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First published online January 10, 2008
doi: 10.1242/10.1242/jcs.020412

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PI3K accelerates, but is not required for, neutrophil chemotaxis to fMLP

¹ Immunology Research Group, Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, T2N 4N1, Canada
² Department of Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5E5, Canada
³ Calistoga Pharmaceuticals, Seattle, WA 98121, USA

View larger version (20K): [in this window] [in a new window]   Fig. 1. The role of PI3K in mediating long-term migration to fMLP. Neutrophils were either uninhibited (UT, C57Bl/6), pan-PI3K-inhibited (LY294002), PI3K inhibited (IC87114), wild type (C57Bl/6) or PI3K–/–. (a,e) Number of migrating human (a) or murine (e) neutrophils to fMLP. (b,f) Directionality of human (b) or murine (f) neutrophil migration to fMLP. (c,g) Speed of human (c) or murine (g) neutrophil migration to fMLP. (d) Migratory distance of (d) human neutrophil over 2 hours or (h) murine neutrophils over 4 hours to fMLP. n>4; *P<0.05, ANOVA with Bonferroni correction.

View larger version (32K): [in this window] [in a new window]   Fig. 2. The role of PI3K in mediating initial responses to fMLP and IL8. Human neutrophils were either uninhibited or treated with a pan-PI3K inhibitor (LY294002). fMLP or IL8 was added at time=0 and the cells monitored for 20 minutes. (a) Percentage of cells that polarized in response to fMLP. (b) Percentage of cells orientated towards the source of fMLP. (c) Speed of migration in response to fMLP. (d) Percentage of cells polarized in response to IL8. (e) Percentage of cells orientated towards the source of IL8. (f) Speed of migration in response to IL8. n=4 for all data.

View larger version (34K): [in this window] [in a new window]   Fig. 3. The role of PI3K and PI3K in mediating initial responses to fMLP. Neutrophils were either uninhibited human cells (uninhibited), uninhibited murine cells (C57Bl/6), treated with a PI3K-specific inhibitor (IC87114) or isolated from PI3K–/– mice. fMLP was added at time=0 and the cells monitored for 20 minutes. (a) Percentage of human cells that polarized in response to fMLP. (b) Percentage of human neutrophils orientated towards fMLP. (c) Speed of human neutrophil migration in response to fMLP. (d) Percentage of murine cells that polarized in response to fMLP. (e) Percentage of murine neutrophils orientated towards the source of fMLP. (f) Speed of murine neutrophil migration in response to fMLP. (g) Percentage of murine cells that polarized in response to fMLP. (h) Percentage of murine neutrophils orientated towards the source of fMLP. (i) Speed of murine neutrophil migration in response to fMLP. (j) Adhesion of murine neutrophils to ICAM1 pre- and post-fMLP stimulation. n=4 for all data.

View larger version (15K): [in this window] [in a new window]   Fig. 4. The role of p38 MAPK during neutrophil chemotaxis to fMLP. (a) Human neutrophils treated with the p38 MAPK inhibitor SB20580 did not chemotax to fMLP in a long-term chemotactic assay. (b) Murine neutrophils treated with the p38 MAPK inhibitor SB203580, or deficient in MK2, did not chemotax to fMLP in a long-term chemotactic assay. (c) Murine neutrophils treated with the p38 MAPK inhibitor SB203580, or deficient in MK2, did not polarize in response to fMLP. n=4 for all data; *P<0.05, ANOVA with Bonferroni's correction.

View larger version (18K): [in this window] [in a new window]   Fig. 5. The role of PI3K in mediating in vivo recruitment and chemotaxis in response to fMLP. Mice are either wild type (C57Bl/6) or PI3K–/– (PI3K KO). (a) Number of rolling cells passing through the blood vessel per minute. (b) Number of neutrophils adhering to the endothelium per 100 µm of blood vessel. (c) Number of neutrophils emigrating from a single blood vessel, within one field of view. (d) Directionality (C.I.) of in vivo neutrophil migration to fMLP. (e) Speed of in vivo neutrophil migration to fMLP. (f) Delay between emigration and initiation of chemotaxis in vivo in response to fMLP. n>4 for all data; *P<0.05, Student's t-test.

View larger version (19K): [in this window] [in a new window]   Fig. 6. In vivo recruitment in CXCR2–/– mice in response to fMLP. (a) Velocity of rolling cells. (b) Number of rolling cells passing through the blood vessel per minute. (c) Number of neutrophils adhering to the endothelium per 100 µm of blood vessel. (d) Number of neutrophils emigrating from a single blood vessel, within one field of view. n=3 for all data; no significant difference, ANOVA with Bonferroni's correction.

View larger version (15K): [in this window] [in a new window]   Fig. 7. Number of adhering cells that emigrate or detach in wild-type and PI3K–/– neutrophils in response to fMLP. Animals were either wild type (C57Bl/6) or PI3K–/– (PI3K KO). Data are expressed as the percentage of adherent cells and are presented as mean ± s.e.m. n=3 for all data; *P<0.05 compared to wild-type, Student's t-test.