Paola Maderna

Annexin-1 and Peptide Derivatives Are Released by Apoptotic Cells and Stimulate Phagocytosis of Apoptotic Neutrophils by Macrophages

The resolution of inflammation is a dynamically regulated process that may be subverted in many pathological conditions. Macrophage (Mφ) phagocytic clearance of apoptotic leukocytes plays an important role in the resolution of inflammation as this process prevents the exposure of tissues at the inflammatory site to the noxious contents of lytic cells. It is increasingly appreciated that endogenously produced mediators, such as lipoxins, act as potent regulators (nanomolar range) of the phagocytic clearance of apoptotic cells. In this study, we have investigated the intriguing possibility that apoptotic cells release signals that promote their clearance by phagocytes. We report that conditioned medium from apoptotic human polymorphonuclear neutrophils (PMN), Jurkat T lymphocytes, and human mesangial cells promote phagocytosis of apoptotic PMN by Mφ and THP-1 cells differentiated to a Mφ-like phenotype. This prophagocytic activity appears to be dose dependent, sensitive to the caspase inhibitor zVAD-fmk, and is associated with actin rearrangement and release of TGF-β1, but not IL-8. The prophagocytic effect can be blocked by the formyl peptide receptor antagonist Boc2, suggesting that the prophagocytic factor(s) may interact with the lipoxin A4 receptor, FPRL-1. Using nanoelectrospray liquid chromatography mass spectrometry and immunodepletion and immunoneutralization studies, we have ascertained that annexin-1 and peptide derivatives are putative prophagocytic factors released by apoptotic cells that promote phagocytosis of apoptotic PMN by M[phi] and differentiated THP-1 cells. These data highlight the role of annexin-1 and peptide derivatives in promoting the resolution of inflammation and expand on the therapeutic anti-inflammatory potential of annexin-1.

Modulation of Phagocytosis of Apoptotic Neutrophils by Supernatant from Dexamethasone-Treated Macrophages and Annexin-Derived Peptide Ac 2–26

Phagocytic clearance of apoptotic leukocytes plays an important role in the resolution of inflammation. The glucocorticoid-inducible protein annexin 1 and annexin 1-derived peptides show potent anti-inflammatory responses in acute and chronic inflammation. In this study, we report that the annexin 1-derived peptide (Ac2–26) significantly stimulates nonphlogistic phagocytosis of apoptotic polymorphonuclear leukocytes (PMNs) by human monocyte-derived macrophages (Mφ). Peptide Ac2–26-stimulated phagocytosis is accompanied by rearrangement of the Mφ actin cytoskeleton. To investigate the potential role of endogenous annexin on clearance of apoptotic cells, Mφ were cultured for 5 days in the presence of dexamethasone. Supernatants collected from dexamethasone-treated Mφ significantly enhanced the ability of naive Mφ to engulf apoptotic PMNs. This effect was blocked by an annexin blocking Ab, by immunodepletion of the supernatants, and by the formyl peptide receptor/lipoxin receptor antagonist Boc1. In addition, we show that bone marrow-derived Mφ from annexin 1-null mice present a 40% decreased phagocytosis of apoptotic PMNs compared with cells taken from littermate controls. In conclusion, these results emphasize the pivotal role of annexin 1 as mediator for clearance of apoptotic cells and expand its potential therapeutic role in controlling inflammatory diseases.

Lipoxins: resolutionary road

The resolution of inflammation is an active process controlled by endogenous mediators with selective actions on neutrophils and monocytes. The initial phase of the acute inflammatory response is characterized by the production of pro‐inflammatory mediators followed by a second phase in which lipid mediators with pro‐resolution activities may be generated. The identification of these mediators has provided evidence for the dynamic regulation of the resolution of inflammation. Among these endogenous local mediators of resolution, lipoxins (LXs), lipid mediators typically formed during cell–cell interaction, were the first to be recognized. More recently, families of endogenous chemical mediators, termed resolvins and protectins, were discovered. LXs and aspirin‐triggered LXs are considered to act as ‘braking signals’ in inflammation, limiting the trafficking of leukocytes to the inflammatory site. LXs are actively involved in the resolution of inflammation stimulating non‐phlogistic phagocytosis of apoptotic cells by macrophages. Furthermore, LXs have emerged as potential anti‐fibrotic mediators that may influence pro‐fibrotic cytokines and matrix‐associated gene expression in response to growth factors. Here, we provide a review and an update of the biosynthesis, metabolism and bioactions of LXs and LX analogues, and the recent studies on their therapeutic potential as promoters of resolution and fibro‐suppressants.

