Extracellular NAD(+) inhibits human neutrophil apoptosis.

Regulation of neutrophil apoptosis plays a critical role in the inflammatory response. Inflammation has previously been shown to increase levels of extracellular ß-nicotinamide adenine dinucleotide (NAD(+)). The present study demonstrates that extracellular NAD(+) at concentrations found in the inflamed tissues profoundly delays spontaneous apoptosis of human neutrophils as was evidenced by inhibition of phosphatidylserine (PS) exposure, DNA fragmentation and caspase-3 activation. The effect was abrogated by NF157, an antagonist of P2Y11 receptor, and was pertussis toxin-insensitive. The NAD(+)-mediated delay of neutrophil apoptosis was reversed by 2',5'-dideoxyadenosine, an inhibitor of adenylyl cyclase, and Rp-8-Br-cAMPS, an inhibitor of type I cAMP-dependent protein kinase A (PKA). Blocking of NAD(+)-induced influx of extracellular Ca(2+) with EGTA did not abolish the pro-survival effect of NAD(+). Extracellular NAD(+) inhibited proteasome-dependent degradation of Mcl-1 upstream of caspase activation and, furthermore, suppressed Bax translocation to the mitochondria and attenuated both dissipation of mitochondrial transmembrane potential (ΔΨm) and cytochrome c release from the mitochondria into the cytosol. Finally, we found that extracellular NAD(+) inhibited spontaneous activation of caspase-9, but not caspase-8, and the pro-survival effect of extracellular NAD(+) was abrogated by the inhibitor of caspase-9, but not by the inhibitor of caspase-8. Together, these results demonstrate that extracellular NAD(+) inhibits neutrophil apoptosis via P2Y11 receptor and cAMP/PKA pathway by regulating Mcl-1 level, Bax targeting to the mitochondria and mitochondrial apoptotic pathway. Thus, extracellular NAD(+) acts as a neutrophil survival factor that can contribute to prolonged neutrophil lifespan in inflammatory response.

Diadenosine diphosphate (Ap2A) delays neutrophil apoptosis via the adenosine A2A receptor and cAMP/PKA pathway.

Diadenosine polyphosphates have been shown to inhibit neutrophil apoptosis, but mechanisms of the antiapoptotic effect are not known. Diadenosine diphosphate (Ap2A) is the simplest naturally occurring diadenosine polyphosphate, and its effect on neutrophil apoptosis has not previously been investigated. Here we report that Ap2A delays spontaneous apoptosis of human neutrophils, and the effect is reversed by the adenosine A2A receptor antagonists SCH442416 and ZM241385. Ap2A induced an elevation of intracellular cAMP and the elevation was blocked by the adenosine A2A receptor antagonists. The antiapoptotic effect of Ap2A was abrogated by 2',5'-dideoxyadenosine, an inhibitor of adenylyl cyclase, and Rp-8-Br-cAMPS, an inhibitor of type I cAMP-dependent protein kinase A (PKA). Together, these results demonstrate that Ap2A delays neutrophil apoptosis via the adenosine A2A receptor and cAMP/PKA signaling axis.

Neutrophil microparticles modulate cytokine production by natural killer cells.

Neutrophil microparticles (NMs) are scarce in the circulation but are highly enriched at sites of inflammation and exert profound effects on immune cells. In the present study, we addressed whether NMs modulate cytokine-producing capacity of natural killer (NK) cells. NMs inhibited the production of IFN-γ and TNF-α but enhanced the release of TGF-ß1 by IL-2/IL-12-activated NK cells. The inhibitory effect of NMs was strongly attenuated by blockade of phosphatidylserine exposed on NMs. Thus, NMs skew the cytokine profile of NK cells from pro-inflammatory toward anti-inflammatory, potentially favoring the resolution of inflammation.

Role of the adhesion molecule CD99 in platelet-neutrophil interactions.

The interaction of platelets with neutrophils plays an important role in inflammation and thrombosis and is coordinated by multiple adhesive interactions. The adhesion molecule CD99 is a key mediator of neutrophil migration across the endothelium but whether it is involved in platelet-neutrophil adhesive interactions has not previously been addressed. We found that platelet CD99 is predominantly localized on the cell surface and is not shed following platelet activation. Blocking of either platelet or neutrophil CD99 significantly diminished neutrophil migration across surface-adherent activated platelets in a quantitatively equivalent manner. In contrast, the blocking of CD99 affected neither neutrophil adhesion to surface-adherent activated platelets nor formation of circulating platelet-neutrophil conjugates. Thus, homophilic CD99 interaction mediates neutrophil transplatelet migration but is not involved or is redundant in neutrophil adhesion to surface-adherent or circulating platelets.

