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Stimulation of Human Neutrophils by Chemotactic Factors Is Associated with the Activation of Phosphatidylinositol 3-Kinase γ*

Open AccessPublished:August 04, 2000DOI:https://doi.org/10.1074/jbc.M001780200
      The activation of human polymorphonuclear neutrophil leukocytes (neutrophils) is associated with an increased synthesis of the highly phosphorylated phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3). The aims of the present investigation were to determine whether the newly described, G protein-dependent phosphatidylinositol 3-kinase (PI3K), p110γ, was involved in the responses to chemotactic factors interacting with G protein-coupled receptors. The presence of p110γ in neutrophils was first established both at the protein and the mRNA level. Stimulation of the cells with fMet-Leu-Phe or interleukin-8 increased the PI3K activity in p110γ, but not p85, immunoprecipitates. The time course of this effect (threshold within less than 5 s, maximal activation at 10–15 s) was consistent with that of the generation of PtdIns(3,4,5)P3. Wortmannin, a PI3K inhibitor, abrogated the effects of fMet-Leu-Phe, which were also significantly inhibited by pertussis toxin. Finally, fMet-Leu-Phe also induced a significant translocation of p110γ to a particulate fraction derived from these cells. These data indicate that p110γ represent the major PI3K activated by fMet-Leu-Phe and interleukin-8 at very early time points following the stimulation of human neutrophils.
      PI3K
      phosphatidylinositol 3-kinase
      fMet-Leu-Phe
      formylmethionyl-leucyl-phenylalanine
      PtdIns
      phosphatidylinositol
      PtdIns(3)P
      phosphatidylinositol 3-monophosphate
      PtdIns(3
      4,5)P3, phosphatidylinositol 3,4,5-trisphosphate
      PI3K
      phosphatidylinositol 3-kinase
      DFP
      diisopropylfluorophosphate
      Me2SO
      dimethyl sulfoxide
      IL-8
      interleukin-8
      PCR
      polymerase chain reaction
      GAPDH
      glyceraldehyde-3-phosphate dehydrogenase
      The activation of human polymorphonuclear neutrophil leukocytes (neutrophils) by chemotactic factors is associated with the generation of polyphosphorylated phosphoinositides through the stimulation of the activity of phosphatidylinositol 3-kinase(s) (PI3Ks)1 (
      • Traynor-Kaplan A.E.
      • Thompson B.L.
      • Harris A.L.
      • Taylor P.
      • Omann G.M.
      • Sklar L.A.
      ,
      • Stephens L.R.
      • Hughes K.T.
      • Irvine R.F.
      ,
      • Corey S.
      • Eguinoa A.
      • Puyanatheall K.
      • Bolen J.B.
      • Cantley L.
      • Mollinedo F.
      • Jackson T.R.
      • Hawkins P.T.
      • Stephens L.R.
      ,
      • Stephens L.
      • Smrcka A.
      • Cooke F.T.
      • Jackson T.R.
      • Sternweis P.C.
      • Hawkins P.T.
      ,
      • Al-Shami A.
      • Bourgoin S.G.
      • Naccache P.H.
      ,
      • Vossebeld P.J.M.
      • Homburg C.H.E.
      • Schweizer R.C.
      • Ibarrola I.
      • Kessler J.
      • Koenderman L.
      • Roos D.
      • Verhoeven A.J.
      ,
      • Coffer P.J.
      • Geijsen N.
      • M'Rabet L.
      • Schweizer R.C.
      • Maikoe T.
      • Raaijmakers J.A.M.
      • Lammers J.W.J.
      • Koenderman L.
      ,
      • Kodama T.
      • Hazeki K.
      • Hazeki O.
      • Okada T.
      • Ui M.
      ). Although the characteristics of the accumulation of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3 (
      • Stephens L.R.
      • Hughes K.T.
      • Irvine R.F.
      ,
      • Corey S.
      • Eguinoa A.
      • Puyanatheall K.
      • Bolen J.B.
      • Cantley L.
      • Mollinedo F.
      • Jackson T.R.
      • Hawkins P.T.
      • Stephens L.R.
      ) and the functional effects of inhibition of PI3Ks by compounds such as wortmannin and LY294002 (
      • Vossebeld P.J.M.
      • Homburg C.H.E.
      • Schweizer R.C.
      • Ibarrola I.