Platelet Activation Induces Cell-Surface Immunoreactive Tissue Factor Expression, Which Is Modulated Differently by Antiplatelet Drugs

Objective—Tissue factor (TF) is the main activator of the coagulation cascade occurring in physiologic and pathologic conditions. Recent data suggest that human platelets might contain TF that is possibly derived from leukocytes. In this study, we investigated whether intraplatelet TF can be exposed on the membrane by platelet agonists. The modulation of this process by antiplatelet drugs has been evaluated as well. Methods and Results—Flow cytometric analysis of unstimulated platelets showed a small amount of membrane-associated immunoreactive TF (irTF) in whole blood, platelet-rich plasma, and washed platelets isolated from healthy subjects. ADP, thrombin receptor-activating peptide, and epinephrine significantly increased functionally active, membrane-associated irTF. ADP induced irTF exposure in a concentration- and time-dependent fashion. Agonist-induced irTF expression was completely inhibited by iloprost but not by aspirin. Interestingly, glycoprotein IIb/IIIa antagonists did not inhibit but rather potentiated the stimulatory effect of ADP on platelet irTF expression. Real-time polymerase chain reaction experiments showed detectable amounts of TF mRNA in unstimulated platelets. Conclusions—These findings indicate that platelet agonists and antiplatelet drugs might modulate platelet-associated irTF expression. Regulated TF expression establishes the potential for a previously unrecognized role for platelets in sustaining thrombus formation and growth via coagulation-mediated mechanisms.

FPR2/ALX receptor expression and internalization are critical for lipoxin A4 and annexin-derived peptide-stimulated phagocytosis

Lipoxins (LXs) are endogenously produced eicosanoids with well‐described anti‐inflammatory and proresolution activities, stimulating nonphlogistic phagocytosis of apoptotic cells by macrophages. LXA4 and the glucocorticoid‐derived annexin A1 peptide (Ac2–26) bind to a common G‐proteincoupled receptor, termed FPR2/ALX. However, direct evidence of the involvement of FPR2/ALX in the anti‐inflammatory and proresolution activity of LXA4 is still to be investigated. Here we describe FPR2/ALX trafficking in response to LXA4 and Ac2–26 stimulation. We have transfected cells with HA‐tagged FPR2/ALX and studied receptor trafficking in unstimulated, LXA4 (1–10 nM)‐ and Ac2–26 (30 µM)‐treated cells using multiple approaches that include immunofluorescent confocal microscopy, immunogold labeling of cryosections, and ELISA and investigated receptor trafficking in agonist‐stimulated phagocytosis. We conclude that PKC‐dependent internalization of FPR2/ALX is required for phagocytosis. Using bone marrow‐derived macrophages (BMDMs) from mice in which the FPR2/ALX ortholog Fpr2 had been deleted, we observed the nonredundant function for this receptor in LXA4 and Ac2–26 stimulated phagocytosis of apoptotic neutrophils. LXA4 stimulated phagocytosis 1.7‐fold above basal (P< 0.001) by BMDMs from wild‐type mice, whereas no effect was found on BMDMs from Fpr2‐/‐ mice. Similarly, Ac2–26 stimulates phagocytosis by BMDMs from wild‐type mice 1.5‐fold above basal (P<0.05). However, Ac2–26 failed to stimulate phagocytosis by BMDMs isolated from Fpr2‐/‐ mice relative to vehicle. These data reveal novel and complex mechanisms of the FPR2/ALX receptor trafficking and functionality in the resolution of inflammation.—Maderna, P., Cottell, D. C., Toivonen, T., Dufton, N., Dalli, J., Perretti, M., Godson, C. FPR2/ALX receptor expression and internalization are critical for lipoxin A4 and annexinderived peptide‐stimulated phagocytosis. FASEB J. 24, 4240–4249 (2010). www.fasebj.org

