LETTERS 86 NATURE CELL BIOLOGY VOLUME 9 | NUMBER 1 | JANUARY 2007 PI(3)Kγ has an important context-dependent role in neutrophil chemokinesis G. John Ferguson 1 , Laura Milne 1 , Suhasini Kulkarni 1 , Takehiko Sasaki 2 , Simon Walker 1 , Simon Andrews 1 , Tom Crabbe 3 , Peter Finan 4 , Gareth Jones 5 , Shaun Jackson 6 , Montserrat Camps 7 , Christian Rommel 7 , Matthias Wymann 8 , Emilio Hirsch 9 , Phillip Hawkins 1,10 and Len Stephens 1,10,11 The directional movement of cells in a gradient of external stimulus is termed chemotaxis and is important in many aspects of development and differentiated cell function. Phophoinositide 3-kinases (PI(3)Ks) are thought to have critical roles within the gradient-sensing machinery of a variety of highly motile cells 1,2 , such as mammalian phagocytes 3 , allowing these cells to respond quickly and efficiently to shallow gradients of soluble stimuli. Our analysis of mammalian neutrophil migration towards ligands such as fMLP shows that, although PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 accumulate in a PI(3)Kγ-dependent fashion at the up-gradient leading-edge, this signal is not required for efficient gradient- sensing and gradient-biased movement. PI(3)Kγ activity is however, a critical determinant of the proportion of cells that can move, that is, respond chemokinetically, in reaction to fMLP. Furthermore, this dependence of chemokinesis on PI(3)Kγ activity is context dependent, both with respect to the state of priming of the neutrophils and the type of surface on which they are migrating. We propose this effect of PI(3)Kγ is through roles in the regulation of some aspects of neutrophil polarization that are relevant to movement, such as integrin- based adhesion and the accumulation of polymerized (F)-actin at the leading-edge. Studies of a number of model cell types, primarily Dictyostelium 4 and HL60 cells 5 , indicate that class I PI(3)K lipid products accumulate at the up-gradient edge of the cells during chemotaxis. These observations — together with others that showed the up-gradient leading-edge accumula- tion of PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 was not absolutely dependent on temporally coincident cytoskeletal rearrangements and that chemotaxis was sensitive to various blocks in PI(3)K signalling — led to the hypoth- esis that class I PI(3)K lipid products, probably derived from PI(3)Kγ in neutrophil-like cells, are important signals that direct chemotaxis. We used a transgenic mouse line expressing an eGFP–PH–PKB reporter 6 in neutrophils, which has no impact on chemotaxis (data not shown), to assess PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 distribution in living neutrophils from eGFP–PH–PKB × p110γ +/+ or eGFP–PH–PKB × p110γ –/– mice (p110γ is the catalytic subunit of PI(3)γ; Fig. 1 and see Supplementary Information, Movies S1–4). Using a published algorithm to quantitatively assess the peripheral distribution of the eGFP reporter, the reporter was shown to polarize to the leading-edge of neutrophils chemotaxing towards fMLP, in a PI(3)Kγ-dependent fashion (Fig. 1b). This localization cannot be accounted for by cytosolic entrapment (Fig. 1c) or increased membrane density (Fig. 1d), indicating that PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 , syn- thesized by PI(3)Kγ, accumulate at the up-gradient leading edge. Numerous studies have reported deficiencies in the chemotaxis of mammalian neutrophils through transwell filters (Boyden-type chambers) in the presence of the non-selective PI(3)K inhibitors, wortmannin and LY294002, or in the absence of PI(3)Kγ 7–10 . We have made similar observa- tions with PI(3)K-deficient neutrophils 11 and through the use of PI(3)K isoform-selective small molecule inhibitors (Fig. 2). Use of a PI(3)Kγ- selective inhibitor, which has little effect on migration of PI(3)Kγ-defi- cient neutrophils through transwell filters at the concentrations used here, showed that this isoform has a critical role in both human and mouse neutrophil migration. Other PI(3)K-selective inhibitors showed that PI(3)Kβ has a smaller, but significant, role in both human and mouse neutrophil migration towards fMLP, but PI3Kδ only has a significant role in human neutrophil migration 12 . In lipopolysaccharide (LPS)-primed human neutrophils (human neutrophils are substantially more sensitive to priming than mouse neutrophils) basal migration was augmented, but remained sensitive to chemoattractants. However, in LPS-primed cells this chemoattractant-stimulated migration (Fig. 2b) became completely insensitive to PI(3)K inhibitors; indicating that the roles described above for PI(3)Ks are context dependent, potentially explaining a large number of contradictory reports of the effects of PI(3)K inhibitors on migration of human neutrophils through transwell filters 7,13 . 1 The Babraham Institute, Cambridge, CB22 3AT, UK. 2 Akita University, Akita, Japan. 3 UCB, Slough, Berkshire, SL1 4EN, UK. 4 Novartis, Cambridge, USA. 5 Kings College London, SE1 1UL, UK. 6 Monash University, Melbourne, Victoria 3004, Australia. 7 Serono Research Institute, Geneva, CH-1211, Switzerland. 8 DKBW University, Basel, CH-4508, Switzerland. 9 University of Torino, 10126, Torino, Italy. 10 These authors contributed equally to this work. 11 Correspondence should be addressed to L.S. (len.stephens@bbsrc.ac.uk) Received 30 October 2006; accepted 17 November 2006; published online 17 December 2006; DOI: 10.1038/ncb1517