      • Kessler J.
      • Koenderman L.
      • Roos D.
      • Verhoeven A.J.
      ,
      • Coffer P.J.
      • Geijsen N.
      • M'Rabet L.
      • Schweizer R.C.
      • Maikoe T.
      • Raaijmakers J.A.M.
      • Lammers J.W.J.
      • Koenderman L.
      ,
      • Vlahos C.J.
      • Matter W.F.
      • Brown R.F.
      • Traynor-Kaplan A.E.
      • Heyworth P.G.
      • Prossnitz E.R.
      • Ye R.D.
      • Marder P.
      • Schelm J.A.
      • Rothfuss K.J.
      • Serlin B.S.
      • Simpson P.J.
      ,
      • Downey G.P.
      • Butler J.R.
      • Brumell J.
      • Borregaard N.
      • Kjeldsen L.
      • Sue-A-Quan A.K.
      • Grinstein S.
      ,
      • Okada K.
      • Takanoohmuro H.
      • Obinata T.
      • Abe H.
      ,
      • Krump E.
      • Sanghera J.S.
      • Pelech S.L.
      • Furuya W.
      • Grinstein S.
      ,
      • Knall C.
      • Worthen G.S.
      • Johnson G.L.
      ,
      • Niggli V.
      • Keller H.
      ,
      • Capodici C.
      • Hanft S.
      • Feoktistov M.
      • Pillinger M.H.
      ,
      • Pellegatta F.
      • Chierchia S.L.
      • Zocchi M.R.
      ,
      • Baggiolini M.
      • Dewald B.
      • Schnyder J.
      • Ruch W.
      • Cooper P.H.
      • Payne T.G.
      ,
      • Araki N.
      • Johnson M.T.
      • Swanson J.A.
      ,
      • Lennartz M.R.
      ) have been described, the nature of the specific PI3Ks involved in the responses to specific neutrophil agonists remains only partially defined.
      The PI3K family comprises three classes depending on substrate specificity and protein structure (
      • Vanhaesebroeck B.
      • Leevers S.J.
      • Panayotou G.
      • Waterfield M.D.
      ,
      • Fruman D.A.
      • Meyers R.E.
      • Cantley L.C.
      ,
      • Wymann M.P.
      • Pirola L.
      ). Of particular interest to neutrophil physiology are class I PI3Ks, which are further divided into class IA and class IB and which are involved in receptor-induced cellular responses. Class IA includes heterodimers consisting of a regulatory p85 subunit (α or β) and one of the catalytic p110 subunit (α, β, or δ). At present, class IB includes only one member, p110γ, which, possibly in association with a regulatory p101 subunit (
      • Stephens L.R.
      • Eguinoa A.
      • Erdjumentbromage H.
      • Lui M.
      • Cooke F.
      • Coadwell J.
      • Smrcka A.S.
      • Thelen M.
      • Cadwallader K.
      • Tempst P.
      • Hawkins P.T.
      ,
      • Krugmann S.
      • Hawkins P.T.
      • Pryer N.
      • Braselmann S.
      ), is regulated preferentially by interaction with heterotrimeric G protein subunits (and more particularly the βγ subunit) (
      • Stephens L.R.
      • Eguinoa A.
      • Erdjumentbromage H.
      • Lui M.
      • Cooke F.
      • Coadwell J.
      • Smrcka A.S.
      • Thelen M.
      • Cadwallader K.
      • Tempst P.
      • Hawkins P.T.
      ,
      • Stoyanov B.
      • Volinia S.
      • Hanck T.
      • Rubio I.
      • Loubtchenkov M.
      • Malek D.
      • Stoyanova S.
      • Van-Haesebroeck B.
      • Dhand R.
      • Nurnberg B.
      • Gierschik P.
      • Seedorf K.
      • Hsuan J.J.
      • Waterfield M.D.
      • Wetzker R.
      ).
      A critical role for p110γ in neutrophil migration has recently been shown in p110γ knock-out mice (
      • Li Z.
      • Jiang H.
      • Xie W.
      • Zhang Z.
      • Smrcka A.V.
      • Wu D.
      ,
      • Hirsch E.
      • Katanaev V.L.
      • Garlanda C.
      • Azzolino O.
      • Pirola L.
      • Silengo L.
      • Sozzani S.
      • Mantovani A.
      • Altruda F.