Differential effects of the autophagy inhibitors 3-methyladenine and chloroquine on spontaneous and TNF-α-induced neutrophil apoptosis.

Autophagy and apoptosis cooperate to modulate cell survival. Neutrophils are short-lived cells and apoptosis is considered to be the major mechanism of their death. In the present study, we addressed whether autophagy regulates neutrophil apoptosis and investigated the effects of autophagy inhibition on apoptosis of human neutrophils. We first showed that the established autophagy inhibitors 3-methyladenine (MA) and chloroquine (CQ) markedly accelerated spontaneous neutrophil apoptosis as was evidenced by phosphatidylserine exposure, DNA fragmentation and caspase-3 activation. Apoptosis induced by the autophagy inhibitors was completely abrogated by a pan-caspase inhibitor Q-VD-OPh. Unexpectedly, both MA and CQ significantly delayed neutrophil apoptosis induced by TNF-α, although the inhibitors did attenuate late pro-survival effect of the cytokine. The effect was specific for TNF-α because it was not observed in the presence of other inflammation-associated cytokines (IL-1ß or IL-8). The autophagy inhibitors did not modulate surface expression of TNF-α receptors in the absence or presence of TNF-α. Both MA and CQ induced a marked down-regulation of a key anti-apoptotic protein Mcl-1 but did not affect significantly the levels of another anti-apoptotic protein Bcl-X(L). Finally, to confirm the effects of the pharmacological inhibition of autophagy by a genetic approach, we evaluated the consequences of siRNA-mediated autophagy suppression in neutrophil-like differentiated HL60 cells. Knockdown of ATG5 in the cells resulted in accelerated spontaneous apoptosis but attenuated TNF-α-induced apoptosis. Together, these data suggest that autophagy regulates neutrophil apoptosis in an inflammatory context-dependent manner and mediates the early pro-apoptotic effect of TNF-α in neutrophils.

Comparative evaluation of the role of the adhesion molecule CD177 in neutrophil interactions with platelets and endothelium.

Neutrophil-specific glycoprotein CD177 is expressed on a subset of human neutrophils and has been shown to be a counter-receptor for platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31). Previous studies have demonstrated that the interaction of CD177 with endothelial PECAM-1 supports neutrophil transendothelial migration resulting in preferential transmigration of the CD177-expressing neutrophil subset. As PECAM-1 is also abundantly expressed on platelets, we addressed a follow-up suggestion that CD177/PECAM-1 adhesive interaction may mediate platelet-neutrophil interactions and CD177-positive neutrophils may have a competitive advantage over CD177-negative neutrophils in binding platelets. Here, we report that CD177-positive and CD177-negative neutrophils do not differ significantly in their capacity to form platelet-neutrophil conjugates as assayed in whole blood and in mixed preparations of isolated platelets and neutrophils. Under flow conditions, neither platelet nor neutrophil activation resulted in preferential binding of platelets to CD177-expressing neutrophils. Furthermore, no significant difference was found in the ability of both neutrophil subsets to adhere to and migrate across surface-adherent activated platelets, whereas predominantly CD177-positive neutrophils migrated across HUVEC monolayers. In addition, we demonstrated that S(536) N dimorphism of PECAM-1, which affects CD177/PECAM-1 interaction, did not influence the equal capacity of the two neutrophil subsets to interact with platelets but influenced significantly the transendothelial migration of CD177-expressing neutrophils. Thus, CD177/PECAM-1 adhesive interaction, while contributing to neutrophil-endothelial cell interaction in neutrophil transendothelial migration, does not contribute to or is redundant in platelet-neutrophil interactions.

Extracellular acidosis promotes neutrophil transdifferentiation to MHC class II-expressing cells.

Inflammation in peripheral tissues is usually associated with local acidosis. In the present study, we demonstrate that extracellular acidification enhances GM-CSF- and IFN-γ-induced expression of HLA-DR, CD80 and CD86 in human neutrophils (neutrophil transdifferentiation), and potentiates antigen-capturing capacities (both endocytosis and phagocytosis) of the transdifferentiated cells. Furthermore, in acidic conditions the transdifferentiated neutrophils have stronger antigen-presenting capacity, inducing more intense proliferation of autologous T lymphocytes in the presence of staphylococcal enterotoxin A. Thus, extracellular acidosis can represent a factor that promotes neutrophil transdifferentiation and potentiates the functional abilities of the transdifferentiated cells in inflammatory foci in vivo.

