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The role of phosphoinositide 3-kinases in neutrophil migration in 3D collagen gels.

Martin KJ, Muessel MJ, Pullar CE, Willars GB, Wardlaw AJ - PLoS ONE (2015)

Bottom Line: AS-605240 markedly reduced CXCL8 induced chemokinetic migration but had no effect on CXCL8 induced chemotactic migration.In contrast PIK-75 inhibited chemotactic migration but not chemokinetic migration.This study suggests that PI3 Kinase is necessary for CXCL8 induced migration in a 3D tissue environment but that chemokinetic and chemotactic migration may be controlled by different isoforms with gamma shown to be important in chemokinesis and alpha important in chemotaxis.

View Article: PubMed Central - PubMed

Affiliation: Institute for Lung Health, Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, United Kingdom.

ABSTRACT
The entry of neutrophils into tissue has been well characterised; however the fate of these cells once inside the tissue microenvironment is not fully understood. A variety of signal transduction pathways including those involving class I PI3 Kinases have been suggested to be involved in neutrophil migration. This study aims to determine the involvement of PI3 Kinases in chemokinetic and chemotactic neutrophil migration in response to CXCL8 and GM-CSF in a three-dimensional collagen gel, as a model of tissue. Using a three-dimensional collagen assay chemokinetic and chemotactic migration induced by CXCL8 was inhibited with the pan PI3 Kinase inhibitor wortmannin. Analysis of the specific Class I PI3 Kinase catalytic isoforms alpha, delta and gamma using the inhibitors PIK-75, PIK-294 and AS-605240 respectively indicated differential roles in CXCL8-induced neutrophil migration. PIK-294 inhibited both chemokinetic and chemotactic CXCL8-induced migration. AS-605240 markedly reduced CXCL8 induced chemokinetic migration but had no effect on CXCL8 induced chemotactic migration. In contrast PIK-75 inhibited chemotactic migration but not chemokinetic migration. At optimal concentrations of GM-CSF the inhibitors had no effect on the percentage of neutrophil migration in comparison to the control however at suboptimal concentrations wortmannin, AS-605240 and PIK-294 inhibited chemokinesis. This study suggests that PI3 Kinase is necessary for CXCL8 induced migration in a 3D tissue environment but that chemokinetic and chemotactic migration may be controlled by different isoforms with gamma shown to be important in chemokinesis and alpha important in chemotaxis. Neutrophil migration in response to suboptimal concentrations of GM-CSF is dependent on PI3 Kinase, particularly the gamma and delta catalytic isoforms.

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Related in: MedlinePlus

Wortmannin inhibits both CXCL8 and GM-CSF-mediated neutrophil migration.Neutrophils were pre-treated for 30 mins with 50nM wortmannin, and then stimulated with a) CXCL8 (100ng/ml) or b) GM-CSF (0.5ng/ml, 50ng/ml). Results are shown as mean ±SEM (n = 7) except for DMSO controls and 0.5ng/ml GM-CSF where n = 3, ***p<0.001.
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pone.0116250.g003: Wortmannin inhibits both CXCL8 and GM-CSF-mediated neutrophil migration.Neutrophils were pre-treated for 30 mins with 50nM wortmannin, and then stimulated with a) CXCL8 (100ng/ml) or b) GM-CSF (0.5ng/ml, 50ng/ml). Results are shown as mean ±SEM (n = 7) except for DMSO controls and 0.5ng/ml GM-CSF where n = 3, ***p<0.001.

Mentions: PI3K is thought to play an important role in cell migration. The pan-inhibitor wortmannin was used initially in order to determine the importance of PI3K in neutrophil motility in our 3D assay. Treatment of neutrophils with 50nM wortmannin for 30 minutes prior to the addition of chemoattractant significantly inhibited CXCL8-induced migration in both the gradient and non-gradient assays (Fig. 3a). In the non-gradient assay treatment of cells with wortmannin (50nM, 30 min pre-treatment) significantly reduced migration in response to stimulation with 0.5ng/ml GM-CSF but had no significant effect on migration in response to stimulation with 50ng/ml GM-CSF (Fig. 3b). The vehicle control for wortmannin (0.3% DMSO) had no significant effect on neutrophil migration induced by either CXCL8 or GM-CSF (Fig. 3a, b). These data highlight that PI3K is necessary for neutrophil migration in 3D gels in response to stimulation with CXCL8 and a sub-optimal concentration of GM-CSF.


