and T_max), ψ_max (maximal PMN), T_max (time point when PMN numbers reach maximum); 2) duration (T₅₀), T₅₀ (time point when PMN numbers reduce to 50% of maximum) and 3), and R_i (the interval between T_max and T₅₀, when 50% of PMNs are lost from the pleural cavity).

The elastase activity was measured in cell extracts prepared in the absence of proteases inhibitors by using an in-house procedure that relies on the use of MeO-Suc-AA-Pro-Val-pNA (M4765; Sigma-Aldrich) as substrate. Cells obtained from pleural cavity of mice were lysed on appropriated buffer (200 mM NaCl, 20 mM Tris-HCl, and 1% Triton X-100 [pH 8]). The lysate was centrifuged at 12,000 rpm in a microcentrifuge for 15 min at 4°C, and 50 μl supernatant was incubated with 50 μl substrate MeO-Suc-AA-Pro-Val-pNA in a 96-well microplate at 37°C for 2 h. A standard curve was performed with p-nitroaniline in accordance to the procedures supplied by the manufacturer (BioVision, Milpitas, CA). The absorbance of samples was analyzed in a spectrophotometer (Spectra Max 190; Molecular Devices) at 405 nm. The results are presented as elastase activity absorbance.

Data were analyzed by one-way ANOVA, and differences between groups were assessed using the Student-Newman-Keuls posttest. A p value < 0.05 was considered significant. All results are presented as the mean ± SEM. Calculations were performed using the Prism 5.0 software (GraphPad Software, San Diego, CA).

A well-established model of LPS-induced pleurisy was used (22, 23). In this model the intrapleural injection of LPS induced a time-dependent influx of leukocytes into the cavity (Fig. 1A). The number of neutrophils peaked at 8 and 24 h and decreased thereafter (Fig. 1A). There was a significant increase of mononuclear cells into the pleural cavity that coincided with the resolution phase of inflammation, as seen by decline of neutrophils number (48 and 72 h) (Fig. 1A). Next, we analyzed the kinetics of elastase, an important protease present in neutrophils. The expression and activity of elastase accompanied the kinetics of neutrophil recruitment in the pleural cavity (Fig. 1B, 1C, respectively).

Because elastase can modulate AnxA1 integrity (13, 14), we investigated the kinetics of accumulation for the active/intact (37 kDa) or inactive/cleaved (33 kDa) forms of AnxA1 (Fig. 1B). As previously shown (22) and in Fig. 1B, in PBS-challenged mice, intact AnxA1 protein (37 kDa) was the main form detected. During the acute phase of LPS-induced neutrophil recruitment (8- and 24-h time points), intact AnxA1 accumulation decreased markedly, and the cleaved species were strongly detected. Intact AnxA1 expression was regained during the resolution phase of inflammation (48 and 72 h) (22). Interestingly, high elastase activity was associated with increased levels of the cleaved form of AnxA1 (33 kDa), whereas the decline of elastase activity was associated to higher levels of intact AnxA1 (37 kDa) (Fig. 1B as compared with Fig. 1C).

Following these analyses, we queried what could be the profile of kinetic of the endogenous elastase inhibitor, SLPI. In PBS-injected mice (Fig. 2A), SLPI was detected in basal setting, disappeared at the peak of neutrophil influx (8 h), and was strongly expressed at the time points of resolution of inflammation (48 and 72 h). Although it has been argued that there is no mouse ortholog to Elafin (2), we were able to detect a predicted band with m.w. consistent with Elafin, whose kinetics was quite similar to the SLPI (data not shown). Importantly, augmented expression of endogenous serine protease inhibitors at the 24-h time point coincided to the early decline of elastase activity (Fig. 1C), suggesting existence of a yin/yang balance between inhibitors and elastase activity.

