Danielle Harbour

Differential expression of adenosine receptors in human neutrophils: up‐regulation by specific Th1 cytokines and lipopolysaccharide

Four types of adenosine receptors have been identified in different tissues and cell types, namely, A1, A2A, A2B, and A3 receptors. We report that A2AR but not A2BR mRNA in freshly isolated polymorphonuclear neutrophils (PMN) is maximally up‐regulated after 4 h stimulation with lipopolysaccharide (LPS) or tumor necrosis factor α (TNF‐α) and to a lesser extent, with interleukin (IL)‐1β. These effects were maintained up to 21 h. Consistent with changes in A2AR mRNA expression, up‐regulation of A2AR protein was also detected after 4 h of LPS or TNF‐α exposure. Up‐regulation of A2AR protein expression was transient and returned to near basal levels after 12 h or 16 h stimulation with TNF‐α or LPS, respectively. Conversely, IL‐1β failed to promote A2AR protein expression. Suppression of thapsigargin‐induced leukotriene synthesis by the selective A2AR agonist CGS‐21680 was found to be more pronounced when PMN were cultured for 4 h with LPS or TNF‐α. In contrast, the up‐regulation of A2AR has no impact on CGS‐21680‐induced inhibition of phospholipase D activation and superoxide production in response to formyl‐Met‐Leu‐Phe. These results demonstrate that the A2AR is up‐regulated by specific T helper cell type 1 cytokines and LPS. Although this could represent a potential feedback mechanism to control inflammation, the effect of A2AR up‐regulation varied depending on the stimulus used to stimulate PMN functional responses after their incubation with proinflammatory mediators.

Occupancy of adenosine A2a receptors promotes fMLP-induced cyclic AMP accumulation in human neutrophils: impact on phospholipase D activity and recruitment of small GTPases to membranes

The aim of this study was to assess in human neutrophils theimplication of an adenosine 3′,5′‐cyclic monophosphate (cAMP)‐dependentpathway in the inhibitory effects of A2a receptor engagement. We foundthat Ro20‐1724, a cAMP phosphodiesterase inhibitor, in the presence ofadenosine deaminase (ADA) or A2a receptor antagonists renderedtransient the fMLP‐induced sustained increases in cAMP levels. The roleof A2a receptor stimulation was demonstrated by the ability of the A2areceptor agonist, CGS21680, to prevent ADA‐mediated reduction of thepersistent cAMP elevation induced by fMLP. Persistent cAMP elevationcorrelated with inhibition of fMLP‐induced PLD activation andrecruitment of Arf, RhoA, and PKC to membranes. The suppressive effectof CGS21680 or isoproterenol, a β‐adrenergic receptor agonist, wasincreased by Ro20‐1724 or by the adenylyl cyclase activator, forskolin, and reversed, at least in part, by the inhibitor of adenylyl cyclase,2′,5′‐dideoxyadenosine. The activator of protein kinase A (PKA),Sp‐cAMP inhibited fMLP‐induced PLD activation and translocation of Arfand RhoA to membranes. In contrast, the suppression by A2a receptorstimulation of fMLP‐induced PLD activation and cofactor recruitment wasantagonized by PKA inhibitors, Rp‐cAMP and H89. In conclusion, A2areceptor occupancy by extracellular adenosine inhibits fMLP‐inducedneutrophil activation via cAMP and PKA‐regulated events.

Cytohesin-1 Regulates the Arf6-Phospholipase D Signaling Axis in Human Neutrophils: Impact on Superoxide Anion Production and Secretion

Polymorphonuclear neutrophil (PMN) stimulation with fMLP stimulates small G proteins such as ADP-ribosylation factors (Arfs) Arf1 and Arf6, leading to phospholipase D (PLD) activation and functions such as degranulation and the oxidative burst. However, the molecular links between fMLF receptors and PLD remain unclear. PMNs express cytohesin-1, an Arf-guanine exchange factor that activates Arfs, and its expression is strongly induced during the acquisition of the neutrophilic phenotype by neutrophil-like cells. The role of cytohesin-1 in the activation of the fMLF-Arf-PLD signaling axis, and the accomplishment of superoxide anion production, and degranulation was investigated in PMNs using the selective inhibitor of cytohesin, Sec 7 inhibitor H3 (secinH3). Cytohesin-1 inhibition with secinH3 leads to Arf6 but not Arf1 inhibition, demonstrating the specificity for Arf6, and fMLF-mediated activation of PLD and of the oxidative burst as well. We observed a decrease in fMLF-mediated protein secretion and expression of cell surface markers corresponding to primary (CD63/myeloperoxidase), secondary (CD66/lactoferrin), and tertiary (matrix metalloproteinase-9) granules in PMNs incubated with secinH3. Similarly, silencing cytohesin-1 or Arf6 in PLB-985 cells negatively affected fMLF-induced activation of PLD, superoxide production, and expression of granule markers on the cell surface. In contrast, stable overexpression of cytohesin-1 in PLB-985 cells enhanced fMLF-induced activation of Arf6, PLD, and NADPH oxidase. The results of this study provide evidence for an involvement of cytohesin-1 in the regulation of the functional responses of human PMNs and link these events, in part at least, to the activation of Arf6.

An essential role for phospholipase D in the recruitment of vesicle amine transport protein-1 to membranes in human neutrophils.

