Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official. Federal government websites often end in . gov or VSports app下载. mil. Before sharing sensitive information, make sure you’re on a federal government site. .

Https

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. V体育官网.

. 2003 Aug 28;550(1-3):94-100.
doi: 10.1016/s0014-5793(03)00828-7.

Rapamycin inhibits GM-CSF-induced neutrophil migration (VSports最新版本)

Julian Gomez-Cambronero  1
Affiliations

"VSports" Rapamycin inhibits GM-CSF-induced neutrophil migration

Julian Gomez-Cambronero. FEBS Lett. .

"V体育平台登录" Abstract

The molecular mechanisms that govern cell movement are the subject of intense study, as they impact biologically and medically important processes such as leukocyte chemotaxis and angiogenesis, among others. We demonstrate that leukocyte chemotaxis is prevented by the macrolide immunosuppressant rapamycin, a specific inhibitor of the mammalian target of rapamycin (mTOR)/ribosomal p70-S6 kinase (p70S6K) pathway. Both neutrophil chemotaxis and chemokinesis elicited by granulocyte-macrophage colony-stimulating factor (GM-CSF) were strongly inhibited by rapamycin with an IC(50) of 0. 3 nM. Inhibition, although at a higher dose, was also observed when the chemoattractant was interleukin-8. As for the mechanism, rapamycin targeted the increase of phosphorylation of p70S6K due to GM-CSF treatment, as demonstrated with specific anti-p70S6K immunoprecipitation and subsequent immunoblotting with anti-T(421)/S(424) antibodies. Rapamycin also inhibited GM-CSF-induced actin polymerization, a hallmark of leukocyte migration. The specificity of the effect of rapamycin was confirmed by the use of the structural analog FK506, which did not have a significant effect on chemotaxis but effectively rescued rapamycin-induced p70S6K inhibition VSports手机版. This was expected from a competitive effect of both molecules on FK506-binding proteins (FKBP). Additionally, GM-CSF-induced chemotaxis was completely (>90%) blocked by a combination of rapamycin and the MAPK kinase (MEK) inhibitor PD-98059. In summary, the results presented here indicate for the first time that rapamycin, at sub-nanomolar concentrations, inhibits GM-CSF-induced chemotaxis and chemokinesis. This serves to underscore the relevance of the mTOR/S6K pathway in neutrophil migration. .

PubMed Disclaimer

Figures (VSports app下载)

Figure 1
Figure 1. Effect of rapamycin on cell migration
(A) Freshly isolated human neutrophil suspensions were resuspended in RPMI-based chemotaxis buffer at the density of 3×106 cells/ml and incubated with the indicated concentrations of rapamycin for 30 minutes at 37 oC. Next, aliquots containing 6×105 cells were placed in the upper “insert” wells of Transwell plates and exposed to either 7 nM GM-CSF or 10 nM IL-8 for 45 minutes at 37 °C under a 5% CO2 atmosphere. (B) Effect of washing rapamycin off (indicated in the figure by “w”). Cells were incubated with the indicated concentrations of rapamycin for 30 minutes and then spun down at 600×g for five minutes. Supernatants were decanted and pellets were resuspended in fresh chemotaxis buffer before moving them to the Transwell plates. (C) Dose-response of rapamycin on IL-8- and GM-CSF-induced chemokinesis and effect of washing off the drug. Chemokinesis was accomplished by placing the cytokine (either 7 nM GM-CSF or 10 nM IL-8) in both the lower wells and the upper inserts wells of Transwell plates. 100% represents 21±1×104 cells/ml for Control, 70±6×104 cells/ml for GM-CSF and 132±8×104 cells/ml for IL-8.
Figure 2
Figure 2. Effect of rapamycin on GM-CSF-stimulated p70S6K phosphorylation and enzymatic activity
Neutrophils were incubated with 10 nM rapamycin for 30 minutes, followed by a short (5 min.) incubation with 20 nM GM-CSF and cell lysates in boiling SDS were generated and immunoprecipitation with anti-p70S6K followed. Resulting immunocomplexes were used for immunoblotting with anti-phospho-T421/S424-p70S6K antibodies (A) or with anti-p70S6K antibodies to show equal protein loading (B). (C) Detection of p70S6K enzymatic activity and its modulation by GM-CSF and rapamycin. Cells were incubated ± rapamycin for 30 min and them subjected to GM-CSF incubation. Cells lysates were derived and immunoprecipitated. Immunocomplex beads were assayed for p70S6K activity against the S6 kinase substrate peptide KKRNRTLTK. Results are the mean ± SE of three independent experiments performed in duplicate. 100% represents 1052 ± 77 dpm.
Figure 3
Figure 3. Effect of rapamycin on GM-CSF-stimulated F-actin polymerization
Several sets of neutrophil suspensions were incubated at 37 °C in the presence or the absence of 7 nM GM-CSF for 5 minutes (A); or with either 50 nM rapamycin or 50 nM FK506 (B) for 30 minutes; or with the indicated concentrations of rapamycin (C,D) or FK506 (E,F) for 30 minutes prior to the addition of GM-CSF and further incubated for 5 minutes. All stimulations were stopped at the end of the indicated lengths of time by fixing cells in formaldehyde. 200 μl-samples of 1×104 cells were incubated with phalloidin-FITC and analyzed by flow cytometry. X-axes: Log fluorescence; y-axes: Relative cell number.
Figure 4
Figure 4. FK506 and MEKi
(A) Effect on neutrophil chemotaxis. Neutrophils were incubated at 37 °C with the indicated concentrations of FK506 (group of bars to the left) or MEKi (group of bars to the right) for 30 minutes followed by an additional 30 minute-incubation with rapamycin. Cells were then placed in the upper wells of Transwell plates and allowed to migrate against GM-CSF (where indicated) for 45 minutes. The differences between control and GM-CSF-induced chemotaxis (*), as well as the inhibition caused by rapamycin over GM-CSF (**) were significant (p<0.01). The further inhibition by MEKi of the rapamycin inhibition (***) was also significant (p<0.05). Treatment with MEKi in the absence of GM-CSF exhibited curves indistinguishable from controls (as in Fig. 3B). (B) Effect on actin polymerization. Cells were treated with either MEKi alone or with a combination of MEKi and Rapamycin, stimulated with GM-CSF for 5 min, fixed, incubated with phalloidin-FITC and analyzed for flow cytometry.
See this image and copyright information in PMC

References

    1. Servant G, Weiner OD, Herzmark P, Balla T, Sedat JW, Bourne HR. Science. 2000;287:1037–1040. - PMC - PubMed
    1. Sasaki T, Irie-Sasaki J, Jones RG, Oliveira-dos-Santos AJ, Stanford WL, Bolon B, Wakeham A, Itie A, Bouchard D, Kozieradzki I, Joza N, Mak TW, Ohashi PS, Suzuki A, Penninger JM. Science. 2000;287:1040–1046. - PubMed (VSports注册入口)
    1. Li Z, Jiang H, Xie W, Zhang Z, Smrcka AV, Wu D. Science. 2000;287:1046–1049. - VSports app下载 - PubMed
    1. Nijhuis E, Lammers JW, Koenderman L, Coffer PJ. J Leukocyte Biol. 2002;71:115–124. - PubMed
    1. Crouch MF. Biochem Biophys Res Commun. 1997;233:193–199. - PubMed

Publication types (VSports)

V体育2025版 - MeSH terms

"VSports最新版本" Substances