G2A Protects Mice against Sepsis by Modulating Kupffer Cell Activation: Cooperativity with Adenosine Receptor 2b.

G2A is a GPCR abundantly expressed in immune cells. G2A-/- mice showed higher lethality, higher plasma cytokines, and an impaired bacterial clearance in response to a murine model of sepsis (cecal ligation and puncture), which were blocked by GdCl3, an inhibitor of Kupffer cells. Anti-IL-10 Ab reversed the impaired bacterial clearance in G2A-/- mice. Indomethacin effectively blocked both the increased i.p. IL-10 levels and the impaired bacterial clearance, indicating that disturbed PG system is the proximal cause of these phenomena. Stimulation with LPS/C5a induced an increase in Escherichia coli phagocytosis and intracellular cAMP levels in G2A+/+ peritoneal macrophages but not G2A-/- cells, which showed more PGE2/nitrite release and intracellular reactive oxygen species levels. Heterologous coexpression of G2A and adenosine receptor type 2b (A2bAR) induced a synergistic increase in cAMP signaling in a ligand-independent manner, with the evidence of physical interaction of G2A with A2bAR. BAY 60-6583, a specific agonist for A2bAR, increased intracellular cAMP levels in Kupffer cells from G2A+/+ but not from G2A-/- mice. Both G2A and A2bAR were required for antiseptic action of lysophosphatidylcholine. These results show inappropriate activation of G2A-/- Kupffer cells to septic insults due to an impaired cAMP signaling possibly by lack of interaction with A2bAR.

Scant Extracellular NAD Cleaving Activity of Human Neutrophils is Down-Regulated by fMLP via FPRL1.

Extracellular nicotinamide adenine dinucleotide (NAD) cleaving activity of a particular cell type determines the rate of the degradation of extracellular NAD with formation of metabolites in the vicinity of the plasma membrane, which has important physiological consequences. It is yet to be elucidated whether intact human neutrophils have any extracellular NAD cleaving activity. In this study, with a simple fluorometric assay utilizing 1,N(6)-ethenoadenine dinucleotide (etheno-NAD) as the substrate, we have shown that intact peripheral human neutrophils have scant extracellular etheno-NAD cleaving activity, which is much less than that of mouse bone marrow neutrophils, mouse peripheral neutrophils, human monocytes and lymphocytes. With high performance liquid chromatography (HPLC), we have identified that ADP-ribose (ADPR) is the major extracellular metabolite of NAD degradation by intact human neutrophils. The scant extracellular etheno-NAD cleaving activity is decreased further by N-formyl-methionine-leucine-phenylalanine (fMLP), a chemoattractant for neutrophils. The fMLP-mediated decrease in the extracellular etheno-NAD cleaving activity is reversed by WRW4, a potent FPRL1 antagonist. These findings show that a much less extracellular etheno-NAD cleaving activity of intact human neutrophils compared to other immune cell types is down-regulated by fMLP via a low affinity fMLP receptor FPRL1.

Sulfur mustard primes human neutrophils for increased degranulation and stimulates cytokine release via TRPM2/p38 MAPK signaling.

Sulfur mustard (2,2'-bis-chloroethyl-sulfide; SM) has been a military threat since the World War I. The emerging threat of bioterrorism makes SM a major threat not only to military but also to civilian world. SM injury elicits an inflammatory response characterized by infiltration of neutrophils. Although SM was reported to prime neutrophils, the mechanism has not been identified yet. In the present study, we investigated the mechanism of SM-induced priming in human neutrophils. SM increased [Ca(2+)](i) in human neutrophils in a concentration-dependent fashion. Transient receptor potential melastatin (TRPM) 2 inhibitors (clotrimazole, econazole and flufenamic acid) and silencing of TRPM2 by shRNA attenuated SM-induced [Ca(2+)](i) increase. SM primed degranulation of azurophil and specific granules in response to activation by fMLP as previously reported. SB203580, an inhibitor of p38 MAPK, inhibited SM-induced priming. Neither PD98057, an ERK inhibitor, nor SP600215, a JNK inhibitor, inhibited SM-induced priming. In addition, SM enhanced phosphorylation of NF-kB p65 and release of TNF-α, interleukin (IL)-6 and IL-8. SB203580 inhibited SM-induced NF-kB phosphorylation and cytokine release. These results suggest the involvement of TRPM2/p38 MAPK pathway in SM-induced priming and cytokines release in neutrophils.

Angiotensin converting enzyme inhibitors and angiotensin II receptor antagonist attenuate tumor growth via polarization of neutrophils toward an antitumor phenotype.

Tumor microenvironments polarize neutrophils to protumoral phenotypes. Here, we demonstrate that the angiotensin converting enzyme inhibitors (ACEis) and angiotensin II type 1 receptor (AGTR1) antagonist attenuate tumor growth via polarization of neutrophils toward an antitumoral phenotype. The ACEis or AGTR1 antagonist enhanced hypersegmentation of human neutrophils and increased neutrophil cytotoxicity against tumor cells. This neutrophil hypersegmentation was dependent on the mTOR pathway. In a murine tumor model, ACEis and AGTR1 antagonist attenuated tumor growth and enhanced neutrophil hypersegmentation. ACEis inhibited tumor-induced polarization of neutrophils to a protumoral phenotype. Neutrophil depletion reduced the antitumor effect of ACEi. Together, these data suggest that the modulation of Ang II pathway attenuates tumor growth via polarization of neutrophils to an antitumoral phenotype.