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in Neutrophil Directional Movement
ICOS Corporation, Bothell, WA 98021
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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in neutrophil chemotaxis, we have developed a potent and selective PI3K inhibitor, IC87114. IC87114 inhibited polarized morphology of neutrophils, fMLP-stimulated PIP3 production and chemotaxis. Tracking analysis of IC87114-treated neutrophils indicated that PI3K activity was required for the directional component of chemotaxis, but not for random movement. Inhibition of PI3K, however, did not block F-actin synthesis or neutrophil adhesion. These results demonstrate that PI3K can play a selective role in the amplification of PIP3 levels that lead to neutrophil polarization and directional migration. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Class I PI3K members have been implicated in PIP3 generation in response to external stimuli in a wide variety of cell types. All four class I PI3Ks (p110
, p110
, p110, and p110
) are expressed in neutrophils (16). Neutrophils from p110 null mice showed a profound defect in fMLP-induced PIP3 generation (17, 18, 19). These neutrophils demonstrated no significant impairment in random movement and activation of Rac, which regulates actin polymerization and lamellipodia formation. Although there was a significant defect in fMLP-induced chemotaxis, it was not completely abolished in the p110 null neutrophils (18, 20). These results suggest that in addition to p110, other members of the class I PI3K probably contribute to neutrophil polarization and chemotaxis. Consistent with these findings, using isoform-specific Abs Vanhaesebroeck et al. (21) reported a specific role of PI3K, but not PI3K, in cytoskeletal reorganization and chemotaxis of a macrophage-like cell line in response to CSF1.
Involvement of PI3K in neutrophil polarization has also been suggested from the observed inhibition of chemotaxis by pharmacological PI3K inhibitors. Both wortmannin and LY294002, two widely used inhibitors of PI3Ks, block PIP3 generation and chemotaxis (19, 22). However, these inhibitors do not distinguish among the four class I PI3Ks and also inhibit protein kinases. These studies have not been able to distinguish whether the inhibitors blocked random or directional locomotion of the neutrophils. To circumvent the limitations of the existing pharmacological probes, we developed isoform-specific PI3K inhibitors. Of the four class I PI3K isoforms only PI3K is preferentially expressed in cells of hemopoietic origin (23, 24). Recently, a selective role of PI3K in immune function has been proposed from the observed defects in B and T cell activation in mice bearing a catalytically inactive p110 mutation (25). We report here, using the selective inhibitor, that PI3K plays an important role in the directional migration of neutrophils, but not in random movement. Thus PI3K could be an attractive target for the development of specific small molecule inhibitors that might be used in the treatment of a variety of inflammatory diseases. In this report, we describe an isoform-specific inhibitor of PI3 kinase that is selective for PI3K. Experiments using the PI3K inhibitor suggest that PI3K plays an important role in the directional migration of neutrophils, but not in random movement.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Various isoforms of PI3K were expressed in SF9 cells as His-tagged proteins using the baculovirus expression system according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA). PI3K assay was performed according to Chantry et al. (23) and Volinia et al. (26) with the following modifications. Phosphatidylinositol-(4,5)-bisphosphate (PIP2) containing phospholipid liposomes were prepared using a mini-extruder fitted with an 0.08-µm filter (Avanti Polar Lipids, Alabaster, AL) according to Mayer et al. (27). Briefly, bovine PIP2 and phosphatidylserine (1:2 molar ratio; Avanti Polar Lipids) were vacuum-dried and resuspended at 1 mM PIP2 in 20 mM HEPES-KOH, pH 7.4, 50 mM NaCl, 5 mM EDTA. The lipid suspension was subjected to a brief sonication, followed by 5 freeze-thaw cycles and then 20 extrusion cycles to produce the liposomes. The assay was conducted in 60-µl reaction volumes in 20 mM HEPES, pH 7.4, buffer containing 1 nM PI3K, 1 µM PIP2, 200 µM ATP, 1 µCi [-32P]ATP, 5 mM MgCl2, plus 50 µg/ml horse IgG (Pierce, Rockford, IL) as carrier protein. The reaction was incubated for 10 min at room temperature, quenched in 140 µl of 1 M K2PO4, 30 mM EDTA, pH 8.0, captured onto a 96-well polyvinylidene difluoride filter plate (Millipore, Bedford, MA) and washed five times with 1 M K2PO4. The filter was allowed to dry completely, and the bound radioactivity was quantitated. For screening the small molecule compound library, all compound dilutions were assayed in a final concentration of 1% (w/w) DMSO. Details of the synthesis of IC87114 will be published in a patent for which an application, no. 20020161014, has been made.