      • Wymann M.P.
      ,
      • Sasaki T.
      • Irie-Sasaki J.
      • Jones R.G.
      • Oliveira-dos-Santos A.J.
      • Stanford W.L.
      • Bolon B.
      • Wakeham A.
      • Itie A.
      • Bouchard D.
      • Kozieradzki I.
      • Joza N.
      • Mak T.W.
      • Ohashi P.S.
      • Suzuki A.
      • Penninger J.M.
      ). Phagocyte chemotaxis in response to fMet-Leu-Phe and C5a was reduced, as was their accumulation in the peritoneal cavity in vivo in response to inflammatory stimuli. In addition, T lymphocyte development and activation were impaired in p110γ−/− mice. Although these data show that neutrophil recruitment in response to fMet-Leu-Phe and C5a is severely diminished in mice genetically deficient in p110γ (
      • Li Z.
      • Jiang H.
      • Xie W.
      • Zhang Z.
      • Smrcka A.V.
      • Wu D.
      ,
      • Hirsch E.
      • Katanaev V.L.
      • Garlanda C.
      • Azzolino O.
      • Pirola L.
      • Silengo L.
      • Sozzani S.
      • Mantovani A.
      • Altruda F.
      • Wymann M.P.
      ,
      • Sasaki T.
      • Irie-Sasaki J.
      • Jones R.G.
      • Oliveira-dos-Santos A.J.
      • Stanford W.L.
      • Bolon B.
      • Wakeham A.
      • Itie A.
      • Bouchard D.
      • Kozieradzki I.
      • Joza N.
      • Mak T.W.
      • Ohashi P.S.
      • Suzuki A.
      • Penninger J.M.
      ), direct evidence that human neutrophils express p110γ has not been provided as yet.
      Partial and conflicting data are available related to the specific species of PI3K activated upon stimulation of human neutrophils by chemotactic factors, which has not been directly examined as yet. Two studies (
      • Stephens L.
      • Eguinoa A.
      • Corey S.
      • Jackson T.
      • Hawkins P.T.
      ,
      • Vlahos C.J.
      • Matter W.F.
      ) have reported data suggesting that p85/p110 is not, or is only minimally, involved in the responses of human neutrophils. A similar conclusion was drawn very recently in murine neutrophils, based on the generation of p110γ−/− knock-out mice (
      • Li Z.
      • Jiang H.
      • Xie W.
      • Zhang Z.
      • Smrcka A.V.
      • Wu D.
      ,
      • Hirsch E.
      • Katanaev V.L.
      • Garlanda C.
      • Azzolino O.
      • Pirola L.
      • Silengo L.
      • Sozzani S.
      • Mantovani A.
      • Altruda F.
      • Wymann M.P.
      ,
      • Sasaki T.
      • Irie-Sasaki J.
      • Jones R.G.
      • Oliveira-dos-Santos A.J.
      • Stanford W.L.
      • Bolon B.
      • Wakeham A.
      • Itie A.
      • Bouchard D.
      • Kozieradzki I.
      • Joza N.
      • Mak T.W.
      • Ohashi P.S.
      • Suzuki A.
      • Penninger J.M.
      ). These data, however, are difficult to reconcile with those of other studies indicating that the formation of PtdIns(3,4,5)P3stimulated by fMet-Leu-Phe was inhibited by tyrosine kinase inhibitors (
      • Ptasznik A.
      • Prossnitz E.R.
      • Yoshikawa D.
      • Smrcka A.
      • Traynor-Kaplan A.E.
      • Bokoch G.M.
      ) or that enhanced PI3K activity was recovered in phosphotyrosine immunoprecipitates from fMet-Leu-Phe-stimulated cells (
      • Coffer P.J.
      • Geijsen N.
      • M'Rabet L.
      • Schweizer R.C.
      • Maikoe T.
      • Raaijmakers J.A.M.
      • Lammers J.W.J.
      • Koenderman L.
      ). On the other hand, it is well established that the formation of PtdIns(3,4,5)P3 stimulated by fMet-Leu-Phe is inhibited by pertussis toxin (
      • Traynor-Kaplan A.E.
      • Thompson B.L.
      • Harris A.L.
      • Taylor P.
      • Omann G.M.
      • Sklar L.A.
      ,
      • Stephens L.R.
      • Hughes K.T.