Although phosphatidic acid (PA) regulates a wide variety of physiological processes, its targets remain poorly characterized in human neutrophils. By co-sedimentation with PA-containing vesicles we identified several PA-binding proteins including vesicle amine transport protein-1 (VAT-1), Annexin A3 (ANXA3), Rac2, Cdc42 and RhoG in neutrophil cytosol. Except for ANXA3, protein binding to PA-containing liposomes was calcium-independent. Cdc42 and RhoG preferentially interacted with PA whereas VAT-1 bound to PA or phosphatidylserine with the same affinity. VAT-1 translocated to neutrophil membranes upon N-formyl-methionyl-leucyl-phenylalanine (fMLF) stimulation. Inhibition of fMLF-induced PLD activity with the Src kinase inhibitor PP2, the selective inhibitor of PLD FIPI, or of PA formation with primary alcohols reduced VAT-1 translocation. In contrast, inhibition of PA hydrolysis with propranolol enhanced fMLF-mediated VAT-1 recruitment to membranes. PMA also redistributed VAT-1 to membranes in a PKC- and PLD-dependent manner. Though fMLF and PMA increased VAT-1 phosphorylation, different kinases appear to be involved. Cell fractionation revealed that a pool of VAT-1 was co-localized with primary, secondary and tertiary granules and plasma membrane markers in resting neutrophils. Stimulation with fMLF enhanced VAT-1 co-localization with CD32a, a plasma membrane marker. Confocal microscopy revealed that VAT-1 decorates granular structures at the cell periphery and double labeling with VAT-1/lactoferrin antibodies showed a partial co-localization with secondary granules in control and fMLF-stimulated cells. Characterization of these putative PA-binding proteins constitutes another step forward for a better understanding of the role of PLD-derived PA in neutrophil physiology.

Characterisation of degranulation and phagocytic capacity of a human neutrophilic cellular model, PLB-985 cells ☆

Exocytosis of neutrophil granules is a major event that converts circulating neutrophils into fully activated cells capable of chemotaxis, phagocytosis and destruction of pathogens. The PLB-985 cell line is a suitable neutrophilic cellular model which is utilised to study the different functional responses of neutrophils. In this study, we characterised the differentiation of PLB-985 cells toward the granulocytic pathway, using three different inducing agents: dbcAMP, DMSO and DMF. The differentiation efficiency was monitored by observation of cell morphology with electron microscopy, and by analysis of the expression of receptors such as FPRL1 and FcgammaRIIA, the distribution or release of granule markers, phagocytic capacity, as well as measurement of fMLF-induced calcium fluxes. Exocytosis and phagocytosis in differentiated cells were weaker as compared to neutrophils. fMLF stimulated primary granule exocytosis in cells differentiated with dbcAMP, DMSO and DMF, whereas the release of the contents of tertiary granules, as well as that of secretory vesicles, was only observed in dbcAMP-differentiated cells. DMSO-differentiated cells exhibited the highest phagocytic capacity. Altogether our results reinforce the fact that depending on the differentiating agent used, PLB-985 cells represent a useful model to study neutrophil functions and to bypass difficulties inherent to these primary cells.

Characterization of cytohesin-1 monoclonal antibodies: expression in neutrophils and during granulocytic maturation of HL-60 cells.

ADP-ribosylation factors (Arf) are small GTP-binding proteins involved in vesicular transport and the activation of phospholipase D (PLD). The conversion of Arf-GDP to Arf-GTP is promoted in vivo by guanine nucleotide exchange factors such as ARNO or cytohesin-1. In order to examine the expression of ARNO and cytohesin-1 in human granulocytes, we generated specific polyclonal and monoclonal antibodies (mAbs). We also overexpressed GFP-ARNO and GFP-cytohesin-1 in RBL-2H3 cells to characterize the specificity and the ability of cytohesin-1 mAbs to immunoprecipitate cytohesin-1. Among the hybridomas secreting cytohesin-1 mAbs, only the clones 2E11, 1E4, 3C8, 6F5, 4C7, 7A3 and 8F7 were found to be specific for cytohesin-1. Furthermore, mAb 2E11 immunoprecipitated GFP-cytohesin-1 but not GFP-ARNO under native conditions. In contrast, mAbs 5D8, 4C3, 2G8, 6G11, 4C3, 6D4, 7B4 and 6F8 detected both cytohesin-1 and ARNO as monitored by immunoblotting. Although mAb 6G11 detected both proteins, this antibody immunoprecipitated GFP-ARNO but not GFP-cytohesin-1 under native conditions. Another antibody, mAb 10A12, also selectively immunoprecipitated GFP-ARNO under native conditions, but the epitope recognized by this mAb is unlikely to be linear as no signal was obtained by immunoblotting. Immunoprecipitation with a cytohesin-1 polyclonal antibody and blotting with cytohesin-1 specific mAbs revealed that cytohesin-1 is highly expressed in neutrophils. Cytohesin-1 can be detected in HL-60 cells but the endogenous protein levels were low in undifferentiated cells. Using the specific cytohesin-1 mAb 2E11 we observed a marked increase in levels of cytohesin-1 expression during dibutyryl-cyclic AMP-induced granulocytic differentiation of HL-60 cells. These data suggest that cytohesin-1, which may have important functions in neutrophil physiology, can be useful as a potential marker for granulocytic differentiation.