p38 mitogen-activated protein kinase (MAPK; Alexis, San Diego, CA) was assayed in the presence of 100 mM HEPES, pH 7.5, 10 mM MgCl2, 25 mM 2-glycerophosphate, 10% glycerol, 1 mM DTT, 10 µM ATP, 2.5 µCi [-32P]ATP, and 4.5 µM myelin basic protein in a 60-µl final volume. SF9 cell-expressed and purified FLAG-tagged checkpoint kinase 1 (CHK1) was assayed in the presence of 20 mM HEPES, pH 7.2, 5 mM MgCl2, 0.1% Nonidet P-40, 4 µM ATP, 1 mM DTT, 0.6 µCi [-32P]ATP, and 20 µM cdc24c peptide in a 60-µl final volume. For the testing of casein kinase 1 (CK1), PKB, protein kinase C (PKC), and PKCII activities the Kinase Profiler system of Upstate Biotechnology (Lake Placid, NY) was used. DNA-PK was assayed according to Anderson and Lees-Miller (28).
Measurement of PIP3 synthesis by neutrophils
Neutrophils were isolated by density gradient centrifugation on Polymorphoprep (Accurate Chemical, NY), washed, and resuspended in 30 mM HEPES, pH 7.4, 110 mM NaCl, 10 mM KCl, 1 mM MgCl2, and 10 mM glucose at 5 x 107 cells/ml. Cells were incubated at 37°C for 90 min in the presence of 1 mCi/ml [32P]orthophosphate, washed three times, and then preincubated at 37°C for 20 min in the presence of the inhibitors. Subsequently, the cells were stimulated for 15 s with either 1 µM fMLP or control, DMSO. Phospholipids were extracted and resolved on TLC according to Ptasznik et al. (29). The TLC-resolved radiolabeled phospholipids were detected and quantified by phosphorimaging (Storm; Molecular Dynamics, Sunnyvale, CA).
Quantitation of F-actin synthesis by flow cytometry
Neutrophils isolated from human blood were treated in suspension with DMSO or IC87114 (5 µM) and then stimulated with fMLP (1 µM) for 30 s at 37°C. The cells were fixed with paraformaldehyde, washed in PBS, permeabilized with 0.1% Triton X-100, and stained with Oregon Green-conjugated phalloidin. The stained neutrophils were washed three times in PBS and analyzed in a FACSCalibur (BD Biosciences, Mountain View, CA).
Visualization of actin redistribution, cell polarization, and spreading
Freshly isolated human neutrophils were pretreated either with DMSO or with IC87114 (final concentration, 5 µM). The neutrophils were then plated on a fibrinogen-coated coverslip and allowed to adhere. Subsequently, the neutrophils were stimulated with 1 µM fMLP or with DMSO only. After 1 h, the cells were fixed for 10 min in 4% paraformaldehyde at 4°C, unbound neutrophils were removed by three washes with PBS, and the bound cells were permeabilized with 0.1% Triton X-100. G-actin was stained with Texas Red-conjugated DNase I and F-actin was stained with Oregon Green-conjugated phalloidin (Molecular Probes, Eugene, OR). The actins were visualized by fluorescence microscopy. Dimensions of the neutrophils were measured using the Metamorph program (Gryphon, San Diego, CA).
Measurement of neutrophil adhesion and migration
The adhesion assays were performed by the following modifications of the procedure described by Sadhu et al. (30). Briefly, 96-well plates were coated overnight with 5 µg/ml ICAM-1/Ig, blocked with 1% HSA (no. 12666; Calbiochem, La Jolla, CA) in RPMI 1640, and 2 x 105 neutrophils (untreated or pretreated with the indicated concentration of IC87114) were added in triplicate wells. After incubation at 37°C for the desired time, 5% CO2 glutaraldehyde was added to a 1% final concentration. Unbound cells were removed by washing the wells in water and the adherent cells were stained with crystal violet. Cell adhesion was quantified by reading the intensity of crystal violet on Spectra MAX (Molecular Devices, Sunnyvale, CA) at 570 nm.
