Taisuke Ohira

Resolvin E1 selectively interacts with leukotriene B4 receptor BLT1 and ChemR23 to regulate inflammation

Resolvin E1 (RvE1) is a potent anti-inflammatory and proresolving mediator derived from omega-3 eicosapentaenoic acid generated during the resolution phase of inflammation. RvE1 possesses a unique structure and counterregulatory actions that stop human polymorphonuclear leukocyte (PMN) transendothelial migration and PMN infiltration in several murine inflammatory models. To examine the mechanism(s) underlying anti-inflammatory actions on PMNs, we prepared [3H]RvE1 and characterized its interactions with human PMN. Results with membrane fractions of human PMN demonstrated specific binding with a Kd of 48.3 nM. [3H]RvE1 specific binding to human PMN was displaced by leukotriene B4 (LTB4) and LTB4 receptor 1 (BLT1) antagonist U-75302, but not by chemerin peptide, a ligand specific for another RvE1 receptor ChemR23. Recombinant human BLT1 gave specific binding with [3H]RvE1 with a Kd of 45 nM. RvE1 selectively inhibited adenylate cyclase with BLT1, but not with BLT2. In human PBMC, RvE1 partially induced calcium mobilization, and blocked subsequent stimulation by LTB4. RvE1 also attenuated LTB4-induced NF-κB activation in BLT1-transfected cells. In vivo anti-inflammatory actions of RvE1 were sharply reduced in BLT1 knockout mice when given at low doses (100 ng i.v.) in peritonitis. In contrast, RvE1 at higher doses (1.0 μg i.v.) significantly reduced PMN infiltration in a BLT1-independent manner. These results indicate that RvE1 binds to BLT1 as a partial agonist, potentially serving as a local damper of BLT1 signals on leukocytes along with other receptors (e.g., ChemR23-mediated counterregulatory actions) to mediate the resolution of inflammation.

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.

Resolvin E1 Receptor Activation Signals Phosphorylation and Phagocytosis

Resolvins are endogenous lipid mediators that actively regulate the resolution of acute inflammation. Resolvin E1 (RvE1; (5S,12R,18R)-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid) is an endogenous anti-inflammatory and pro-resolving mediator derived from eicosapentaenoic acid that regulates leukocyte migration and enhances macrophage phagocytosis of apoptotic neutrophils to resolve inflammation. In the inflammatory milieu, RvE1 mediates counter-regulatory actions initiated via specific G protein-coupled receptors. Here, we have identified RvE1-specific signaling pathways initiated by the RvE1 receptor ChemR23. RvE1 stimulated phosphorylation of Akt that was both ligand- and receptor-dependent. RvE1 regulated Akt phosphorylation in a time (0–15 min)- and dose-dependent (0.01–100 nm) manner in human ChemR23-transfected Chinese hamster ovary cells. RvE1 stimulated phosphorylation of both Akt and a 30-kDa protein, a downstream target of Akt, identified using a phospho-Akt substrate antibody. The 30-kDa protein was identified as ribosomal protein S6, a translational regulator, and its phosphorylation was inhibited by a phosphatidylinositol 3-kinase (PI3K) inhibitor (wortmannin) and an ERK inhibitor (PD98059) but not by a p38-MAPK inhibitor (SB203580). Ribosomal protein S6 is a downstream target of the PI3K/Akt signaling pathway as well as the Raf/ERK pathway. In ChemR23-expressing differentiated HL60 cells, RvE1 also stimulated the phosphorylation of ribosomal protein S6. In addition, RvE1 enhanced phagocytosis of zymosan A by human macrophages, which are inhibited by PD98059 and rapamycin (mTOR inhibitor). These results indicate that RvE1 initiates direct activation of ChemR23 and signals receptor-dependent phosphorylation. These phosphorylation-signaling pathways identified for RvE1 receptor-ligand interactions underscore the importance of endogenous pro-resolving agonists in resolving acute inflammation.