To verify their therapeutic potential, SLPI (recombinant human) and Elafin (synthetic peptide) were injected at the peak of inflammation (8 h post-LPS), and the inflammatory status was determined at the 24-h time point. Treatment of mice with both polypeptides decreased neutrophil numbers into the pleural cavity after 24 h of challenge (Fig. 2B). Interestingly, such an effect was accompanied by the appearance of apoptotic neutrophils in the pleural cavity (Fig. 2C) and associated to inhibition of neutrophils elastase and decrease of the prosurvival pathways NF-κB (evaluated by IκB-α phosphorylation) and Mcl-1 (Fig. 2D). We also evaluated the effectiveness of a short treatment protocol with SLPI and Elafin (4-h LPS + 4-h antiprotease) and observed that they were also effective in decreasing neutrophils counts in the pleural cavity (PBS, 0.09 ± 0.041; LPS, 8.6 ± 0.99; LPS + SLPI, 4.5 ± 0.61; LPS + Elafin, 5.3 ± 0.19; number of neutrophils × 10⁵/cavity; n = 5 mice/group; p < 0.001, when comparing LPS × PBS; and p < 0.01, when comparing LPS × LPS + SLPI or LPS + Elafin). Importantly, both treatments were able to prevent AnxA1 degradation (Fig. 2D). The short treatment protocol was also carried out using Eglin c (a synthetic inhibitor of elastase and cathepsin G) and compared it with Elafin peptide. Administration of Elafin or Eglin c similarly decreased neutrophil numbers in the pleural cavity, and such an event was associated with appearance of apoptotic neutrophil in the pleural cavity, as shown by morphological criteria (Supplemental Fig. 1A–C). Importantly, there were increased numbers of macrophages containing apoptotic bodies after antiprotease treatment (indicated by arrowheads in representative images of the Supplemental Fig. 1C). Taken together, these results indicate a temporal expression of anti-elastase at times when resolution begin and thereafter, suggesting that endogenous protease inhibitors function as a control checkpoint to regulate the inflammatory response. In accordance with this possibility, exogenous therapy with elastase inhibitors was able to promote resolution of neutrophilic inflammation, and this was associated to a proapoptotic program in neutrophils.

Next, we tested whether synthetic small molecule inhibitors could be effective in preventing AnxA1 cleavage, providing a translational potential to these data. The selective inhibitor SIV was injected 8 h after LPS (at the peak of inflammation). SIV produced a dose-dependent inhibition of neutrophil counts at 24 h after LPS (Fig. 3A). These effects on cell numbers were associated with a dose-dependent decrease in elastase expression (Fig. 3B) together with prevention of AnxA1 cleavage, as monitored by Western blot and quantified by densitometry analysis (Fig. 3C). The dose of 5 mg/kg was selected to establish the resolution indices for SIV in this model. Mice received an injection of LPS, and 8 h later, a systemic injection of SIV and cells was collected at 8, 24, 36, and 48 h after LPS. The treatment of mice with SIV shortened R_i: R_i LPS ∼ 26 h; R_i LPS+SIV ∼18 h (Fig. 4A). Noteworthy, short treatment of LPS-inflamed mice with SIV was able to decrease elastase expression, increase intact levels of AnxA1 (37-kDa form), and partially inhibit AnxA1 degradation (33-kDa band) in pleural exudates at 2 and 4 h after injection of the compound (Fig. 4B). Moreover, the measurement of AnxA1 in the supernatant of pleural exudates show increased AnxA1 content after SIV treatment, which may be the result of the increased AnxA1 externalization (PBS, 1 ± 0.49; LPS, 1.9 ± 0.2; LPS + SIV, 3.8 ± 0.4; total AnxA1 in arbitrary units normalized against PBS-Group, n = 3 mice/group; p < 0.05, when comparing LPS × LPS + SIV). These results indicate that pharmacological treatment with SIV promoted resolution of LPS-induced neutrophilic inflammation probably by increasing AnxA1 expression and preventing its cleavage.