      • Irvine R.F.
      ). Furthermore, it has also been shown that the introduction of GTP into permeabilized neutrophils led to the accumulation of PtdIns(3,4,5)P3 (
      • Stephens L.
      • Jackson T.
      • Hawkins P.T.
      ). These two sets of data suggest that heterotrimeric GTP-binding proteins regulate the formation of the highly phosphorylated phosphoinositides. It is presently unclear, however, whether the implication of heterotrimeric GTP binding proteins in this response is direct or indirect.
      The present study was initiated to examine the potential involvement of the class IB PI3K p110γ in the responses of native human neutrophils isolated from the peripheral blood to chemotactic factors. To test this possibility as directly as possible, the effects of fMet-Leu-Phe and interleukin-8 (IL-8) on the activity and subcellular distribution of immunoprecipitated p110γ were examined. The results obtained provide evidence that chemotactic factors rapidly and transiently stimulate the activity of p110γ in a pertussis toxin-sensitive manner at a time when fMet-Leu-Phe does not increase the PI3K activity associated with p85 immunoprecipitates. Furthermore, neutrophil stimulation with chemotactic factors results in a translocation of p110γ to a particulate, membrane-enriched fraction of these cells.

      DISCUSSION

      Although the G protein-activated isoform of PI3K, p110γ, has been cloned and sequenced (
      • Stephens L.R.
      • Eguinoa A.
      • Erdjumentbromage H.
      • Lui M.
      • Cooke F.
      • Coadwell J.
      • Smrcka A.S.
      • Thelen M.
      • Cadwallader K.
      • Tempst P.
      • Hawkins P.T.
      ,
      • Stoyanov B.
      • Volinia S.
      • Hanck T.
      • Rubio I.
      • Loubtchenkov M.
      • Malek D.
      • Stoyanova S.
      • Van-Haesebroeck B.
      • Dhand R.
      • Nurnberg B.
      • Gierschik P.
      • Seedorf K.
      • Hsuan J.J.
      • Waterfield M.D.
      • Wetzker R.
      ), its biochemical and domain characteristics described in detail (see reviews in Refs.
      • Fruman D.A.
      • Meyers R.E.
      • Cantley L.C.
      ,
      • Wymann M.P.
      • Pirola L.
      ,
      • Walker E.H.
      • Perisic O.
      • Ried C.
      • Stephens L.
      • Williams R.L.
      ), and its tissue distribution examined (e.g., Refs.
      • Li Z.
      • Jiang H.
      • Xie W.
      • Zhang Z.
      • Smrcka A.V.
      • Wu D.
      and
      • Bernstein H.G.
      • Keilhoff C.
      • Reiser M.
      • Freese S.
      • Wetzker R.
      ), its role and function in native or unengineered cells remains only very partially investigated and in only a very few cases,e.g., NK cells (
      • Al-Aoukaty A.
      • Rolstad B.
      • Maghazachi A.A.
      ) and Jurkat cells (
      • Sotsios Y.
      • Whittaker G.C.
      • Westwick J.
      • Ward S.G.
      ) and very recently in knock-out mice (
      • Li Z.
      • Jiang H.
      • Xie W.
      • Zhang Z.
      • Smrcka A.V.
      • Wu D.
      ,
      • Hirsch E.
      • Katanaev V.L.
      • Garlanda C.
      • Azzolino O.
      • Pirola L.
      • Silengo L.
      • Sozzani S.
      • Mantovani A.
      • Altruda F.
      • Wymann M.P.
      ,
      • Sasaki T.
      • Irie-Sasaki J.
      • Jones R.G.
      • Oliveira-dos-Santos A.J.
      • Stanford W.L.
      • Bolon B.
      • Wakeham A.
      • Itie A.
      • Bouchard D.
      • Kozieradzki I.
      • Joza N.
      • Mak T.W.
      • Ohashi P.S.
      • Suzuki A.
      • Penninger J.M.
      ). In the present study, the presence of p110γ in isolated human peripheral blood neutrophils was demonstrated as was its activation and translocation to a particulate fraction upon stimulation by pathophysiologically relevant agonists interacting with G protein-coupled receptors.
      The presence of p110γ in human neutrophils was examined first by immunoblotting and by immunoprecipitation. The rabbit antiserum against amino acids 742–757 of the sequence of human p110γ (
      • Stoyanov B.