Neutrophil migration under agarose was studied according to a modified protocol described by Nelson et al. (31). Briefly, 6-well plates (no. 3516; Costar, Cambridge, MA) were coated with ICAM-1/Ig (25 µg/ml in bicarbonate buffer, pH 9.3), overnight at 4°C. After washing, 1% agarose solution (with or without the inhibitor in RPMI 1640 and 0.5% BSA) at 50°C was layered on the ICAM-1-coated surface. Plates were cooled at 4°C before punching holes in the agarose layer to form wells (one central well surrounded by six peripheral wells). Human neutrophils were obtained as described above, and resuspended in 0.5% BSA (in RPMI 1640) at 5 x 106/ml. After combining equal volumes of neutrophil suspension and medium (either with DMSO, or IC87114 in DMSO), 10 µl of the neutrophil suspension were aliquoted into each of the peripheral wells. To the central well 10 µl of fMLP (5 µM) was added. Plates were incubated at 37°C in the presence of 5% CO2 for 2–3 h to allow neutrophil migration. Less than 5% of input cells migrated during the time period of the experiment. Migration was terminated by the addition of glutaraldehyde (1%) in each of the wells. After removing the agarose layer, wells were washed with distilled water and dried. Using a Diaphot inverted microscope (1x objective; Nikon, Melville, NY) equipped with a camera and digital processor (Dage-MTI, Michigan City, IN) that was connected to a PowerMac 8500/120 (Apple Computer, Cupertino, CA), three recordings per experimental condition of neutrophil migration were collected using the NIH Image Scion 1.61 program (Bethesda, MD). Using Microsoft Excel and Table Curve 4 (Redmond, WA), data were plotted to obtain migration index (MI) values for each of the conditions. MI was defined as the area under a curve representing the number of neutrophils (OD) vs the distance of migration.
For video recordings of neutrophil migration under agarose, 20x objective with phase contrast was used with the NIH Image Scion 1.61 program. Data were collected at around 2 h from the time of cell addition into the wells. At this time, a sufficient number of cells migrated for subsequent analysis under each experimental condition without interference artifacts from the edge of the well. Positions of cells were recorded every 15 s. A total of 25 consecutive frames were used for the analysis of neutrophil movements per condition. Using the NIH Image Object 1.62p4.1 program, 10 cells that were present in all 25 frames were traced and analyzed with Microsoft Excel. Total distance of movement was calculated using the formula
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Statistics
For data analysis, the Student t test was performed using SigmaStat 2.03 software (SPSS (Jandel Scientific), Chicago, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Of the four class I PI3K isoforms, PI3K is preferentially expressed in cells of hemopoietic lineage. To determine the potential role of PI3K in leukocyte migration we identified an inhibitor of this isoform in a screen of our diverse chemical library. IC87114 (Fig. 1a), an analog of the original inhibitor, was synthesized and tested for PI3K selectivity relative to the other class I PI3Ks (Fig. 1b). The IC50 of IC87114 for PI3K inhibition was 0.5 µM whereas the IC50 values for PI3K, PI3K, and PI3K were >100, 75, and 29 µM, respectively (Fig. 1b). Thus IC87114 is 58-fold more selective for PI3K relative to PI3K, and over 100-fold selective relative to PI3K and PI3K. In contrast, the widely used PI3K inhibitor LY294002 demonstrated IC50 values that differed by only 
10-fold among the four class I PI3Ks (Fig. 1c), consistent with earlier reports stating that LY294002 is a nonselective PI3K inhibitor (32). Importantly, there is no concentration of LY294002 that will selectively antagonize any single class I PI3Ks. In contrast, IC87114 selectively antagonizes PI3K over at least a concentration range of 0.3–10 µM.
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(Akt 1), PKC, and PKCII by IC87114 was observed (Table I). These results show that IC87114 is a highly selective antagonist of PI3K.
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-dependent PIP3 biosynthesis in fMLP-stimulated neutrophils
In neutrophils, chemoattractants can activate both the G-regulated PI3K (p110) and the p85-associated PI3Ks (p110, p110, and p110) (29, 33, 34, 35). The p85-associated PI3Ks can be regulated by protein tyrosine kinases (PTKs) of the Src family (29, 36). We tested the effects of PI3K and PTK inhibitors on fMLP-stimulated PIP3 generation in human neutrophils. Neutrophil suspensions were pretreated with the inhibitor and then exposed to fMLP (1 µM) for 15 s to stimulate fMLP synthesis. Consistent with earlier reports, the pan PI3K inhibitor LY294002 blocked fMLP-stimulated PIP3 biosynthesis by almost 80% (Fig. 2). Similar to the report by Ptasznik et al. (29), the broad-spectrum PTK inhibitor genistein also blocked 80% of PIP3 biosynthesis induced by fMLP (Fig. 2b). These observations indicate that the p85-associated PI3Ks contribute significantly in fMLP-stimulated PIP3 biosynthesis in a PTK-dependent manner. Although neutrophils express all three p85-associated PI3Ks as well as PI3K (16), the PI3K-selective inhibitor IC87114 potently inhibited PIP3 biosynthesis by 60–65% (Fig. 2). IC87048, an analog of IC87114 and a weak inhibitor of PI3K (IC50 >50 µM), was much less effective in the inhibition of PIP3 biosynthesis. These data suggest a significant role for PI3K in production of PIP3 by neutrophils in response to fMLP stimulation.