To assess whether serine protease inhibitors shows similar effects by accelerating resolution in another experimental model of neutrophilic inflammation, we performed a set of experiments using a murine model of gout. This model is characterized by an intense recruitment of neutrophils after a single injection of MSU crystals into the knee (24, 30). Interestingly, the treatment of mice with serine protease inhibitors (Elafin, SLPI, and SIV) decreased the numbers of leukocytes into the knee cavity (Supplemental Fig. 2A) associated with increased numbers of apoptotic neutrophils (Supplemental Fig. 2B).

Next, we evaluated potential mechanism(s) underlying the proresolving effects of SIV. The neutrophil elastase inhibitor was injected 4 h after LPS, and neutrophil numbers determined 4 h later. As shown in Fig. 5A, SIV (5 mg/kg) efficiently decreased neutrophil numbers in the pleural cavity and this was associated with reduced elastase activity in the cavity (PBS, 0.08 ± 0.02 absorbance; LPS, 1.4 ± 0.1 absorbance; LPS + SIV, 0.7 ± 0.1 absorbance, n = 4 mice/group; p < 0.01 when comparing LPS × LPS + SIV). Such an effect was prevented by using a pan-caspase inhibitor (zVAD-fmk), indicating a caspase dependency in SIV-induced resolution (Fig. 5A). Treatment with zVAD alone did not alter the kinetics of neutrophil recruitment (Fig. 5A). More importantly, SIV induced dose-dependent apoptosis of neutrophils in the pleural cavity, as quantified by morphological (Fig. 5B) and biochemical criteria, including increase of caspase-3 cleavage, decrease of Mcl-1 and NF-κB (evaluated by P-IκB-α), (Fig. 5C, 5D), and flow cytometry (Fig. 5E).

Neutrophils are exposed to inflammatory mediators at sites of inflammation that may extend their life span by delaying apoptosis (28). Because SIV induced apoptosis in vivo, in a milieu exposure to prosurvival factors, we investigated the ability of SIV to counteract the prosurvival effects of LPS. Treatment of human neutrophils with SIV dose-dependently increased levels of intact AnxA1 in neutrophils (Supplemental Fig. 3A) and induced neutrophil apoptosis, as evaluated by increased percentage of apoptotic neutrophils (Supplemental Fig. 3B, 3C - representative figures) and caspase-3 cleavage (Supplemental Fig. 3A), when comparing LPS-treated cells with LPS + SIV. Noteworthy, the treatment of human neutrophils with the prosurvival LPS decreases the spontaneous apoptosis of cultured neutrophil (Supplemental Fig. 3B, 3C). In addition, Elafin and SLPI were also able to override the survival-inducing effects of LPS and promoted neutrophil apoptosis in vitro (Supplemental Fig. 4A). Apoptosis was abolished by pretreatment with zVAD. Protease inhibitors inhibited total elastase activity in presence or absence of zVAD (Supplemental Fig. 4B). Taken together, these findings indicate that elastase inhibitors can effectively induce or accelerate a proapoptotic program in neutrophils leading to resolution of inflammation.

Next, we evaluated the leukocyte population of LPS-challenged mice after treatment with SIV, based on a recent description of three macrophage populations: M1 (F4/80^lowGr1⁺Cd11b^med), M2 (F4/80^highGr1⁻Cd11b^high), and resolution-promoting macrophages Mres (F4/80^medCd11b^low) (26, 27, 31). The gating strategy was performed as shown previously (26). LPS injection increased the number of M1 macrophages as previously shown (31), which was decreased with SIV treatment (data not shown). M2 macrophages were detected on PBS-injected mice and at 24 h but significantly increased after SIV treatment (Fig. 6A). Interestingly, the number of Mres was only increased after SIV treatment (Fig. 6B). In keeping with the proefferocytic ability of these macrophages, efferocytosis of apoptotic neutrophils was also increased as evaluated by flow cytometry (Fig. 6C) and by counting the percentage of macrophages contained apoptotic bodies from cytospin preparations (data not shown). These results indicate that the resolution induced by SIV is associated with an accumulation of M2 and Mres macrophages and clearance of apoptotic cells into the pleural cavity.

