Adenosine receptor occupancy suppresses chemoattractant-induced phospholipase D activity by diminishing membrane recruitment of small GTPases.

Adenosine (Ado) is an important autocrine modulator of neutrophil functions. In this study, we determined the effects of endogenous Ado on fMet-Leu-Phe (fMLP)-induced phospholipase D (PLD) activity in neutrophils. The removal of extracellular Ado by Ado deaminase (ADA) or the blockade of its action by the A2a receptor antagonists 8-(3-chlorostyryl) caffeine (CSC) or CGS15943 markedly increased fMLP-induced PLD activation. The concentration-dependent stimulatory effects of CSC and CGS15943 were abolished by a pretreatment of neutrophil suspensionswith ADA. In contrast, the selective A2a receptor agonist CGS21680 suppressed fMLP-induced PLD activation. Furthermore, inhibition by CGS21680 of fMLP-induced PLD activity was reversed by CSC or CGS15943. The removal of Ado by ADA or the blockade of its action by CSC or CGS15943, markedly increased the membrane recruitment of cytosolic protein kinase Calpha (PKCalpha), RhoA, and ADP-ribosylation factor (ARF) in response to fMLP. As shown for PLD activity, the stimulatory effect of Ado receptor antagonists on PLD cofactors translocation was abolished by a pretreatment of the cells with ADA. Moreover, the membrane translocation of both PKCalpha, RhoA, and ARF in response to fMLP was attenuated by CGS21680 and this effect of the A2a receptor agonist was antagonized by CSC or CGS15943. These data demonstrate that Ado released by neutrophils in the extracellular milieu inhibits PLD activation by blocking membrane association of ARF, RhoA, and PKCalpha through Ado A2a receptor occupancy. (Blood. 2000;95:519-527)

Crystal-induced neutrophil activation VI. Involvment of FcgammaRIIIB (CD16) and CD11b in response to inflammatory microcrystals.

The inflammatory reaction associated with the deposition of monosodium urate (MSU) crystals in synovial spaces is known to be due to interactions with polymorphonuclear neutrophils mediated by presently unidentified surface structures. In this study, we have observed that antibodies directed against CD16 (VIFcRIII) and CD11b (VIM12) selectively and potently inhibit the activation of neutrophils by MSU crystals. The responses affected include the stimulation of tyrosine phosphorylation, activation of the tyrosine kinase syk, tyrosine phosphorylation of the proto-oncogene Cbl, mobilization of calcium, and stimulation of the activity of phospholipase D and of the production of superoxide anions. Tyrosine phosphorylation responses to MSU crystals develop during the Me2SO4-induced differentiation of HL-60 cells in parallel with the surface expression of CD16. These data strongly support the hypothesis that inflammatory microcrystals interact opportunistically with CD16 initially, and that the signal transduction pathways activated thereby depend on CD11b. An examination of the relevance of the hypothesis that an uncontrolled activation of CD16/CD11b may play a role in inflammatory reactions associated with a dysregulation of neutrophil function (other than crystal arthropathies) appears warranted on the basis of the present results.

Agonist-specific tyrosine phosphorylation of Cbl in human neutrophils.

The effects of soluble and particulate agonists on the tyrosine phosphorylation levels of the proto-oncogene Cbl in human neutrophils were examined. Experimental conditions allowing the maintenance of Cbl as well as of its tyrosine phosphorylation status were first established. Their use allowed us to observe that Cbl was tyrosine phosphorylated in response to some (FcgammaRII ligation, opsonized bacteria and zymosan, granulocyte-macrophage colony-stimulating factor, monosodium urate, and calcium pyrophosphate microcrystals), but not all (fMet-Leu-Phe, interleukin-8) neutrophil agonists. Cbl was also shown to account for a varying proportion of the 120-kDa phosphoprotein(s) observed in response to the above stimuli. These data establish that Cbl is present in human neutrophils and that its level of tyrosine phosphorylation is modulated by some of these cells' agonists, and in particular by phagocytic particles. Furthermore, the signaling pathways activated by chemotactic factors and the other neutrophil stimuli tested in this investigation diverge at or downstream from the tyrosine phosphorylation of Cbl.