Lipoxins Induce Actin Reorganization in Monocytes and Macrophages But Not in Neutrophils: Differential Involvement of Rho GTPases

Lipoxins (LXs) are endogenously produced eicosanoids that inhibit neutrophil trafficking and stimulate nonphlogistic phagocytosis of apoptotic neutrophils by monocyte-derived macrophages. In this study we assessed the effect of LXs on cell ultrastructure and actin reorganization in human leukocytes and investigated the signaling events that subserve LX bioactivity in this context. LXA(4) (10(-9) mol/L), the stable synthetic analogues 15-(R/S)-methyl-LXA(4) and 16-phenoxy-LXA(4) (10(-11) mol/L), but not the LX precursor 15-(S)-HETE, induced marked changes in ultrastructure and rearrangement of actin in monocytes and macrophages. In contrast, LXA(4) did not modify actin distribution in neutrophils under basal conditions and after stimulation with leukotriene B(4). Blockade of Rho kinases by the inhibitor Y-27632 prevented LXA(4)-triggered actin reorganization in macrophages. To investigate the role of the specific small GTPases in LX-induced actin rearrangement we used THP-1 cells differentiated to a macrophage-like phenotype. THP-1 cells stimulated with LXs, but not with 15-(S)-HETE, showed an increase in membrane-associated RhoA and Rac by immunoblotting. Additionally, a twofold increase in Rho activity was seen in response to LXA(4). LX-induced actin rearrangement and RhoA activation were inhibited by the cell permeable cAMP analogue 8-Br-cAMP, whereas Rp-cAMP, an inhibitor of protein kinase A, mimicked the effect of LXA(4). These data demonstrate that LXs stimulate RhoA- and Rac-dependent cytoskeleton reorganization, contributing to the potential role of LXs in the resolution of inflammation.

Lipoxin A4: Anti-Inflammatory and Anti-Angiogenic Impact on Endothelial Cells

Lipoxins (LX) are a class of eicosanoid that possesses a wide spectrum of antiinflammatory and proresolution bioactions. Here we have investigated the impact of the endogenously produced eicosanoid LXA4 on endothelial cell inflammatory, proliferative, and antigenic responses. Using HUVECs we demonstrate that LXA4 inhibits vascular endothelial growth factor (VEGF)-stimulated inflammatory responses including IL-6, TNF-α, IFN-γ and IL-8 secretion, as well as endothelial ICAM-1 expression. Interestingly, LXA4 up-regulated IL-10 production from HUVECs. Consistent with these antiinflammatory and proresolution responses to LXA4, we demonstrate that LXA4 inhibited leukotriene D4 and VEGF-stimulated proliferation and angiogenesis as determined by tube formation of HUVECs. We have explored the underlying molecular mechanisms and demonstrate that LXA4 pretreatment is associated with the decrease of VEGF-stimulated VEGF receptor 2 (KDR/FLK-1) phosphorylation and downstream signaling events including activation of phospholipase C-γ, ERK1/2, and Akt.

Lipoxin A4 attenuates adipose inflammation

Aging and adiposity are associated with chronic low‐grade inflammation, which underlies the development of obesity‐associated complications, including type 2 diabetes mellitus (T2DM). The mechanisms underlying adipose inflammation may include macrophage infiltration and activation, which, in turn, affect insulin sensitivity of adipocytes. There is a growing appreciation that specific lipid mediators (including lipoxins, resolvins, and protectins) can promote the resolution of inflammation. Here, we investigated the effect of lipoxin A4 (LXA4), the predominant endogenously generated lipoxin, on adipose tissue inflammation. Using adipose tissue explants from perigonadal depots of aging female C57BL/6J mice (Animalia, Chordata, Mus musculus) as a model of age‐associated adipose inflammation, we report that LXA4 (1 nM) attenuates adipose inflammation, decreasing IL‐6 and increasing IL‐10 expression (P<0.05). The altered cytokine milieu correlated with increased GLUT‐4 and IRS‐1 expression, suggesting improved insulin sensitivity. Further investigations revealed the ability of LXA4 to rescue macrophage‐induced desensitization to insulin‐stimulated signaling and glucose uptake in cultured adipocytes, using vehicle‐stimulated cells as controls. This was associated with preservation of Akt activation and reduced secretion of proinflammatory cytokines, including TNF‐α. We therefore propose that LXA4 may represent a potentially useful and novel therapeutic strategy to subvert adipose inflammation and insulin resistance, key components of T2DM.—Börgeson, E., McGillicuddy, F. C., Harford, K. A., Corrigan, N., Higgins, D. F., Maderna, P., Roche, H. M., Godson C. Lipoxin A4 attenuates adipose inflammation. FASEB J. 26, 4287–4294 (2012). www.fasebj.org