Because AnxA1-derived peptides engineered to resist serine protease cleavage promote resolution of inflammation more potently than wild-type peptides, next, we compared the effects of SIV with those of CR-AnxA1_2–50 (19). Fig. 7A shows that at 5 mg/kg SIV decreased neutrophil numbers into the pleural cavity to the same extent of the natural AnxA1 peptide (150 μM/mouse). However, CR-AnxA1_2–50 was more effective than either treatment. Noteworthy, all treatments decreased Mcl-1 and NF-κB activation, two important survival pathways in neutrophils (Fig. 7B) (32).

Finally, these partially similar pharmacological effects of SIV and AnxA1 peptide and the modulation exerted by elastase on AnxA1 brought us to test whether endogenous AnxA1 could be involved in proresolving actions of SIV. Mice were treated with a neutralizing anti-AnxA1 Ab given alongside a prophylactic protocol, ahead of SIV administration. Neutralization of AnxA1 with a blocking Ab abolished the modulation exerted by SIV on neutrophil accumulation (Fig. 7C) and prevented SIV-induced decrease on Mcl-1 accumulation and IκB-α phosphorylation (Fig. 7D). Moreover, we investigated the effect of SIV following inhibition of AnxA1 receptor by using BOC-1 (a nonselective AnxA1 receptor antagonist) and found that under this situation SIV lost its effectiveness on the promotion of inflammation resolution (Fig. 7E). Taken together, these results clearly suggest a direct functional correlation between elastase inhibitors and the dynamic of AnxA1 accumulation, suggesting an engagement of the endogenous proresolving AnxA1 system in the resolution of inflammation promoted by antiproteases.

Proteases regulate a wide variety of essential physiological functions, including protein catabolism, cell growth and migration, blood coagulation, inflammation, and modulation of pharmacologically active peptides. Thus, the finely tuned natural equilibrium between proteases and their inhibitors is essential for the maintenance of homeostasis. Hence, an imbalance of the function of proteolytic enzymes is a common feature of inflammatory diseases (33). Recent studies have shown that therapeutic inhibition of proteases, including neutrophil-derived elastase, may be a promising therapeutic strategy in view of the powerful anti-inflammatory effects of these inhibitors in various preclinical models of diseases (2).

We have reported the importance of AnxA1 in driving the resolution of the acute inflammatory response (12, 32). Importantly, we detected an active process of AnxA1 cleavage to its 33-kDa breakdown product during the peak of acute pleurisy (22). The impact of this phenomenon appears not to be irrelevant because recent studies have indicated that modulation of AnxA1 cleavage may be a new strategy to control inflammatory diseases, as seen with AnxA1 CR mutants and shorter modified peptides (17–19). However, the role of antiproteases, more specifically, elastase inhibitors, in protecting AnxA1 cleavage in vivo and its association to resolution of acute inflammation has not been established. In this follow-up study, we investigated the role of synthetic (SIV; Eglin c) and natural (Elafin; SLPI) elastase inhibitors on the resolution of LPS-induced neutrophilic inflammation. In this study, we applied a dynamic model of pleural inflammation and reported that pharmacological treatment with both natural and synthetic antiproteases promoted resolution of inflammation. Such an effect was also seen in a model of gout. Mechanistically, treatment with antiproteases induced AnxA1 expression and caspase-dependent neutrophil apoptosis associated with NF-κB inhibition and Mcl-1 decrease. Moreover, treatment of inflamed mice with SIV promotes reprogramming of macrophages to the phenotypes that are more prone to resolution and efferocytosis of apoptotic neutrophils. Finally, the effect of SIV was AnxA1 dependent because it was abolished by inhibiting AnxA1 with a neutralizing Ab and by blocking its receptor, suggesting that endogenous AnxA1 is involved in the proresolving actions of antiproteases.