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activity in neutrophil spreading and polarization
To determine the requirement of PI3K in fMLP-induced F-actin synthesis, untreated or IC87114-treated neutrophils were stimulated with fMLP and the changes in F-actin content were quantitated by flow cytometry. Stimulation of neutrophils with fMLP resulted in an 3-fold increase in F-actin content per cell. Pretreatment of the cells with IC87114 (5 µM) resulted only in a minor reduction of F-actin. Pairwise comparison shows that the differences in the mean fluorescence intensities between the untreated and fMLP-treated neutrophils (p < 0.01) and that of the untreated and IC87114 + fMLP-treated neutrophils (p < 0.001) are significant. However, the difference in mean fluorescence intensities between fMLP-treated and IC87114 + fMLP-treated samples was not significant. These results suggest that PI3K does not play a major role in fMLP-induced F-actin synthesis (Fig. 3).
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M2, and then were stimulated with a uniform concentration of fMLP. In the unstimulated population, the majority of the adherent neutrophils possessed a rounded morphology (mean length 9 ± 2 µm) and demonstrated colocalization (yellow-orange fluorescence) of G- (red fluorescence) and F- (green fluorescence) actin with an occasional spreading of cells and actin redistribution (Fig. 4a). In the fMLP-treated population, cells had a highly elongated morphology (mean length 17.7 ± 5 µm) with extensions of lamellipodia. In these cells, there was a distinct separation between the F- and G-actins (Fig. 4b). F-actin was localized almost exclusively at the lamellipodia while G-actin was concentrated in the cell body. Cells pretreated with the PI3K inhibitor IC87114 showed a more rounded morphology (mean length 10 ± 2 µm) with markedly few lamellipodia but demonstrated occasional long, spindly uropods containing F-actin (Fig. 4c). However, the sequestration of F- and G-actins was still observed in these cells. Quantitation of the spread of the neutrophils shows that the untreated cells had a mean area of 52.16 ± 18 µm2. Treatment of the neutrophils with fMLP resulted in a significant increase in the mean surface area to 81.38 ± 17 µm2 (p < 0.01). However, prior exposure of the fMLP-treated cells to IC87114 reduced their mean surface area to 50.65 ± 14 µm2 which is similar to the values obtained with the untreated cells (p = 0.84). These results suggest that although PI3K does not significantly inhibit F-actin synthesis and reorganization, it plays an important role in fMLP-stimulated neutrophil spreading and polarization.
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activity regulates neutrophil chemotaxis but not random locomotion
Because cell polarization is an essential element of migration against a concentration gradient of stimulus, we determined the effect of IC87114 on neutrophil migration in the "under-agarose" assay (31, 37). This technique, together with video recording and computer analysis, permitted us to quantify the resulting cell movements. As shown in Fig. 5a, the number of neutrophils migrated on ICAM-1-coated plates in 3 h was higher in the untreated sample, compared with the IC87114- (1 µM) treated sample. Not only were the total number of migrating neutrophils reduced, but the distance of migration was also diminished. We quantified the effect of IC87114 on neutrophil migration using a MI, which integrates both the number of cells and their migration distances. Thus, neutrophil migration at 1 µM IC87114 was inhibited by 75% as compared with the control (MIcontrol = 157, 672; MIIC87114 = 37, 578; Fig. 5b). Using MI values from different experiments we show that the PI3K inhibitor IC87114 blocked fMLP-induced neutrophil chemotaxis on ICAM-1 in a concentration-dependant manner (Fig. 5c). The EC50 value for IC87114-mediated inhibition of chemotaxis was 0.375 ± 0.072 µM (n = 4).
