Nicos A. Petasis

Cutting Edge: Lipoxins Rapidly Stimulate Nonphlogistic Phagocytosis of Apoptotic Neutrophils by Monocyte-Derived Macrophages

Lipoxins (LX) are lipoxygenase-derived eicosanoids generated during inflammation. LX inhibit polymorphonuclear neutrophil (PMN) chemotaxis and adhesion and are putative braking signals for PMN-mediated tissue injury. In this study, we report that LXA4 promotes another important step in the resolution phase of inflammation, namely, phagocytosis of apoptotic PMN by monocyte-derived macrophages (Mφ). LXA4 triggered rapid, concentration-dependent uptake of apoptotic PMN. This bioactivity was shared by stable synthetic LXA4 analogues (picomolar concentrations) but not by other eicosanoids tested. LXA4-triggered phagocytosis did not provoke IL-8 or monocyte chemoattractant protein-1 release. LXA4-induced phagocytosis was attenuated by anti-CD36, αvβ3, and CD18 mAbs. LXA4-triggered PMN uptake was inhibited by pertussis toxin and by 8-bromo-cAMP and was mimicked by Rp-cAMP, a protein kinase A inhibitor. LXA4 attenuated PGE2-stimulated protein kinase A activation in Mφ. These results suggest that LXA4 is an endogenous stimulus for PMN clearance during inflammation and provide a novel rationale for using stable synthetic analogues as anti-inflammatory compounds in vivo.

Resolvins and Protectins in Inflammation-Resolution

The critical role of inflammatory processes in health and disease has long been recognized,1 yet the detailed molecular mechanisms and biological events that regulate the progression and resolution of inflammation remain of interest. A number of recent investigations have provided strong evidence that the resolution of inflammation is not a passive process, as believed earlier.2–4 Instead, resolution is a biosynthetically active process, regulated by biochemical mediators and receptor-signaling pathways, and driven by specialized pro-resolving mediators (SPM). In particular, following a number of findings by Serhan and his group, the authors and their collaborators introduced and systematically investigated a number of SPM derived from polyunsaturated fatty acids (PUFA), including lipoxins, E-series resolvins, D-series resolvins, protectins/neuroprotectins, and, most recently, maresins. This review summarizes efforts on the resolvins and protectins with an emphasis on the corresponding biochemical pathways. Additional reviews covering different aspects of these anti-inflammatory and pro-resolving lipid mediators,5 including immunology,6,7 pathology,8 biochemistry,9 pharmacology10 and chemistry11 are also available.6–9,12 1.1 The Inflammatory Response and the Resolution of Inflammation Localized acute inflammation is part of the host’s normal protective response to tissue injury and infection by invading microbial pathogens.1 Although this inflammatory response to a range of harmful stimuli is protective to the host, if kept uncontrolled it can result in a wide range of acute, chronic and systemic inflammatory disorders. Indeed, some of the most common and difficult to treat diseases are linked to excessive, uncontrollable, or chronic inflammation, including: cardiovascular disease, rheumatoid arthritis, periodontal disease, asthma, diabetes, inflammatory bowel disease (IBD), as well as neurological disorders such as Alzheimer’s disease and age-related macular degeneration (AMD).1,13,14 Although the involvement of inflammatory pathways in the initiation of all of these diseases is well established, the specific role by which inflammation contributes to their pathogenesis is not fully understood. The recent findings that the resolution of inflammation is an active process2–4,15 have provided new insights and created new paradigms for understanding and treating these conditions. The key role of a number of lipid mediators in the initiation of the inflammatory response and the subsequent progression towards resolution is illustrated in Fig. 1. Among the first signaling events following microbial infection or tissue injury, is the release of pro-inflammatory lipid mediators, such as leukotrienes (LTs) and prostaglandins (PGs) that launch a series of signaling cascades with the ultimate goal of destroying the invading pathogens and repairing the damaged tissue.16–18 Thus, the biosynthesis and release of the potent chemotactic agent leukotriene B4 (LTB4) promotes the recruitment of neutrophils (PMNs) to the inflamed tissue, while the formation of prostaglandins E2 and D2 (PGE2 and PGD2)15 further accelerates the inflammatory process, ultimately resulting in a condition of acute inflammation. Despite its critical host-protective function, acute inflammation is not sustainable over a prolonged period of time, giving rise to disruptive conditions of chronic inflammation that may be responsible for the pathogenesis of a wide range of diseases, that can be attributed to a failure of resolution.19 Typically, the therapeutic treatment of such conditions involves the inhibition of pro-inflammatory mediators, but in many cases such approaches are often not very effective. Figure 1 From initiation of acute inflammation to resolution: The inflammatory response to microbial infection and tissue injury, and the role of selected cell types and specialized pro-resolving lipid mediators. The recognition of the proactive nature of the resolution of inflammation has revealed alternative therapeutic paradigms based on resolving acute inflammation and preventing the onset of chronic inflammation.19 Indeed, a number of endogenous lipid mediators identified are able to act in this manner, suggesting a lipid mediator class switching3 from the initial actions of pro-inflammatory lipid mediators, i.e. leukotriene and prostaglandins, to the anti-inflammatory and pro-resolving actions of lipoxins, resolvins, protectins and maresins, which are discussed in this review. Each family of these pro-resolving mediators exert specialized actions, including blocking neutrophil recruitment, promoting the recruitment and activation of monocytes, as well as mediating the nonphlogistic phagocytosis and lymphatic clearance of apoptotic neutrophils by activated macrophages. Eventually, through the combined actions of these mediators, the resolution of inflammation is completed and homeostasis is reached. In this regard, it was important to introduce precise definitions for resolution mechanisms and indices19–21 that also permitted the first information that certain agents can be resolution-toxic.22–24