The regulation of the activity of potentially harmful proteases secreted by leukocytes during inflammation is important for the prevention of excessive tissue injury (34). SLPI is a serine proteinase inhibitor constitutively expressed in mucosal tissues and immune cells, including monocytes, macrophages, and neutrophils (35, 36) that exerts pleiotropic activities in different biological systems (7). For example, SLPI promotes cutaneous wound healing, cell proliferation of epithelial cells, prevents HIV infection, exhibits antimicrobial and antifungal functions, inhibits NF-κB activation, and modulates macrophage functions (6, 37). The protective effect of SLPI as an anti-inflammatory mediator has been documented in inflammatory lung diseases, including chronic obstructive pulmonary disease, cystic fibrosis (38), and allergic asthma (37).

Importantly, SLPI-deficient mice have exacerbated susceptibly to endotoxin-induced shock (39). Another endogenous antiprotease, known as skin-derived antileukoprotease (SKALP) or Elafin, has similar anti-inflammatory actions (40). Elafin is a secreted protein expressed in epithelial cells such as skin and lung epithelium but also by immune cells, including neutrophils (41) and macrophages (42). Elafin inhibits the activation of proinflammatory transcription factors AP-1 and NF-κB and, like SLPI, possesses antimicrobial and fungicidal properties (40). The anti-inflammatory effects of Elafin have been established in a number of studies and animal models, including lung inflammatory disease induced by LPS, chronic obstructive pulmonary disease, cardiac dysfunction, and intestinal diseases (3, 6). In the current study, both SLPI and Elafin potently accelerated resolution with significant reduction of neutrophils numbers. The observation that the kinetics of anti-protease expression paralleled that of macrophages suggest that—in these settings—this cell type is their most likely source. The physiological function of anti-protease was complemented by the efficacy of exogenous administration of these protease inhibitors with evident positive impact on neutrophil apoptosis and macrophage efferocytosis. These results identify specific cellular processes as major event/target of anti-protease physiopharmacology. Akin with these findings is the study that indicated higher SLPI production from murine macrophages during the clearance of apoptotic cells (36). A more recent report showed that SLPI is a pivotal mediator of anti-inflammatory response in acetaminophen-induced acute liver failure by modulating the monocyte/macrophage function, and this included a reduced production of proinflammatory cytokines and increased phagocytosis of necrotic debris (43). Therefore, our results do show the relevance and effects of antiproteases in the context of inflammation resolution and add to the literature by suggesting that it may indeed be useful to development of protease inhibitors to control overexuberant inflammatory reactions.

It is noteworthy that SIV, a synthetic specific neutrophil elastase inhibitor, is clinically used as an anti-inflammatory agent for acute lung injury and acute respiratory distress syndrome (2). In preclinical models, SIV reduces markers of tissue injury and systemic inflammation, including ischemia reperfusion injury (44), sepsis (45), and acute lung injury (46, 47). In our set of experiments, treatment of animals with SIV dose-dependently reduced neutrophil accumulation into the pleural cavity, an effect associated to reduced resolution indices and R_i. Importantly, and to our knowledge for the first time, we showed that SIV induced caspase-dependent neutrophil apoptosis and AnxA1 expression in neutrophils, highlighting the pivotal proresolving protein AnxA1 as an important player in the mechanism of action of this drug. It is likely that engagement of AnxA1 may be a common feature of known anti-inflammatory drugs, and data with glucocorticoids (48) and, more recently, chromones (49) appear to corroborate the existence of this shared protective pathway.