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L2 and M2 (data not shown). To determine whether the effect of IC87114 on neutrophil migration is due to inhibition of integrin binding to ICAM-1, neutrophils were allowed to adhere to ICAM-1-coated substrate in the presence of fMLP. Neutrophil adhesion was then measured at 30-min intervals. As shown in Fig. 5c, after 2 h, IC87114, at concentrations as high as 20 µM, did not inhibit fMLP-stimulated neutrophil adhesion to ICAM-1. Similar results were obtained at other time points (data not shown), suggesting that neutrophil migration was at least 40-fold more sensitive to IC87114 relative to adhesion. These results demonstrate that under our experimental conditions the effect of IC87114 on migration was not a result of blocking neutrophil adhesion.
The observed inhibition of neutrophil chemotaxis by IC87114 could be due to a disruption of either the basic cell movement machinery, or of the components involved specifically in the directional migration. To distinguish between these two possibilities, we analyzed both directional (chemotaxis toward fMLP) and random movement (chemokinesis). After neutrophils were exposed to a gradient of fMLP for 2 h, cell movements were video recorded (see Materials and Methods), and the individual neutrophil paths were traced and analyzed. An example of such analyses is shown in Fig. 6a. In this particular example, the average speed of neutrophils either in the absence of any inhibitor, or in the presence of 1 µM of the inhibitor was almost identical (2000 ± 310 µm/h vs 2130 ± 180 µm/h, respectively). However, the average speed of neutrophil directional movement in the presence of IC87114 was greatly reduced compared with the control cells. (Fig. 6a). The speed of directional migration of untreated cells was 1530 ± 360 µm/h, while that of IC87114-treated cells was 920 ± 1000 µm/h. (The apparently large SD in the latter case is due to the net negative migration of 4 of 10 cells.) Such analyses showed that IC87114 did not affect chemokinesis, although it significantly inhibited directional movement (EC50 
1 µM; Fig. 6b). These observations clearly demonstrate that rather than affecting the processes underlying adhesion and basic movement, PI3K plays a significant and selective role in chemotaxis by influencing cell polarization and directional movement.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. To our knowledge, this is the first selective small molecule inhibitor of a PI3K isoform. Selective inhibition of PI3K activity demonstrates that this target is responsible for a significant portion of fMLP-induced PIP3 generation in neutrophils, and that it is critical to chemotaxis, but not adhesion. Inhibition of PI3K resulted in an 60–65% reduction of PIP3 synthesis when neutrophils are exposed to fMLP in suspension. This is consistent with the finding that PI3K can be directly activated by G, and hence produce the initial burst of PIP3 (33, 34, 38). A role for PI3K in fMLP-induced neutrophil PIP3 generation has been further documented recently by Cadwallader et al. (39). However, inhibition of PIP3 generation by the tyrosine kinase inhibitors genistein and radicicol have also been demonstrated (29, 39) suggesting a role for the p85-associated PI3Ks as well in this process. The role of PI3Ks in cell polarization and chemotaxis has been demonstrated in diverse systems such as neutrophils and D. discoideum (9, 10, 14, 15). Similarly using the broad-spectrum PI3K inhibitor LY294002, a role of the class I PI3Ks in cell spreading has been demonstrated (40). Our data using IC87114 suggest that PI3K plays an important role in neutrophil spreading.
In comparison to its effect on the initial level of fMLP-stimulated PIP3 production in suspension, the effect of IC87114 on neutrophil migration is much more pronounced. This indicates a specific critical requirement of PI3K in PIP3 synthesis during neutrophil migration. Previously, using isoform-specific Abs, PI3K activity was shown to be important for chemotaxis of macrophage-like cells in response to CSF1 (21). These and the data presented in this study suggest that PI3K probably plays an important role in chemotaxis in a variety of hemopoietic cells. We have further studied the role of PI3K in the two components of chemotaxis, random migration and directed movement. Our analyses, using IC87114 as a selective probe, demonstrate that during fMLP-induced chemotaxis PI3K activity is not required for random locomotion; however, it appears to be essential for cell polarization and CD18 integrin-dependent chemotactic migration. Thus, PI3K plays a unique role in neutrophil chemotaxis by selectively influencing the directional component.