Urokinase receptor (uPAR) regulates complement receptor 3 (CR3)-mediated neutrophil phagocytosis.

Urokinase receptor (uPAR) associates in cis with complement receptor 3 (CR3). In the present study, we addressed whether this coupling regulates CR3-mediated phagocytosis. CR3-mediated attachment of iC3b-opsonized sheep red blood cells to human neutrophils and internalization of these cells were reduced by removal of cell-bound uPAR by phosphatidylinositol-specific phospholipase C and reconstituted in the presence of soluble uPAR. The attachment and internalization were suppressed in the presence of anti-uPAR polyclonal antibody, proteolytically inactive urokinase and saccharides that disrupt interaction of uPAR with CR3. Thus, uPAR acts as a cofactor for iC3b binding to CR3 and regulates CR3-mediated phagocytosis.

Activated human neutrophils rapidly release the chemotactically active D2D3 form of the urokinase-type plasminogen activator receptor (uPAR/CD87).

The urokinase-type plasminogen activator receptor (uPAR/CD87) exists both in cell-bound and soluble forms. Neutrophils contain extensive intracellular pools of uPAR that are translocated to the plasma membrane upon activation. In the present study, we investigated the ability of human neutrophils to shed uPAR from cell surface following activation and addressed the possible involvement of the released receptor in the inflammatory response. We first observed that the spontaneous release of suPAR by resting neutrophils was strongly and rapidly (within minutes) enhanced by calcium ionophore ionomycin and to a lesser extent when cells were primed with TNF-alpha and then stimulated with fMLP or IL-8. We demonstrated that suPAR is produced by resting and activated neutrophils predominantly as a truncated form devoid of N-terminal D1 domain (D2D3 form) that lacks GPI anchor. Migration of formyl peptide receptor-like 1 (FPRL1)-transfected human embryonic kidney (HEK) 293 cells toward the supernatants harvested from activated neutrophils was significantly diminished when D2D3 form of suPAR was immunodepleted from the supernatants. We conclude that activated neutrophils release the chemotactically active D2D3 form of suPAR that acts as a ligand of FPRL1. Interestingly, we present evidence that GPI-specific phospholipase D (GPI-PLD) that has previously been shown to shed uPAR in cancer cells is not involved in suPAR release from human neutrophils. We suggest that production of the chemotactically active D2D3 form of suPAR by activated human neutrophils in vivo could contribute to the recruitment of monocytes and other formyl peptide receptors-expressing cells to the sites of acute inflammation where neutrophil accumulation and activation occur.

Cytokine-mediated induction of MHC class II in human neutrophils is dependent on NADPH oxidase activity.

In response to selected cytokines, neutrophils are induced to express MHC class II and acquire properties of antigen-presenting cells. Here we show that (a) GM-CSF- and IFN-γ-mediated induction of MHC class II in human neutrophils is associated with intracellular ROS up-regulation, (b) cell-permeable ROS scavengers MnTMPyP and polyethylene glycol-conjugated superoxide dismutase and NADPH oxidase inhibitors diphenylene iodonium and apocynin abrogate both the cytokine-mediated ROS elevation and the induction of MHC class II and (c) neutrophils from chronic granulomatous disease patients which lack NADPH oxidase activity fail to express MHC class II in response to the cytokines. Thus, NADPH oxidase activity is required for the cytokine-mediated induction of MHC class II expression in neutrophils.

Anti-adhesive proteins and resolution of neutrophil-mediated inflammation.

The onset of inflammatory response is associated with neutrophil recruitment into infected or injured tissues, whereas the resolution of inflammation, on the contrary, requires blockade of further recruitment and clearance of the recruited cells. Adhesion of circulating neutrophils to the endothelium represents a crucial step in their recruitment into the inflamed tissues. Previous studies have identified a number of proteins which can interfere with neutrophil-endothelial adhesive interactions and act as natural inhibitors of the leukocyte egress from the circulation. Essentially, these proteins are up-regulated in inflammation and, therefore, have potential pro-resolving activity. Knockout animals deficient in the anti-adhesive proteins demonstrated increased magnitude and duration of acute inflammation indicating that the proteins play a non-redundant role in dampening the inflammatory response. This paper is a review of the experimental data supporting the hypothesis that the up-regulation of the anti-adhesive proteins may represent an anti-inflammatory mechanism that contributes to the resolution of inflammation.

