Siglec-9 transduces apoptotic and nonapoptotic death signals into neutrophils depending on the proinflammatory cytokine environment

Abstract

We report about new apoptotic and nonapoptotic death pathways in neutrophils that are initiated via the surface molecule sialic acid-binding immunoglobulin-like lectin (Siglec)-9. In normal neutrophils, Siglec-9 ligation induced apoptosis. Inflammatory neutrophils obtained from patients with acute septic shock or rheumatoid arthritis demonstrated increased Siglec-9, but normal Fas receptor-mediated cytotoxic responses when compared with normal blood neutrophils. The increased Siglec-9-mediated death was mimicked in vitro by short-term preincubation of normal neutrophils with proinflammatory cytokines, such as granulocyte/macrophage colony-stimulating factor (GM-CSF), interferon-␣ (IFN-␣), and IFN-␥, and was demonstrated to be caspase independent. Experiments using scavengers of reactive oxygen species (ROS) or neutrophils unable to generate ROS indicated that both Siglec-9-mediated caspase-dependent and caspase-independent forms of neutrophil death depend on ROS. Interestingly, the caspaseindependent form of neutrophil death was characterized by cytoplasmic vacuolization and several other nonapoptotic morphologic features, which were also seen in neutrophils present in joint fluids from rheumatoid arthritis patients. Taken together, these data suggest that apoptotic (ROS-and caspasedependent) and nonapoptotic (ROSdependent) death pathways are initiated in neutrophils via Siglec-9. The new insights have important implications for the pathogenesis, diagnosis, and treatment of inflammatory diseases such as sepsis and rheumatoid arthritis. IntroductionNeutrophils are important effector cells in inflammation. Delayed neutrophil apoptosis has been associated with several acute and chronic inflammatory diseases and appears to be mediated, at least partially, by the overexpression of neutrophil survival cytokines such as granulocyte-colony-stimulating factor (G-CSF), 1-3 granulocyte/macrophage colony-stimulating factor (GM-CSF), 1,2,4,5 and macrophage migration inhibitory factor (MIF). 6 Any failure in the process of neutrophil apoptosis likely results in the initiation of an inflammatory response and/or in the maintenance of an already existing inflammation. Therefore, studying neutrophil apoptosis under normal and inflammatory conditions seems to be important.The induction of neutrophil apoptosis during the resolution of a neutrophilic inflammatory response can be mimicked by culturing the cells with insufficient amounts of survival factors, a process, which is called spontaneous apoptosis. Spontaneous neutrophil apoptosis can be enhanced by Fas receptor stimulation. 7 Similar observations have been made in eosinophil in vitro cultures. Eosinophils undergo spontaneous apoptosis, which can be inhibited by survival cytokines such as interleukin 5 (IL-5) or GM-CSF. 8 Eosinophils also express functional Fas receptors that accelerate in vitro cell death. 9,10 Sialic acid-binding immunoglobulin-like lectins (Siglecs) belong to the immunoglobulin (Ig) supergene family and are characterized by the presence of an N-terminal V...

Neutrophil apoptosis mediated by nicotinic acid receptors (GPR109A)

“…Mature blood neutrophils were isolated from peripheral blood of healthy donors by Ficoll-Hypaque centrifugation. 11,15,35,36 Briefly, peripheral blood mononuclear cells were separated by centrifugation on Ficoll-Hypaque (Seromed-Fakola AG, Basel, Switzerland). The lower phase, mainly granulocytes and erythrocytes, was treated with erythrocyte lysis solution (155 mmol/l NH 4 Cl, 10 mmol/l KHCO 3 , and 0.1 mmol/l EDTA, pH 7.3).…”

“…Cell death was assessed by uptake of 1 mM ethidium bromide and flow cytometric analysis (FACSCalibur). 11,15,35,36 To determine whether cell death was apoptosis, DNA fragmentation and redistribution of PS were measured. 11,15,35,36 Immunoprecipitation.…”

“…11,15,35,36 To determine whether cell death was apoptosis, DNA fragmentation and redistribution of PS were measured. 11,15,35,36 Immunoprecipitation. Neutrophils (5 Â 10 6 ) were lysed in a buffer containing 0.2% Nonidet P-40, and protein samples were immunoprecipitated with 2 mg of anti-14-3-3 Ab followed by precipitation with A-Sepharose.…”

“…Gel electrophoresis and immunoblotting were performed as described previously. 11,15,16,35,36 Briefly, after electrotransfer of the separated proteins, the filters were incubated overnight with anti-caspase-3 Ab, anti-Mcl-1 mAb, or anti-phospho-Bad Abs (all 1/1000) at 41C in TBS/0.1% Tween 20/5% nonfat dry milk. For loading controls, stripped filters were incubated with anti-GAPDH mAb (1/2000) or anti-Bad Ab (1/1000).…”

“…Therefore, it is important to study how inflammation normally resolves and under which conditions this process does not work. Although some surface receptors on neutrophils have been identified that transduce death signals, such as Fas 10 and Siglec-9, 11 other death ligands may additionally contribute to the limitation of neutrophil numbers under inflammatory conditions. Additional knowledge in this field may provide novel approaches to anti-inflammatory pharmacotherapy.…”