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

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.

Fig 1. Neutrophil migration is dose dependent in both the gradient and non-gradient assays.

a) migration in response to CXCL8, *significantly greater as compared to control cells, p<0.05, n = 3 except for non gradient control and 100ng/ml where n = 5. b) The direction of movement for each neutrophil in the non gradient and gradient assay in response to 100ng/ml of CXCL8 is illustrated in the vector diagrams which show chemokinetic migration in the non-gradient assay and a chemotactic pattern of migration in the gradient assay. c) Neutrophil migration to GM-CSF is dose dependent in both the non gradient and gradients assays. *significantly greater as compared to control cells, p<0.05, n = 3 except for gradient control and 50ng/ml where n = 7 and 6 respectively. d). The direction of movement for each neutrophil in the non gradient and gradient assay in response to 50ng/ml of GM-CSF is illustrated in the vector diagrams, These show that the pattern of migration to GM-CSF was chemokinetic in both the gradient and non-gradient assays.

Fig 2. Neutrophils express the class I PI3K catalytic subunits α,δ and γ.

RNA was isolated from unstimulated neutrophils. RT-PCR was carried out using primers for each of the four class I PI3-kinase catalytic isoforms and β-actin as a control. n = 1 representative of three experiments. The identity of each of the bands was confirmed by excising the band and sequencing the DNA product.

Fig 3. 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.

Fig 4. An antagonist of PI3Kγ inhibits CXCL8 mediated chemokinetic but not chemotactic migration and GM-CSF mediated chemokinetic migration at a sub-optimal concentration of GM-CSF.

Neutrophils were treated with 10μM PI3Kγ selective inhibitor for 30 mins prior to the addition of CXCL8 (100ng/ml, a) or GM-CSF (0.5ng/ml, 50ng/ml, b). Data shown are mean ±SEM (n = 3) except for non gradient CXCL8 where n = 4 and GM-CSF where n = 6 ***p<0.001.

Fig 5. An antagonist of PI3Kδ inhibits both chemotactic and chemokinetic migration mediated by CXCL8 and GM-CSF-.

Neutrophils were treated with 1μM and 10μM of the PI3Kδ selective inhibitor PIK-274 for 30 mins prior to the addition of CXCL8 (100ng/ml, a) or 0.5ng/ml GM-CSF (b). Data shown are mean ±SEM (n = 3) except for gradient DMSO and 1μM of PIK-274 where n = 8. *p<0.05, ***p<0.001.

Fig 6. An antagonist of PI3Kα inhibits CXCL8 induced chemotactic migration in a dose and time dependent manner.

a) Neutrophils were pre-treated for 30 mins with varying concentrations (from 0.1μM to 10μM) of the PI3Kα selective inhibitor PIK75, and then stimulated with 100ng/ml CXCL8 in the gradient assay. b) Neutrophils were pre-treated with 1μM of PIK-75 for 30 minutes or 2 hours, prior to construction of the migration gel and the addition of 100ng/ml of CXCL8 in the gradient assay. Results are shown as mean ±SEM; n = 4, except for CXCL8, 1μM and the corresponding DMSO control where n = 11 ***p<0.001, **p<0.01. Antagonists of PI3Kδ and α have additive effects on inhibition of CXCL8 mediated neutrophil chemotaxis. Neutrophils were pre-treated for 30 mins with either 2μM of the PI3Kα selective inhibitor, PIK-75, 2μM of the PI3Kδ selective inhibitor, PIK-274 or 1μM of each and then stimulated with 100ng/ml CXCL8. Results are shown as mean ±SEM (n = 4) *p<0.05. (Fig. 6c)

Fig 7. CXCL8 and GM-CSF induce the phosphorylation of Akt.

Neutrophils were treated with wortmannin (50nM) or the PI3K (1M), (10M) and (10μM) selective inhibitors for 2 min in combination with 500ng/ml of CXCL8 (a,b) or 50ng/ml GM-CSF (c,d). Western blots were carried out using the antibodies Phosphorylated Akt and total Akt (a,c). Densitometry was performed on the western blot films (b,d). The western blots shown are an example of 1 of the 3 carried out which all demonstrated the same pattern. In Fig. 7b CXCL8+DMSO significantly increased phosphorylation of Akt compared to the unstimulated control (p<0.05) and PIK-75 (PI3kinase α) caused significant inhibition of phosphorylation (p<0.05). In Fig. 7d the PI3kinase δ inhibitor PIK-294 significantly inhibited GM-CSF+DMSO mediated phosphorylation (p<0.05)