Granulocyte-macrophage colony-stimulating factor-activated signaling pathways in human neutrophils. I. Tyrosine phosphorylation-dependent stimulation of phosphatidylinositol 3-kinase and inhibition by phorbol esters.

Phosphatidylinositol 3-kinase (PI3-kinase) is a cytosolic enzyme that plays key roles in mediating signaling through many receptors. The heterodimeric form of PI3-kinase is made up of a regulatory subunit, p85, and a catalytic subunit, p110. Although granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to activate PI3-kinase, the mechanisms by which this activation is mediated and regulated are incompletely understood. Here we show that treatment of human neutrophils with GM-CSF induced both time- and concentration-dependent increases in the level of tyrosine phosphorylation of p85. The ability of GM-CSF to activate PI3-kinase was abolished by pretreating the cells with erbstatin, a tyrosine kinase inhibitor. The simultaneous treatment of the cells with GM-CSF and phorbol esters such as phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) significantly inhibited both the tyrosine phosphorylation of p85 and the activation of PI3-kinase. The inhibitory effects of phorbol esters were not induced by their inactive analogues and they were selective to the stimulation of tyrosine phosphorylation of p85 since phorbol esters did not alter the enhancement of the pattern of tyrosine phosphorylation of other cellular proteins, including that of Jak2 induced by GM-CSF. However, PMA significantly inhibited the in situ tyrosine phosphorylation and the activation of lyn observed in response to GM-CSF. The results suggest that the activation of PI3-kinase by GM-CSF is mediated by the tyrosine phosphorylation of p85 and that this activation is downregulated by PKC possibly via the inhibition of lyn.

Role of protein kinase C alpha, Arf, and cytoplasmic calcium transients in phospholipase D activation by sodium fluoride in osteoblast-like cells.

The effect of fluoride on phospholipase D (PLD) activation was studied using in vitro culture of Saos-2, MG-63 osteosarcoma cells, and normal osteoblast-like cells derived from human bone explants. Millimolar concentrations of NaF induced a significant accumulation of phosphatidylethanol (PEt) in Saos-2 cells but not in MG-63 and normal osteoblast-like cells. PLD activation was evident at 15 mM and concentration-dependent up to 50 mM. This stimulation was inhibited by deferoxamine, a chelator of Al3+, suggesting that PLD activation involves fluoride-sensitive G proteins. A good correlation was found between the levels of intracellular free Ca2+ and the activation of PLD. The time courses of the two responses were nearly identical. The ability of NaF to induce both responses was largely dependent on the presence of extracellular calcium. The calcium ionophore A23187 reproduced the effect of NaF, and this effect was antagonized by EGTA, suggesting that PLD activation was, at least in part, a calcium-regulated event. Phorbol 12-myristate 13-acetate (PMA) also stimulated PLD activity in human bone cells. Protein kinase C alpha (PKC alpha) and epsilon were expressed in Saos-2 cells. Acute pretreatment of cells with PMA reduced concomitantly the amounts of PKC alpha, but not of PKC epsilon, and the subsequent activation of PLD elicited by PKC activators. The PLD response to NaF was not attenuated but rather enhanced by down-regulation of PKC alpha. Therefore, PKC-alpha-induced PLD activation is unlikely to mediate the effect of NaF. Moreover, PMA and NaF showed a supraadditive effect on PLD activation in Saos-2 cells. This stimulation, in contrast to NaF alone, was not reduced by EGTA. Hence, mobilization of calcium by NaF cannot account for the enhanced PLD activation in response to PMA stimulation. Membrane Arf and RhoA contents were assessed by Western immunoblot analyses. Membranes derived from NaF-stimulated Saos-2 cells contained more Arf and RhoA when compared with membranes derived from control or PMA-stimulated cells. Translocation of the small GTPases was calcium-independent. We conclude that PLD activation by NaF in Saos-2 cells includes a fluoride-sensitive G protein, increases in the levels of intracellular calcium, and Arf/RhoA redistribution to membranes. The results also indicate that the NaF-induced Arf/RhoA translocation exerts in concert with PMA-activated PKC alpha a synergistic effect on the activation of PLD in Saos-2 cells.