Enhanced superoxide release and elevated protein kinase C activity in neutrophils from diabetic patients: association with periodontitis

Inflammation and oxidative stress are important factors in the pathogenesis of diabetes and contribute to the pathogenesis of diabetic complications. Periodontitis is an inflammatory disease that is characterized by increased oxidative stress, and the risk for periodontitis is increased significantly in diabetic subjects. In this study, we examined the superoxide (O2−)‐generating reduced nicotinamide adenine dinucleotide phosphate‐oxidase complex and protein kinase C (PKC) activity in neutrophils. Fifty diabetic patients were grouped according to glycemic control and the severity of periodontitis. Neutrophils from diabetic patients with moderate [amount of glycated hemoglobin (HbA1c) between 7.0% and 8.0%] or poor (HbA1c >8.0%) glycemic control released significantly more O2− than neutrophils from diabetic patients with good glycemic control (HbA1c <7.0%) and neutrophils from nondiabetic, healthy individuals upon stimulation with 4β‐phorbol 12‐myristate 13‐acetate or N‐formyl‐Met‐Leu‐Phe. Depending on glycemic status, neutrophils from these patients also exhibited increased activity of the soluble‐ and membrane‐bound forms of PKC, elevated amounts of diglyceride, and enhanced phosphorylation of p47‐phox during cell stimulation. In addition, we report a significant correlation between glycemic control (HbA1c levels) and the severity of periodontitis in diabetic patients, suggesting that enhanced oxidative stress and increased inflammation exacerbate both diseases. Thus, hyperglycemia can lead to a novel form of neutrophil priming, where elevated PKC activity results in increased phosphorylation of p47‐phox and O2− release.

Priming of neutrophil oxidative burst in diabetes requires preassembly of the NADPH oxidase.

Hyperglycemia associated with diabetes mellitus results in the priming of neutrophils leading to oxidative stress that is, in part, responsible for diabetic complications. p47phox, a NADPH oxidase cytosolic subunit, is a key protein in the assembly of the NADPH oxidase leading to superoxide generation. Little is known about the priming mechanism of oxidative pathways in neutrophils of people with diabetes. In this study, the kinetics of p47phox activation was investigated by comparing neutrophils from diabetic and healthy subjects, and the mechanism of hyperglycemia‐induced changes was studied by using neutrophil‐like HL‐60 cells as a model. In resting neutrophils from diabetic subjects, p47phox prematurely translocates to the cell membrane and preassembles with p22phox, a NADPH oxidase membrane subunit. This premature p47phox translocation and preassembly with p22phox were also observed in HL‐60 cells cultured with high glucose (HG; 25 mM) and with the specific ligand for the receptor for advanced glycation end products (RAGE), S100B. Phosphorylation of ERK1/2, but not p38 MAPK, was the primary signaling pathway, as evidenced by PD98059 suppressing the translocation of p47phox in HL‐60 cells incubated with HG and S100B. HL‐60 cells cultured in HG and S100B exhibited a 1.8‐fold increase in fMLP‐induced superoxide generation compared with those cultured in normal glucose (5.5 mM). These data suggest that HG and increased AGE prime neutrophils and increase oxidative stress inducing the translocation of p47phox to the cell membrane and preassembly with p22phox by stimulating a RAGE‐ERK1/2 pathway.

An endogenous regulator of inflammation, resolvin E1, modulates osteoclast differentiation and bone resorption

The inflammation‐resolving lipid mediator resolvin E1 (RvE1) effectively stops inflammation‐induced bone loss in vivo in experimental periodontitis. It was of interest to determine whether RvE1 has direct actions on osteoclast (OC) development and bone resorption.

A Stable Aspirin-Triggered Lipoxin A4 Analog Blocks Phosphorylation of Leukocyte-Specific Protein 1 in Human Neutrophils

Lipoxins and their aspirin-triggered 15-epimers are endogenous anti-inflammatory agents that block neutrophil chemotaxis in vitro and inhibit neutrophil influx in several models of acute inflammation. In this study, we examined the effects of 15-epi-16-(p-fluoro)-phenoxy-lipoxin A4 methyl ester, an aspirin-triggered lipoxin A4-stable analog (ATLa), on the protein phosphorylation pattern of human neutrophils. Neutrophils stimulated with the chemoattractant fMLP were found to exhibit intense phosphorylation of a 55-kDa protein that was blocked by ATLa (10–50 nM). This 55-kDa protein was identified as leukocyte-specific protein 1, a downstream component of the p38-MAPK cascade in neutrophils, by mass spectrometry, Western blotting, and immunoprecipitation experiments. ATLa (50 nM) also reduced phosphorylation/activation of several components of the p38-MAPK pathway in these cells (MAPK kinase 3/MAPK kinase 6, p38-MAPK, MAPK-activated protein kinase-2). These results indicate that ATLa exerts its anti-inflammatory effects, at least in part, by blocking activation of the p38-MAPK cascade in neutrophils, which is known to promote chemotaxis and other proinflammatory responses by these cells.