The role of phosphoinositide 3-kinases in neutrophil migration in 3D collagen gels.

Martin KJ, Muessel MJ, Pullar CE, Willars GB, Wardlaw AJ - PLoS ONE (2015)

Wortmannin inhibits both CXCL8 and GM-CSF-mediated neutrophil migration.Neutrophils were pre-treated for 30 mins with 50nM wortmannin, and then stimulated with a) CXCL8 (100ng/ml) or b) GM-CSF (0.5ng/ml, 50ng/ml). Results are shown as mean ±SEM (n = 7) except for DMSO controls and 0.5ng/ml GM-CSF where n = 3, ***p<0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4320071&req=5

pone.0116250.g003: Wortmannin inhibits both CXCL8 and GM-CSF-mediated neutrophil migration.Neutrophils were pre-treated for 30 mins with 50nM wortmannin, and then stimulated with a) CXCL8 (100ng/ml) or b) GM-CSF (0.5ng/ml, 50ng/ml). Results are shown as mean ±SEM (n = 7) except for DMSO controls and 0.5ng/ml GM-CSF where n = 3, ***p<0.001.
Mentions: PI3K is thought to play an important role in cell migration. The pan-inhibitor wortmannin was used initially in order to determine the importance of PI3K in neutrophil motility in our 3D assay. Treatment of neutrophils with 50nM wortmannin for 30 minutes prior to the addition of chemoattractant significantly inhibited CXCL8-induced migration in both the gradient and non-gradient assays (Fig. 3a). In the non-gradient assay treatment of cells with wortmannin (50nM, 30 min pre-treatment) significantly reduced migration in response to stimulation with 0.5ng/ml GM-CSF but had no significant effect on migration in response to stimulation with 50ng/ml GM-CSF (Fig. 3b). The vehicle control for wortmannin (0.3% DMSO) had no significant effect on neutrophil migration induced by either CXCL8 or GM-CSF (Fig. 3a, b). These data highlight that PI3K is necessary for neutrophil migration in 3D gels in response to stimulation with CXCL8 and a sub-optimal concentration of GM-CSF.

Bottom Line: AS-605240 markedly reduced CXCL8 induced chemokinetic migration but had no effect on CXCL8 induced chemotactic migration.In contrast PIK-75 inhibited chemotactic migration but not chemokinetic migration.This study suggests that PI3 Kinase is necessary for CXCL8 induced migration in a 3D tissue environment but that chemokinetic and chemotactic migration may be controlled by different isoforms with gamma shown to be important in chemokinesis and alpha important in chemotaxis.

View Article: PubMed Central - PubMed

Affiliation: Institute for Lung Health, Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, United Kingdom.

ABSTRACT
The entry of neutrophils into tissue has been well characterised; however the fate of these cells once inside the tissue microenvironment is not fully understood. A variety of signal transduction pathways including those involving class I PI3 Kinases have been suggested to be involved in neutrophil migration. This study aims to determine the involvement of PI3 Kinases in chemokinetic and chemotactic neutrophil migration in response to CXCL8 and GM-CSF in a three-dimensional collagen gel, as a model of tissue. Using a three-dimensional collagen assay chemokinetic and chemotactic migration induced by CXCL8 was inhibited with the pan PI3 Kinase inhibitor wortmannin. Analysis of the specific Class I PI3 Kinase catalytic isoforms alpha, delta and gamma using the inhibitors PIK-75, PIK-294 and AS-605240 respectively indicated differential roles in CXCL8-induced neutrophil migration. PIK-294 inhibited both chemokinetic and chemotactic CXCL8-induced migration. AS-605240 markedly reduced CXCL8 induced chemokinetic migration but had no effect on CXCL8 induced chemotactic migration. In contrast PIK-75 inhibited chemotactic migration but not chemokinetic migration. At optimal concentrations of GM-CSF the inhibitors had no effect on the percentage of neutrophil migration in comparison to the control however at suboptimal concentrations wortmannin, AS-605240 and PIK-294 inhibited chemokinesis. This study suggests that PI3 Kinase is necessary for CXCL8 induced migration in a 3D tissue environment but that chemokinetic and chemotactic migration may be controlled by different isoforms with gamma shown to be important in chemokinesis and alpha important in chemotaxis. Neutrophil migration in response to suboptimal concentrations of GM-CSF is dependent on PI3 Kinase, particularly the gamma and delta catalytic isoforms.

Show MeSH
Related in: MedlinePlus