Central Role for G Protein-Coupled Phosphoinositide 3-Kinase γ in Inflammation
Tóm tắt
Phosphoinositide 3-kinase (PI3K) activity is crucial for leukocyte function, but the roles of the four receptor-activated isoforms are unclear. Mice lacking heterotrimeric guanine nucleotide-binding protein (G protein)–coupled PI3Kγ were viable and had fully differentiated neutrophils and macrophages. Chemoattractant-stimulated PI3Kγ −/− neutrophils did not produce phosphatidylinositol 3,4,5-trisphosphate, did not activate protein kinase B, and displayed impaired respiratory burst and motility. Peritoneal PI3Kγ-null macrophages showed a reduced migration toward a wide range of chemotactic stimuli and a severely defective accumulation in a septic peritonitis model. These results demonstrate that PI3Kγ is a crucial signaling molecule required for macrophage accumulation in inflammation.
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Tài liệu tham khảo
A genomic DNA fragment of 11 kb encompassing exons 1 through 8 of the mouse PI3Kγ was used to integrate the IRES-LacZ cassette followed by the PGK-neomycin resistance gene from the pWH9 plasmid (provided by R. Fässler Lund University Sweden) in exon 2 105 base pairs downstream of the first coding ATG. Five independently targeted R1 ES cell clones were identified by Southern blot hybridization. No evidence for random integration was detected (17).
All ES cells clones were injected into C57BL/6 blastocysts. For genotyping of mice DNA derived from tail biopsies was amplified by polymerase chain reaction with two primers sets (1: 5′-GGAGAACTATGAACAACCGG-3′ 5′-CAACTTCCAGTAATGCAGGC-3′; 2: 5′-CTGCTCTTTACTGAAGGCTC-3′ 5′-CAACTTCCAGTAATGCAGGC-3′) that detect the WT and targeted allele respectively. Phenotypic analysis was performed on two lines derived from independent clones and results were confirmed in a 129sv-C57BL/6 mixed and 129sv inbred genetic background. The IRES-LacZ reporter gene under the control of the PI3Kγ promoter was expressed in peripheral blood leukocytes and in spleen macrophages [(17); for expression studies see
Bernstein H. G., Keilhoff G., Reiser M., Freese S., Wetzker R., Cell Mol. Biol. 6, 973 (1998);
]. Animal experiments were carried out in accordance with institutional guidelines.
Bone marrow cells were flushed from femurs with ice-cold RPMI 1640 medium (Gibco/BRL) supplemented with 10% fetal calf serum and antibiotics. Erythrocytes were removed by isotonic lysis and white blood cells were loaded onto a Percoll 60/80% discontinuous step gradient. PMNs were collected from the 60/80% interface after a 30-min centrifugation (4°C 520 g ). Splenocytes were depleted of macrophages (3-hour incubation at 37°C 5% CO 2 in Iscove's modified Dulbecco's medium (IMDM medium Life Technologies). Macrophages were obtained by washing the peritoneal cavity of mice 5 days after intraperitoneal injection of 1 ml of 3% thioglycollate (Difco). Antibodies to PI3K were from A. Klippel (Berlin Germany; PI3Kα) R. Wetzker (Jena Germany; PI3Kγ) B. Vanhaesebroeck (London UK; PI3Kδ) and St. Cruz Biotechnology (PI3Kβ).
E. Hirsch and M. P. Wymann unpublished data.
D. R.. Alessi and
; B. Tilton M. Andjelkovic S. A. Didichenko B. A. Hemmings M. Thelen J. Biol. Chem. 272 28096 (1997).
IL-8 (50 nM) stimulated a significant [ P < 0.05 analysis of variance (ANOVA)] increase in adhesion from 27.1% (SE = 4.8% n = 10; percentage of total administered cells) to 43.5% (SE = 6.0% n = 14) in WT but not in PI3Kγ-null neutrophils [unstimulated 27.8 ± 3.3% ( n = 10) versus 27.0 ± 2.9% ( n = 14) in the presence of IL-8]. C5a- and f MLP-induced adhesion were not significantly reduced by the loss of PI3Kγ. Teflon-coated 12-well glass slides (Marienfeld) were coated with fibronectin (20 μg/ml; Sigma) solution. Calcein-AM (Molecular Probes)–loaded PMNs (20 μl) were applied to the glass slides. After stimulation nonadherent cells were removed by washing. Fluorescence of attached cells was measured in a Bio-Tek FL600 fluorescence plate reader (excitation 485 nm 20-nm slit; emission 530 nm 25-nm slit).
Kownatzki E., Kapp A., Uhrich S., Clin. Exp. Immunol. 74, 143 (1988);
Supplemental Web data are presented on Science Online at www.sciencemag.org/feature/data/1044275.shl.
We thank L. Barberis M. F. Brizzi G. Bulgarelli-Leva G. Frascaroli W. Luini G. Montrucchio M. Laffargue and R. Calvez for their support and assistance. We also thank G. Tarone P. Defilippi L. Fumagalli and G. Topley for critically reading the manuscript; B. Vanhaesebroeck R. Wetzker A. Klippel and B. Hemmings for antibodies; C. Dahinden for C5a; and B. Moser for chemokines. Supported by the Progetto Finalizzato Biotecnologie CNR (project biotechnology) Associazione Italiana per la Ricerca sul Cancro (AIRC) San Salvatore Foundation Telethon grant E 635 and Swiss National Science Foundation grant 31-50506.97.