In p110-/- mice, neutrophils are severely defective in fMLP-stimulated PIP3 biosynthesis, suggesting a critical requirement of PI3K in the response of neutrophils to fMLP (17, 18, 19). However, the p110 null neutrophils are still able to migrate although at a slower rate (18). Recently it has been demonstrated that the defect in p110 null mice is primarily at the level of chemotaxis rather than chemokinesis (20). We propose that PI3K plays an important role during the initial burst of fMLP-induced PIP3 biosynthesis, and that PI3K plays a critical role in amplification of PIP3 production leading to polarization and chemotaxis. This is consistent with the observation that exogenous PIP3 can induce endogenous PIP3 production that is necessary for neutrophil polarization (14). The initial burst of PIP3 production could activate GTP exchange factors of the Rho/Rac family of small GTPases that can in turn activate PI3K. In particular, PI3K could be activated by Rac via the recently identified GTP exchange factor, P-Rex1, that is simultaneously activated by PIP3 and G (41). Stimulation of neutrophils by fMLP is also known to activate Ras (42) as well as the Src family tyrosine kinases (SFKs) such as Fgr (43, 44). Upon activation, Ras can activate PI3K by direct association with the p110 catalytic subunit (24). It has been shown that Hck, another member of SFKs, can also be activated by Gi (45). Fgr and Hck can activate PI3Ks via tyrosine phosphorylation of the associated p85 subunit (36). Thus, in fMLP-stimulated neutrophils PI3K can be activated by the components of heterotrimeric G proteins (Gi and G), the Ras superfamily of G proteins as well as SFKs. These signaling pathways leading to activation of PI3K and amplification of the PIP3 level are schematically presented in Fig. 7. In our model, PI3K activity is an important initial source of PIP3 in response to stimulation of G protein-coupled receptors. Signaling through these receptors leads to the activation of the Ras superfamily of GTPases and SFKs that in turn activate PI3K leading to a positive feedback loop amplifying PIP3 levels. Chan et al. (46) have demonstrated the existence of a motif on the p85 subunit that can mediate activation of the associated PI3Ks by the small GTPases H-Ras and Rac1 as well as Src, which further supports our model.
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L2 and M2 can activate SFKs like Fgr and Hck (36, 44, 47) which, as discussed above, can activate PI3K via the p85 subunit. Thus, through both chemoattractant receptors and integrin engagement pathways, PI3K can serve as a signal amplifier for neutrophil chemotaxis by further enhancing or maintaining the PIP3 level beyond that produced by PI3K.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Chanchal Sadhu, ICOS Corporation, 22021 20th Avenue SE, Bothell, WA 98021. E-mail address: csadhu{at}icos.com
3 Abbreviations used in this paper: PIP3, phosphatidylinositol (3,4,5) trisphosphate; PH, pleckstrin homology; CK1, casein kinase 1; PKB, protein kinase B; PI3K, phosphoinositide 3-kinase; PIP2, phosphatidylinositol-(4,5)-bisphosphate; PKC, protein kinase C; CHK1, checkpoint kinase 1; PTK, protein tyrosine kinase; SFK, Src family tyrosine kinase; MI, migration index; MAPK, mitogen-activated protein kinase.
Received for publication September 6, 2002. Accepted for publication December 18, 2002.
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 M. Nakhaei-Nejad, A. M. Hussain, Q.-X. Zhang, and A. G. Murray Endothelial PI 3-kinase activity regulates lymphocyte diapedesis Am J Physiol Heart Circ Physiol, December 1, 2007; 293(6): H3608 - H3616. [Abstract] [Full Text] [PDF]
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H. Ji, F. Rintelen, C. Waltzinger, D. Bertschy Meier, A. Bilancio, W. Pearce, E. Hirsch, M. P. Wymann, T. Ruckle, M. Camps, et al. Inactivation of PI3K{gamma} and PI3K{delta} distorts T-cell development and causes multiple organ inflammation Blood, October 15, 2007; 110(8): 2940 - 2947. [Abstract] [Full Text] [PDF]
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L. Liu, K. D. Puri, J. M. Penninger, and P. Kubes Leukocyte PI3K{gamma} and PI3K{delta} have temporally distinct roles for leukocyte recruitment in vivo Blood, August 15, 2007; 110(4): 1191 - 1198. [Abstract] [Full Text] [PDF]