Neutrophil lifespan is increased by antiapoptotic factors, such as Mcl-1 (myeloid cell leukemia-1), which in general have their expression inversely correlated with the degree of neutrophil apoptosis (50). In our work we showed that protease inhibitors were able to induce resolution of inflammation and this was associated with decreased levels of Mcl-1 in pleural exudates. In fact, it is already demonstrated that Mcl-1 downregulation contributes to anti-inflammatory and proresolution effects leading to resolution of inflammation (23, 26, 28, 51, 52). Altogether, these data reinforce the idea that intervention on neutrophil survival could be a potential pharmacological strategy to control inflammatory diseases.

An important determinant of inflammation resolution is the efficient removal of apoptotic neutrophils by macrophages (53). Macrophages are usually classified as either classically (M1) or alternatively activated (M2). Under a proinflammatory environment macrophages usually have proinflammatory phenotype (M1) that has little efferocytic capacity and increased capacity to engulf (phagocytose) foreign organisms. Inflammatory macrophages are skewed to the M2 phenotype in a resolution milieu and they produce IL-10 and TGF-β, which have anti-inflammatory actions (54, 55). M2 macrophages are prone to efferocytose neutrophils (56), and uptake of apoptotic cells during the resolution of inflammation leads to their conversion to a proresolving CD11b low phenotype (27). It has been shown that murine macrophages secrete an increased amount of SLPI when encountering apoptotic cells, which may help to attenuate potential inflammation during clearance of these cells (36). Moreover, it was shown that Elafin prevented CD14 cleavage by elastase and restored apoptotic cell recognition by macrophages (57). Importantly, our in vivo data show that, in addition to inducing apoptosis of neutrophils, antiproteases were able to decrease macrophages with a proinflammatory phenotype and to increase number of proresolving macrophages.

Intact AnxA1 (37 kDa) is the biologically active form of the protein endowed with anti-inflammatory properties, but at sites of inflammation, it can be cleaved to its inactive form of 33 kDa by neutrophil-derived proteases (13, 14). Thus, modulation of endogenous AnxA1 pool might be an important mechanism to resolve inflammatory responses. This can be even of more acute importance because the 33-kDa form of AnxA1 displays proinflammatory effects by promoting ERK1/2 activation and neutrophil transendothelial migration (16). To our knowledge, the first concept that the 33-kDa AnxA1 form might have proinflammatory properties has been suggested by findings in fluid samples from cystic fibrosis patients (15). Recently, we demonstrated in a model of acute inflammation, greater accumulation of AnxA1 cleaved (33 kDa) during the peak of neutrophilic inflammation (22). Akin to these findings, and as reported above, CR-AnxA1 displays a greater anti-inflammatory effect over time compared with the parent protein (17) and accelerated resolution in an animal model of inflammatory arthritis (18).

Finally the current study tested a peptide derived from the AnxA1 N-terminal region, hence the active portion of the protein with respect to proresolving actions, mutated in its cleavage site, termed CR-AnxA1_2–50. This peptide is resistant to the action of elastase and PR3 and is more effective in inflammatory peritonitis and acute myocardial infarct (19). In analogy to SIV—which increased levels of intact AnxA1 in neutrophils—CR-AnxA1_2–50 was highly effective in reducing pleurisy. This peptide is also very potent with an active dose in the low nanomole range (19, 20). It has been shown that the inhibition of AnxA1 function by protein neutralization or the blockade of AnxA1 receptor are useful strategies to revert anti-inflammatory and proresolving effects mediated by AnxA1 in different inflammatory conditions (22, 25, 49, 58). In this study, we showed that the blockade of AnxA1 was able to reverse the resolution induced by SIV, suggesting that the proresolving effects of SIV are dependent on AnxA1. Collectively, these findings suggest that AnxA1 can be a mediator of the SIV proresolving actions, and modulation of endogenous pool of AnxA1 might be an important approach to impact on ongoing or recurrent inflammatory status by driving endogenous proresolution pathways and processes.

We thank Frankcinéia Assis and Ilma Marçal for technical assistance. We also thank Dr. Gustavo Menezes for providing the anti-neutrophil elastase.

The authors have no financial conflicts of interest.