Regulation of the NADPH-oxidase complex of phagocytic leukocytes. Recent insights from structural biology, molecular genetics, and microscopy.

The NADPH-oxidase complex is a multisubunit enzyme complex that catalyzes the formation of superoxide (O2−) by phagocytic leukocytes. This paper reviews some of the major advances in understanding the assembly and regulation of this enzyme system that have occurred during the past decade. For example, novel domains/motifs have been identified in p47-phox (PX and super SH3 domains) and p67-phox (tetratricopeptide repeat motifs). X-ray crystallography and NMR spectroscopy have provided detailed structural data on these domains and how p47-phox and p67-phox interact with p22-phox and activated Rac, respectively. Site-directed mutagenesis and knockout experiments have identified the critical phosphorylation sites in p47-phox, revealed an “activation domain” in p67-phox, and demonstrated that a specific pathway exists for activating Rac to participate in oxidase assembly/activation. Cytochemistry and immunofluorescence microscopy have provided new insights into the assembly of the oxidase and reveal a level of complexity not previously appreciated.

p21-Activated Kinase 2 in Neutrophils Can Be Regulated by Phosphorylation at Multiple Sites and by a Variety of Protein Phosphatases

The p21-activated kinase(Pak) 2 undergoes rapid autophosphorylation/activation in neutrophils stimulated with a variety of chemoattractants (e.g., fMLP). Phosphorylation within the activation loop (Thr402) and inhibitory domain (Ser141) is known to increase the activity of Pak in vitro, whereas phosphorylation within the Nck (Ser20) and Pak-interacting guanine nucleotide exchange factor (Ser192 and Ser197) binding sites blocks the interactions of Pak 2 with these proteins. A panel of phosphospecific Abs was used to investigate the phosphorylation of Pak 2 in neutrophils at these sites. Pak 2 underwent rapid (≤15 s) phosphorylation at Ser20, Ser192/197, and Thr402 in neutrophils stimulated with fMLP. Phosphorylation at Ser192/197 and Thr402 were highly transient events, whereas that at Ser20 was more persistent. In contrast, Pak 2 was constitutively phosphorylated at Ser141 in unstimulated neutrophils and phosphorylation at this site was less sensitive to cell stimulation than at other residues. Studies with selective inhibitors suggested that a variety of phosphatases might be involved in the rapid dephosphorylation of Pak 2 at Thr402 in stimulated neutrophils. This was consistent with biochemical studies which showed that the activation loop of GST-Pak 3, which is homologous to that in Pak 2, was a substrate for protein phosphatase 1, 2A, and a Mg2+/Mn2+-dependent phosphatase(s) which exhibited properties different from those of the conventional isoforms of protein phosphatase 2C. The data indicate that Pak 2 undergoes a complex pattern of phosphorylation in neutrophils and that dephosphorylation at certain sites may involve multiple protein phosphatases that exhibit distinct modes of regulation.

Protein phosphorylation in neutrophils monitored with phosphospecific antibodies.

Protein phosphorylation in neutrophils was monitored with two phosphospecific antibodies (pAbs) [termed pPKC(S1) Ab and pPKC(S2) Ab] that recognize products of protein kinase C (PKC) and other Arg/Lys-directed Ser/Thr protein kinases. The pPKC(S1) Ab bound preferentially to p-Ser/p-Thr residues with Arg or Lys in the -3 and -5 positions or the -2 and -3 positions, whereas the pPKC(S2) Ab bound preferentially to p-Ser with Arg or Lys in the -2 and +2 positions and with a hydrophobic residue at the +1 position. Phosphorylated pleckstrin, myristoylated alanine-rich C-kinase substrate (MARCKS), the 47-kDa subunit of the phagocyte oxidase (p47-phox) and numerous unidentified proteins that underwent phosphorylation during neutrophil stimulation were readily detected with these pAbs. Priming effects of tumor necrosis factor alpha (TNF-alpha) and the susceptibility of certain reactions in neutrophils to inhibitors of protein kinases could also be easily investigated with these reagents. Compared to the commonly used 32P-labeling/autoradiographic method, Western blotting with pAbs was found to be a faster, safer, more specific and in many cases more sensitive approach for monitoring protein phosphorylation events in neutrophils. These pAbs may facilitate the identification of several new phosphorylation reactions involved in neutrophil stimulation.