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 C. Burelout, P. H. Naccache, and S. G. Bourgoin Dissociation between the translocation and the activation of Akt in fMLP-stimulated human neutrophils--effect of prostaglandin E2 J. Leukoc. Biol., June 1, 2007; 81(6): 1523 - 1534. [Abstract] [Full Text] [PDF]
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L. Verkoczy, B. Duong, P. Skog, D. Ait-Azzouzene, K. Puri, J. L. Vela, and D. Nemazee Basal B Cell Receptor-Directed Phosphatidylinositol 3-Kinase Signaling Turns Off RAGs and Promotes B Cell-Positive Selection J. Immunol., May 15, 2007; 178(10): 6332 - 6341. [Abstract] [Full Text] [PDF]
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F. Asperti-Boursin, E. Real, G. Bismuth, A. Trautmann, and E. Donnadieu CCR7 ligands control basal T cell motility within lymph node slices in a phosphoinositide 3-kinase- independent manner J. Exp. Med., May 14, 2007; 204(5): 1167 - 1179. [Abstract] [Full Text] [PDF]
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C. D. Wrann, N. A. Tabriz, T. Barkhausen, A. Klos, M. van Griensven, H. C. Pape, D. O. Kendoff, R. Guo, P. A. Ward, C. Krettek, et al. The Phosphatidylinositol 3-Kinase Signaling Pathway Exerts Protective Effects during Sepsis by Controlling C5a-Mediated Activation of Innate Immune Functions J. Immunol., May 1, 2007; 178(9): 5940 - 5948. [Abstract] [Full Text] [PDF]
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A. Hirayama, R. Adachi, S. Otani, T. Kasahara, and K. Suzuki Cofilin plays a critical role in IL-8-dependent chemotaxis of neutrophilic HL-60 cells through changes in phosphorylation J. Leukoc. Biol., March 1, 2007; 81(3): 720 - 728. [Abstract] [Full Text] [PDF]
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M. M. Al-Alwan, K. Okkenhaug, B. Vanhaesebroeck, J. S. Hayflick, and A. J. Marshall Requirement for Phosphoinositide 3-Kinase p110{delta} Signaling in B Cell Antigen Receptor-Mediated Antigen Presentation J. Immunol., February 15, 2007; 178(4): 2328 - 2335. [Abstract] [Full Text] [PDF]
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F. Shiroki, S. Matsuda, T. Doi, M. Fujiwara, Y. Mochizuki, T. Kadowaki, H. Suzuki, and S. Koyasu The p85{alpha} Regulatory Subunit of Class IA Phosphoinositide 3-Kinase Regulates beta-Selection in Thymocyte Development J. Immunol., February 1, 2007; 178(3): 1349 - 1356. [Abstract] [Full Text] [PDF]
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C. Grabher, A. Cliffe, K. Miura, J. Hayflick, R. Pepperkok, P. Rorth, and J. Wittbrodt Birth and life of tissue macrophages and their migration in embryogenesis and inflammation in medaka J. Leukoc. Biol., January 1, 2007; 81(1): 263 - 271. [Abstract] [Full Text] [PDF]
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N. Lehman, M. Di Fulvio, N. McCray, I. Campos, F. Tabatabaian, and J. Gomez-Cambronero Phagocyte cell migration is mediated by phospholipases PLD1 and PLD2 Blood, November 15, 2006; 108(10): 3564 - 3572. [Abstract] [Full Text] [PDF]
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 K. Horiguchi, T. Hanada, Y. Fukui, and A. H. Chishti Transport of PIP3 by GAKIN, a kinesin-3 family protein, regulates neuronal cell polarity J. Cell Biol., July 31, 2006; 174(3): 425 - 436. [Abstract] [Full Text] [PDF]
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 C. Keller, R. Hoffmann, R. Lang, S. Brandau, C. Hermann, and S. Ehlers Genetically Determined Susceptibility to Tuberculosis in Mice Causally Involves Accelerated and Enhanced Recruitment of Granulocytes Infect. Immun., July 1, 2006; 74(7): 4295 - 4309. [Abstract] [Full Text] [PDF]
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B. W. LeBlanc, J. E. Albina, and J. S. Reichner The effect of PGG-{beta}-glucan on neutrophil chemotaxis in vivo J. Leukoc. Biol., April 1, 2006; 79(4): 667 - 675. [Abstract] [Full Text] [PDF]
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 K. S. Lee, H. K. Lee, J. S. Hayflick, Y. C. Lee, and K. D. Puri Inhibition of phosphoinositide 3-kinase {delta} attenuates allergic airway inflammation and hyperresponsiveness in murine asthma model FASEB J, March 1, 2006; 20(3): 455 - 465. [Abstract] [Full Text] [PDF]
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A. Bilancio, K. Okkenhaug, M. Camps, J. L. Emery, T. Ruckle, C. Rommel, and B. Vanhaesebroeck Key role of the p110{delta} isoform of PI3K in B-cell antigen and IL-4 receptor signaling: comparative analysis of genetic and pharmacologic interference with p110{delta} function in B cells Blood, January 15, 2006; 107(2): 642 - 650. [Abstract] [Full Text] [PDF]