Circulating CD35(-)/CD49d(+) neutrophils in influenza virus infection patients.

Release of immature neutrophils from bone marrow into circulation accompanies acute inflammatory states. Previous studies have identified a number of antigens that are differentially expressed on mature neutrophils and immature forms. We analyzed expression of these antigens on circulating neutrophils of influenza virus infection patients. We identified a minor neutrophil subpopulation with decreased granularity and the antigen surface expression pattern (CD35(-)/CD49d(+)) which defines metamyelocytes. CD35(-)/CD49d(+) neutrophils were detected in 35% of the patients but not in healthy individuals and correlated positively with blood metamyelocyte count. The sorted CD35(-)/CD49d(+) neutrophils displayed morphological features of metamyelocytes. Compared with a major (CD35(+)/CD49d(-)) neutrophil subpopulation, CD35(-)/CD49d(+) neutrophils demonstrated significantly diminished functional capacities (both phagocytosis and respiratory burst). Thus, CD35(-)/CD49d(+) neutrophils represent a phenotypically and functionally distinct subpopulation of the cells and can potentially be used to quantify immature neutrophil release in inflammation.

Participation of the urokinase-type plasminogen activator receptor (uPAR) in neutrophil transendothelial migration.

The mechanisms underlying migration of neutrophils across endothelium are not completely understood. The urokinase-type plasminogen activator receptor (uPAR) plays a key role in neutrophil adhesion and migration. In the present study, we addressed whether uPAR regulates neutrophil transendothelial migration. We first showed that siRNA-mediated knockdown of uPAR in human umbilical vein endothelial cells (HUVECs) did not affect neutrophil migration across HUVEC monolayers indicating that endothelial uPAR does not regulate neutrophil transmigration. In contrast, the transmigration was significantly inhibited by Fab' fragment of anti-uPAR monoclonal antibody and proteolytically inactive urokinase (uPA), whereas inhibition of proteolytical activity of endogenous uPA (with amiloride or plasminogen activator inhibitor-1) did not affect the transmigration. Both the anti-uPAR Fab' fragment and proteolytically inactive uPA did not exert significant effects upon the transmigration conducted in the presence of F(ab')(2) fragment of blocking antibody to integrin Mac-1 indicating that uPAR regulates Mac-1-dependent transmigration. Mac-1-dependent, but not Mac-1-independent, transmigration was significantly reduced in the presence of N-acetyl-d-glucosamine and d-mannose, the saccharides that disrupt uPAR/Mac-1 association, but was unaffected in the presence of control saccharides (d-sorbitol and sucrose). We conclude that physical association of uPAR with Mac-1 mediates the regulatory effect of uPAR over the transmigration. Finally, we provide evidence that the functional cooperation between uPAR and Mac-1 is essential at both adhesion and diapedesis steps of neutrophil migration across endothelium. Thus, uPAR expressed on neutrophil plasma membrane regulates transendothelial migration independently of uPA proteolytical activity and acting as a cofactor for integrin Mac-1.

Release of the soluble urokinase-type plasminogen activator receptor (suPAR) by activated neutrophils in rheumatoid arthritis.

Soluble form of the urokinase-type plasminogen activator receptor (suPAR) is markedly increased in biological fluids during different inflammatory conditions. It has previously been observed that the highest suPAR concentrations in inflammatory exudates tend to be associated with the presence of high number of neutrophils. Guided by this observation and our recent finding that activated neutrophils release suPAR we investigated whether neutrophils can be a source of suPAR during the inflammatory response in vivo. To address this question we conducted the comparative analysis of neutrophils isolated from the paired samples of synovial fluid (SF) and peripheral blood (PB) of rheumatoid arthritis patients. Freshly isolated SF neutrophils released significantly (p < 0.01) higher amounts of suPAR compared with PB neutrophils. We demonstrated that neutrophils from both sources release predominantly the truncated D2D3 form of suPAR. Migration of formyl peptide receptor-like 1 (FPRL1)-transfected human embryonic kidney (HEK) 293 cells toward the supernatants harvested from in vitro cultured SF neutrophils was significantly diminished when D2D3 form of suPAR was immunodepleted from the supernatants. Taken together, these data demonstrate that neutrophils, first, contribute to or are responsible for the generation of the increased suPAR levels during the inflammatory response and, second, release the chemotactically active form of suPAR that might be involved in the recruitment of formyl peptide receptors-expressing leukocytes into the inflamed tissues.