Resolvin D2 is a potent regulator of leukocytes and controls microbial sepsis

A growing body of evidence indicates that resolution of acute inflammation is an active process. Resolvins are a new family of lipid mediators enzymatically generated within resolution networks that possess unique and specific functions to orchestrate catabasis, the phase in which disease declines. Resolvin D2 (RvD2) was originally identified in resolving exudates, yet its individual contribution in resolution remained to be elucidated. Here, we establish RvD2’s potent stereoselective actions in reducing excessive neutrophil trafficking to inflammatory loci. RvD2 decreased leukocyte–endothelial interactions in vivo by endothelial-dependent nitric oxide production, and by direct modulation of leukocyte adhesion receptor expression. In mice with microbial sepsis initiated by caecal ligation and puncture, RvD2 sharply decreased both local and systemic bacterial burden, excessive cytokine production and neutrophil recruitment, while increasing peritoneal mononuclear cells and macrophage phagocytosis. These multi-level pro-resolving actions of RvD2 translate to increased survival from sepsis induced by caecal ligation and puncture and surgery. Together, these results identify RvD2 as a potent endogenous regulator of excessive inflammatory responses that acts via multiple cellular targets to stimulate resolution and preserve immune vigilance.

Resolvin D1 binds human phagocytes with evidence for proresolving receptors

Endogenous mechanisms that act in the resolution of acute inflammation are essential for host defense and the return to homeostasis. Resolvin D1 (RvD1), biosynthesized during resolution, displays potent and stereoselective anti-inflammatory actions, such as limiting neutrophil infiltration and proresolving actions. Here, we demonstrate that RvD1 actions on human polymorphonuclear leukocytes (PMNs) are pertussis toxin sensitive, decrease actin polymerization, and block LTB4-regulated adhesion molecules (β2 integrins). Synthetic [3H]-RvD1 was prepared, which revealed specific RvD1 recognition sites on human leukocytes. Screening systems to identify receptors for RvD1 gave two candidates—ALX, a lipoxin A4 receptor, and GPR32, an orphan—that were confirmed using a β-arrestin-based ligand receptor system. Nuclear receptors including retinoid X receptor-α and peroxisome proliferator-activated receptor-α, -δ, -γ were not activated by either resolvin E1 or RvD1 at bioactive nanomolar concentrations. RvD1 enhanced macrophage phagocytosis of zymosan and apoptotic PMNs, which increased with overexpression of human ALX and GPR32 and decreased with selective knockdown of these G-protein-coupled receptors. Also, ALX and GPR32 surface expression in human monocytes was up-regulated by zymosan and granulocyte-monocyte–colony-stimulating factor. These results indicate that RvD1 specifically interacts with both ALX and GPR32 on phagocytes and suggest that each plays a role in resolving acute inflammation.