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C. S. Abrams A little grease helps the cell to stick Blood, July 1, 2005; 106(1): 4 - 5. [Full Text] [PDF]
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K. D. Puri, T. A. Doggett, C.-Y. Huang, J. Douangpanya, J. S. Hayflick, M. Turner, J. Penninger, and T. G. Diacovo The role of endothelial PI3K{gamma} activity in neutrophil trafficking Blood, July 1, 2005; 106(1): 150 - 157. [Abstract] [Full Text] [PDF]
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V. Pinho, D. G. Souza, M. M. Barsante, F. P. Hamer, M. S. De Freitas, A. G. Rossi, and M. M. Teixeira Phosphoinositide-3 kinases critically regulate the recruitment and survival of eosinophils in vivo: importance for the resolution of allergic inflammation J. Leukoc. Biol., May 1, 2005; 77(5): 800 - 810. [Abstract] [Full Text] [PDF]
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Y. Kotone-Miyahara, K. Yamashita, K.-K. Lee, S. Yonehara, T. Uchiyama, M. Sasada, and A. Takahashi Short-term delay of Fas-stimulated apoptosis by GM-CSF as a result of temporary suppression of FADD recruitment in neutrophils: evidence implicating phosphatidylinositol 3-kinase and MEK1-ERK1/2 pathways downstream of classical protein kinase C J. Leukoc. Biol., November 1, 2004; 76(5): 1047 - 1056. [Abstract] [Full Text] [PDF]
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A. I. Chernyavsky, J. Arredondo, L. M. Marubio, and S. A. Grando Differential regulation of keratinocyte chemokinesis and chemotaxis through distinct nicotinic receptor subtypes J. Cell Sci., November 1, 2004; 117(23): 5665 - 5679. [Abstract] [Full Text] [PDF]
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 J. Franca-Koh and P. N. Devreotes Moving Forward: Mechanisms of Chemoattractant Gradient Sensing Physiology, October 1, 2004; 19(5): 300 - 308. [Abstract] [Full Text] [PDF]
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K. Reif, K. Okkenhaug, T. Sasaki, J. M. Penninger, B. Vanhaesebroeck, and J. G. Cyster Cutting Edge: Differential Roles for Phosphoinositide 3-Kinases, p110{gamma} and p110{delta}, in Lymphocyte Chemotaxis and Homing J. Immunol., August 15, 2004; 173(4): 2236 - 2240. [Abstract] [Full Text] [PDF]
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D. G. Cronshaw, C. Owen, Z. Brown, and S. G. Ward Activation of Phosphoinositide 3-Kinases by the CCR4 Ligand Macrophage-Derived Chemokine Is a Dispensable Signal for T Lymphocyte Chemotaxis J. Immunol., June 15, 2004; 172(12): 7761 - 7770. [Abstract] [Full Text] [PDF]
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K. D. Puri, T. A. Doggett, J. Douangpanya, Y. Hou, W. T. Tino, T. Wilson, T. Graf, E. Clayton, M. Turner, J. S. Hayflick, et al. Mechanisms and implications of phosphoinositide 3-kinase {delta} in promoting neutrophil trafficking into inflamed tissue Blood, May 1, 2004; 103(9): 3448 - 3456. [Abstract] [Full Text] [PDF]
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Y. Liu, S. K. Shaw, S. Ma, L. Yang, F. W. Luscinskas, and C. A. Parkos Regulation of Leukocyte Transmigration: Cell Surface Interactions and Signaling Events J. Immunol., January 1, 2004; 172(1): 7 - 13. [Full Text] [PDF]
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 A. Kashishian, H. Douangpanya, D. Clark, S. T. Schlachter, C. T. Eary, J. G. Schiro, H. Huang, L. E. Burgess, E. A. Kesicki, and J. Halbrook DNA-dependent protein kinase inhibitors as drug candidates for the treatment of cancer Mol. Cancer Ther., December 1, 2003; 2(12): 1257 - 1264. [Abstract] [Full Text] [PDF]
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N. G. Shenoy, G. J. Gleich, and L. L. Thomas Eosinophil Major Basic Protein Stimulates Neutrophil Superoxide Production by a Class IA Phosphoinositide 3-Kinase and Protein Kinase C-{zeta}-Dependent Pathway J. Immunol., October 1, 2003; 171(7): 3734 - 3741. [Abstract] [Full Text] [PDF]
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S. Merlot and R. A. Firtel Leading the way: directional sensing through phosphatidylinositol 3-kinase and other signaling pathways J. Cell Sci., September 1, 2003; 116(17): 3471 - 3478. [Abstract] [Full Text] [PDF]
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