      • Volinia S.
      • Hanck T.
      • Rubio I.
      • Loubtchenkov M.
      • Malek D.
      • Stoyanova S.
      • Van-Haesebroeck B.
      • Dhand R.
      • Nurnberg B.
      • Gierschik P.
      • Seedorf K.
      • Hsuan J.J.
      • Waterfield M.D.
      • Wetzker R.
      ), which was raised and utilized in this study, consistently detected a doublet of approximately 110 kDa (Figs. 1 and 5). The specificity of the detection was established by displacement experiments with the immunizing peptide. Additional evidence for the presence of p110γ was obtained from the ability to amplify by reverse transcription-PCR sequences of the expected base pair lengths. The nature of the p110γ doublet detected in neutrophils is unclear at present, although it may correspond to the two isoforms of G protein-activated PI3Ks isolated by Stephens et al. (
      • Stephens L.
      • Hawkins P.T.
      • Eguinoa A.
      • Cooke F.
      ). Alternatively, the doublet may represent presently unidentified post-translational modifications of p110γ. A similar doublet was seen in the undifferentiated or Me2SO-differentiated human promyelocytic cell line, PLB-985 (data not shown).
      Rapid increases in the activity of immunoprecipitated p110γ were seen following the addition of two unrelated chemotactic factors, namely fMet-Leu-Phe and IL-8. Both of these neutrophil agonists interact with G protein-coupled receptors (
      • Boulay F.
      • Tardiff M.
      • Brouchon L.
      • Vignais P.
      ,
      • Holmes W.E.
      • Lee J.
      • Kuang W.-J.
      • Rice G.C.
      • Wood W.I.
      ,
      • Murphy P.M.
      • Tiffany H.L.
      ,
      • Thomas K.M.
      • Taylor L.
      • Navarro J.
      ). The time course of the stimulation of the activity of p110γ corresponded closely to that of the accumulation of PtdIns(3,4,5)P3 in intact cells (
      • Traynor-Kaplan A.E.
      • Thompson B.L.
      • Harris A.L.
      • Taylor P.
      • Omann G.M.
      • Sklar L.A.
      ,
      • Stephens L.R.
      • Hughes K.T.
      • Irvine R.F.
      ,
      • Eberle M.
      • Traynor-Kaplan A.E.
      • Sklar L.A.
      • Norgauer J.
      ), with transient responses detectable within the first 5 s and peaking at 10 to 15 s after the addition of either chemotactic factor. This was followed by a return to basal levels of activity within 60 s of stimulation. Thus, the activation of p110γ occurred with a time course that makes it compatible with a role in the synthesis of PtdIns(3,4,5)P3and the very early signaling events in these cells. The inhibition of the stimulation of the activity of p110γ by pertussis toxin provides evidence that this effect is mediated by members of the Gifamily. It should be noted that the magnitude of the increases in the activity of p110γ observed in the present study are likely to represent underestimates of the actual effects, as the efficacy of the antiserum to precipitate p110γ under native conditions was relatively low. Whether this low level was attributable to the low affinity of the antiserum, to the poor accessibility of the immunizing peptide sequence under native conditions, or to masking effects because of protein interactions remains to be examined and will require the development and characterization of new antibodies against other epitopes of p110γ.
      Under the conditions used in the present study, no stimulation of the activity of p85-associated PI3K activity could be detected within the first minute of stimulation with fMet-Leu-Phe. This result is in accord with those of Vlahos and Matter (
      • Vlahos C.J.
      • Matter W.F.
      ), who found no increased PI3K activity in antiphosphotyrosine immunoprecipitates from fMet-Leu-Phe-stimulated human neutrophils, with those of Stephenset al. (
      • Stephens L.
      • Eguinoa A.
      • Corey S.
      • Jackson T.
      • Hawkins P.T.
      ), who concluded that the p85/p110-dependent pathway of synthesis of PtdIns(3,4,5)P3 played only a minor role in the increases in this polyphosphoinositide following stimulation by G protein-coupled agonists in myeloid cells, and with the complete inhibition of the chemotactic factor-stimulated generation of PtdIns(3,4,5)P3 in murine p110γ−/−neutrophils (
      • Li Z.
      • Jiang H.
      • Xie W.
      • Zhang Z.
      • Smrcka A.V.
      • Wu D.