Anti-Inflammatory Actions of Neuroprotectin D1/Protectin D1 and Its Natural Stereoisomers: Assignments of Dihydroxy-Containing Docosatrienes

Protectin D1, neuroprotectin D1 when generated by neural cells, is a member of a new family of bioactive products generated from docosahexaenoic acid. The complete stereochemistry of protectin D1 (10,17S-docosatriene), namely, chirality of the carbon-10 alcohol and geometry of the conjugated triene, required for bioactivity remained to be assigned. To this end, protectin D1/neuroprotectin D1 (PD1) generated by human neutrophils during murine peritonitis and by neural tissues was separated from natural isomers and subjected to liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry. Comparisons with six 10,17-dihydroxydocosatrienes prepared by total organic and biogenic synthesis showed that PD1 from human cells carrying potent bioactivity is 10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid. Additional isomers identified included trace amounts of Δ15-trans-PD1 (isomer III), 10S,17S-dihydroxy-docosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid (isomer IV), and a double dioxygenation product 10S,17S-dihydroxy-docosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid (isomer I), present in exudates. 18O2 labeling showed that 10S,17S-diHDHA (isomer I) carried 18O in the carbon-10 position alcohol, indicating sequential lipoxygenation, whereas PD1 formation proceeded via an epoxide. PD1 at 10 nM attenuated (∼50%) human neutrophil transmigration, whereas Δ15-trans-PD1 was essentially inactive. PD1 was a potent regulator of polymorphonuclear leukocyte (PMN) infiltration (∼40% at 1 ng/mouse) in peritonitis. The rank order at 1- to 10-ng dose was PD1 ≈ PD1 methyl ester ≫ Δ15-trans-PD1 > 10S,17S-diHDHA (isomer I). 10S,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid (isomer VI) proved ≥ PD1 in blocking PMN infiltration, but was not a major product of leukocytes. PD1 also reduced PMN infiltration after initiation (2 h) of inflammation and was additive with resolvin E1. These results indicate that PD1 is a potent stereoselective anti-inflammatory molecule.

Resolvin D1 and Its Aspirin-triggered 17R Epimer STEREOCHEMICAL ASSIGNMENTS, ANTI-INFLAMMATORY PROPERTIES, AND ENZYMATIC INACTIVATION

We recently uncovered two new families of potent docosahexaenoic acid-derived mediators, termed D series resolvins (Rv; resolution phase interaction products) and protectins. Here, we assign the stereochemistry of the conjugated double bonds and chirality of alcohols present in resolvin D1 (RvD1) and its aspirin-triggered 17R epimer (AT-RvD1) with compounds prepared by total organic synthesis. In addition, docosahexaenoic acid was converted by a single lipoxygenase in a “one-pot” reaction to RvD1 in vitro. The synthetic compounds matched the physical and biological properties of those enzymatically generated. RvD1 proved to be 7S,8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid, AT-RvD1 matched 7S,8R,17R-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid, and they both stopped transendothelial migration of human neutrophils (EC50 ∼30 nm). In murine peritonitis in vivo, RvD1 and AT-RvD1 proved equipotent (at nanogram dosages), limiting polymorphonuclear leukocyte infiltration in a dose-dependent fashion. RvD1 was converted by eicosanoid oxidoreductase to novel 8-oxo- and 17-oxo-RvD1 that gave dramatically reduced bioactivity, whereas enzymatic conversion of AT-RvD1 was sharply reduced. These results establish the complete stereochemistry and actions of RvD1 and AT-RvD1 as well as demonstrate the stereoselective basis for their enzymatic inactivation. RvD1 regulates human polymorphonuclear leukocyte transendothelial migration and is anti-inflammatory. When its carbon 17S alcohol is enzymatically converted to 17-oxo-RvD1, it is essentially inactive, whereas the 17R alcohol configuration in its aspirin-triggered form (AT-RvD1) resists rapid inactivation. These results may contribute to the beneficial actions of aspirin and ω-3 fish oils in humans.