Acute Serum Amyloid A Induces Migration, Angiogenesis, and Inflammation in Synovial Cells In Vitro and in a Human Rheumatoid Arthritis/SCID Mouse Chimera Model

Serum amyloid A (A-SAA), an acute-phase protein with cytokine-like properties, is expressed at sites of inflammation. This study investigated the effects of A-SAA on chemokine-regulated migration and angiogenesis using rheumatoid arthritis (RA) cells and whole-tissue explants in vitro, ex vivo, and in vivo. A-SAA levels were measured by real-time PCR and ELISA. IL-8 and MCP-1 expression was examined in RA synovial fibroblasts, human microvascular endothelial cells, and RA synovial explants by ELISA. Neutrophil transendothelial cell migration, cell adhesion, invasion, and migration were examined using transwell leukocyte/monocyte migration assays, invasion assays, and adhesion assays with or without anti–MCP-1/anti–IL-8. NF-κB was examined using a specific inhibitor and Western blotting. An RA synovial/SCID mouse chimera model was used to examine the effects of A-SAA on cell migration, proliferation, and angiogenesis in vivo. High expression of A-SAA was demonstrated in RA patients (p < 0.05). A-SAA induced chemokine expression in a time- and dose-dependent manner (p < 0.05). Blockade with anti-scavenger receptor class B member 1 and lipoxin A4 (A-SAA receptors) significantly reduced chemokine expression in RA synovial tissue explants (p < 0.05). A-SAA induced cell invasion, neutrophil–transendothelial cell migration, monocyte migration, and adhesion (all p < 0.05), effects that were blocked by anti–IL-8 or anti–MCP-1. A-SAA–induced chemokine expression was mediated through NF-κB in RA explants (p < 0.05). Finally, in the RA synovial/SCID mouse chimera model, we demonstrated for the first time in vivo that A-SAA directly induces monocyte migration from the murine circulation into RA synovial grafts, synovial cell proliferation, and angiogenesis (p < 0.05). A-SAA promotes cell migrational mechanisms and angiogenesis critical to RA pathogenesis.

Lipoxins and Annexin-1: Resolution of Inflammation and Regulation of Phagocytosis of Apoptotic Cells

Phagocytosis of apoptotic cells plays a pivotal role in developmental processes and in the resolution of inflammation. Failed or delayed clearance of apoptotic cells can result in chronic inflammation. Furthermore, clearance of apoptotic cells leads to release of anti-inflammatory cytokines. Recent evidence has shown that endogenous mediators can regulate such processes. In this article, we will review the recognition and signaling mechanisms involved in the phagocytosis of apoptotic cells as well as the role of endogenous compounds that play a relevant role in the modulation of inflammation. The first of these endogenous local mediators to be described are lipoxins (LXs). LXs and aspirin-triggered LXs (ATLs) are considered to act as “braking signals” in inflammation, limiting the entrance of leukocytes to the site of inflammation through inhibition of neutrophil and eosinophil trafficking. LXs are actively involved in resolution of inflammation, stimulating nonphlogistic phagocytosis of apoptotic cells by macrophages. LXA4 and ATLs elicit cellular responses by interacting with a G protein -coupled receptor (ALXR) that is expressed in various cell types. ALXR, originally identified as a low-affinity N-formyl-methionyl-leucyl-phenylalanine receptor-like 1, can bind pleiotropic ligands, i.e., both lipid and peptides, including the glucocorticoid-inducible protein, annexin-1. Interestingly, a role for annexin-1 in phagocytosis has recently emerged. Understanding the role and mechanism of the powerful anti-inflammatory and proresolution actions of endogenous compounds can be a useful tool in the development of potential therapeutics in resolving inflammatory diseases.