      ,
      • Hirsch E.
      • Katanaev V.L.
      • Garlanda C.
      • Azzolino O.
      • Pirola L.
      • Silengo L.
      • Sozzani S.
      • Mantovani A.
      • Altruda F.
      • Wymann M.P.
      ,
      • Sasaki T.
      • Irie-Sasaki J.
      • Jones R.G.
      • Oliveira-dos-Santos A.J.
      • Stanford W.L.
      • Bolon B.
      • Wakeham A.
      • Itie A.
      • Bouchard D.
      • Kozieradzki I.
      • Joza N.
      • Mak T.W.
      • Ohashi P.S.
      • Suzuki A.
      • Penninger J.M.
      ). It should be noted that this conclusion does not exclude an indirect and secondary activation of p85/p110 by fMet-Leu-Phe, as the latter may occur as a result of the stimulation of the activity of various Src family tyrosine kinases including Lyn (
      • Ptasznik A.
      • Prossnitz E.R.
      • Yoshikawa D.
      • Smrcka A.
      • Traynor-Kaplan A.E.
      • Bokoch G.M.
      ,
      • Gaudry M.
      • Gilbert C.
      • Barabé F.
      • Poubelle P.E.
      • Naccache P.H.
      ), which is also induced by chemotactic factors (
      • Rollet E.
      • Caon A.C.
      • Roberge C.J.
      • Liao N.W.
      • Malawista S.E.
      • McColl S.R.
      • Naccache P.H.
      ). These secondary effects on src kinases may underlie the apparent discrepancy between the above results and those of Ptasznik et al. (
      • Ptasznik A.
      • Prossnitz E.R.
      • Yoshikawa D.
      • Smrcka A.
      • Traynor-Kaplan A.E.
      • Bokoch G.M.
      ), who attenuated the formation of PtdIns(3,4,5)P3 induced by fMet-Leu-Phe using tyrosine kinase inhibitors.
      Finally, evidence also was obtained that the subcellular distribution of p110γ was altered upon stimulation with a statistically significant proportion of the enzyme translocating to a particulate fraction. The latter presumably contains various cellular membranes, including the plasma membrane, in which the physiological substrate of p110γ, namely PtdIns(4, 5)P2, is present. It is worthwhile to note that the kinetics of the translocation of p110γ corresponds closely with that of the formation of PtdIns(3,4,5)P3 (
      • Traynor-Kaplan A.E.
      • Thompson B.L.
      • Harris A.L.
      • Taylor P.
      • Omann G.M.
      • Sklar L.A.
      ,
      • Stephens L.R.
      • Hughes K.T.
      • Irvine R.F.
      ,
      • Eberle M.
      • Traynor-Kaplan A.E.
      • Sklar L.A.
      • Norgauer J.
      ) and of the stimulation of its activity (Fig. 2), thereby supporting its causal significance. A similar translocation of p110γ to a membrane-containing fraction has previously been described in chemokine-stimulated NK cells, although with significantly slower kinetics (
      • Al-Aoukaty A.
      • Rolstad B.
      • Maghazachi A.A.
      ).
      In conclusion, the results of the current study present evidence that the activity of p110γ is rapidly stimulated upon activation of G protein-coupled receptors in human peripheral blood neutrophils. The characteristics of this effect indicate that the stimulation of p110γ underlies, at least in part, the previously described rapid increases in the levels of PtdIns(3,4,5)P3 induced by chemotactic factors and possibly also the effects of PI3K inhibitors on various neutrophil functions (chemotaxis, phagocytosis, oxidative burst). The causal positioning of the stimulation of p110γ in the various signaling pathways summoned upon neutrophil activation remains to be directly examined. The possibility that it may play a role in the mediation of the recruitment of tyrosine kinases of the Tec family, which contain phosphoinositide-interacting pH domains (
      • Okoh M.P.
      • Vihinen M.
      ), is particularly intriguing in view of the sensitivity of the stimulation of tyrosine phosphorylation in human neutrophils to PI3K inhibitors such as wortmannin and LY294002 (
      • Naccache P.H.
      • Caon A.C.
      • Gilbert C.
      • Gaudry M.
      • Roberge C.J.
      • Poubelle P.E.
      • Bourgoin S.
      ).

      Acknowledgments

      We thank Nathalie Thibault for expert help with the translocation experiments.

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