Reduced Inflammation and Tissue Damage in Transgenic Rabbits Overexpressing 15-Lipoxygenase and Endogenous Anti-inflammatory Lipid Mediators

PGs and leukotrienes (LTs) mediate cardinal signs of inflammation; hence, their enzymes are targets of current anti-inflammatory therapies. Products of arachidonate 15-lipoxygenases (LO) types I and II display both beneficial roles, such as lipoxins (LXs) that stereoselectively signal counterregulation, as well as potential deleterious actions (i.e., nonspecific phospholipid degradation). In this study, we examined transgenic (TG) rabbits overexpressing 15-LO type I and their response to inflammatory challenge. Skin challenges with either LTB4 or IL-8 showed that 15-LO TG rabbits give markedly reduced neutrophil (PMN) recruitment and plasma leakage at dermal sites with LTB4. PMN from TG rabbits also exhibited a dramatic reduction in LTB4-stimulated granular mobilization that was not evident with peptide chemoattractants. Leukocytes from 15-LO TG rabbits gave enhanced LX production, underscoring differences in lipid mediator profiles compared with non-TG rabbits. Microbe-associated inflammation and leukocyte-mediated bone destruction were assessed by initiating acute periodontitis. 15-LO TG rabbits exhibited markedly reduced bone loss and local inflammation. Because enhanced LX production was associated with an increased anti-inflammatory status of 15-LO TG rabbits, a stable analog of 5S,6R,15S-trihydroxyeicosa-7E,9E,11Z,13E-tetraenoic acid (LXA4) was applied to the gingival crevice subject to periodontitis. Topical application with the 15-epi-16-phenoxy-para-fluoro-LXA4 stable analog (ATLa) dramatically reduced leukocyte infiltration, ensuing bone loss as well as inflammation. These results indicate that overexpression of 15-LO type I and LXA4 is associated with dampened PMN-mediated tissue degradation and bone loss, suggesting that enhanced anti-inflammation status is an active process. Moreover, they suggest that LXs can be targets for novel approaches to diseases, e.g., periodontitis and arthritis, where inflammation and bone destruction are features.

RvE1 protects from local inflammation and osteoclast- mediated bone destruction in periodontitis

Periodontitis is a well‐appreciated example of leukocyte‐mediated bone loss and inflammation that has pathogenic features similar to those observed in other inflammatory diseases such as arthritis. Resolvins are a new family of bioactive products of omega‐3 fatty acid transformation circuits initiated by aspirin treatment that counter proinflammatory signals. Because it is now increasingly apparent that local inflammation plays a critical role in many diseases, including cardiovascular disease, atherosclerosis, and asthma, experiments were undertaken to evaluate the actions of the newly described EPA‐derived Resolvin E1 (RvE1) in regulation of neutrophil tissue destruction and resolution of inflammation. The actions of an aspirin‐triggered lipoxin (LX) analog and RvE1 in a human disease, localized aggressive periodontitis (LAP), were determined. Results indicate that neutrophils from LAP are refractory to anti‐inflammatory molecules of the LX series, whereas LAP neutrophils respond to RvE1. In addition, RvE1 specifically binds to human neutrophils at a site that is functionally distinct from the LX receptor. Consistent with these potent actions, topical application of RvE1 in rabbit periodontitis conferred dramatic protection against inflammation induced tissue and bone loss associated with periodontitis.

Defective lipoxin-mediated anti-inflammatory activity in the cystic fibrosis airway.

In cystic fibrosis, dysregulated neutrophilic inflammation and chronic infection lead to progressive destruction of the airways. The underlying mechanisms have remained unclear. Lipoxins are anti-inflammatory lipid mediators that modulate neutrophilic inflammation. We report here that lipoxin concentrations in airway fluid were significantly suppressed in patients with cystic fibrosis compared to patients with other inflammatory lung conditions. We also show that administration of a metabolically stable lipoxin analog in a mouse model of the chronic airway inflammation and infection associated with cystic fibrosis suppressed neutrophilic inflammation, decreased pulmonary bacterial burden and attenuated disease severity. These findings suggest that there is a pathophysiologically important defect in lipoxin-mediated anti-inflammatory activity in the cystic fibrosis lung and that lipoxins have therapeutic potential in this lethal autosomal disease.

The boronic acid Mannich reaction : a new method for the synthesis of geometrically pure allylamines

Abstract Reaction of vinyl boronic acids with the adducts of secondary amines and paraformaldehyde gives tertiary allylamines with the same geometry. This simple and practical method was used for the synthesis of geometrically pure naftifine, a potent antifungal agent.