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Keywords:

  • Neutrophils;
  • Chemokines;
  • Transgenic/knockout;
  • Inflammation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Class I phosphoinositide 3-kinases (PI3K) are known to play a significant role in neutrophil chemotaxis. However, the relative contributions of different PI3K isoforms, and how these impact on lung inflammation, have not been addressed. In vitro studies using wild-type and PI3Kγ knockout neutrophils demonstrated the major role of the γ isoform in chemotactic but not chemokinetic events. This was confirmed by a model of direct chemokine instillation into the airways in vivo. Within all studies, a low yet significant degree of neutrophil movement in the absence of PI3Kγ could be observed. No role for the δ isoform was demonstrated both in vitro and in vivo using PI3Kδ kinase-dead knock-in mice. Moreover, further studies using the broad-spectrum PI3K inhibitors wortmannin or LY294002 showed no other class I PI3K isoforms to be involved in these chemotactic processes. Here, we identify a contributory PI3K-independent mechanism of neutrophil movement, yet demonstrate PI3Kγ as the pivotal mediator through which the majority of neutrophils migrate into the lung in response to chemokines. These data resolve the complexities of chemokine-induced neutrophilia and PI3K signaling and define the γ isoform as a promising target for new therapeutics to treat airway inflammatory diseases.

Abbreviations:
fMLP:

Formyl-Met-Leu-Phe

Gβγ:

G protein βγ subunit

GPCR:

G protein coupled receptor

MIP-2:

Macrophage inflammatory protein-2

KC:

Keratinocyte-derived chemokine

KI:

Knock-in

KO:

Knockout

PI(3,4,5)P3:

Phosphatitylinositol 3,4,5-trisphosphate

PI3K:

Phosphoinositide 3-kinase

PKB:

Protein kinase B (Akt)

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

The activation of cells by a wide variety of stimuli leads to rapid changes in 3-phosphorylated inositol lipids through the action of a family of enzymes known as phosphoinositide 3-kinases (PI3K). The PI3K have been classified into three groups according to their primary sequence and domain structure, mode of regulation and substrate specificity in vitro1. Class I PI3K have been implicated in the regulation of a diverse array of cellular responses including cell survival, mitogenesis, cell movement, secretion, glucose transport and neurite outgrowth 2. The class IA PI3K subgroup consist of three catalytic subunits, p110α, β and δ, which form heterodimers with one of five Src homology 2 (SH2) domain-containing regulatory subunits, p85α, p85β, p55γ, p55α and p50α. The class IA heterodimer can be recruited either directly to cell surface receptors, e.g. growth factor receptors, or indirectly by adaptor molecules such as Shc, Grb2 or IRS-1 2. Class IA distribution is widespread; p110α and p110β are expressed ubiquitously, whereas p110δ has been detected in leukocytes and in breast tissue and melanocytes 3. Class IB consists of one member, a heterodimer of p110γ and the regulatory subunit p101, and is activated by G protein βγ subunits (Gβγ) following the stimulation of G protein-coupled receptors (GPCR) 4, 5.

Both class IA and IB PI3K catalyze the formation of phosphatitylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] in vitro1, and all class I isoforms are inhibited by the PI3K inhibitors wortmannin and LY294002. Using these inhibitors, a number of effects of leukocyte activation have been shown to be PI3K dependent, such as superoxide production 6, actin reorganization and chemotaxis in neutrophils 7, eosinophil migration and degranulation 8. The varied cellular responses following PI3K activation are controlled in part by proteins containing pleckstrin homology (PH) domains, which bind directly to PI(3,4,5)P3 and relocate signaling complexes to the plasma membrane 9. These PH domain-containing proteins include the serine/threonine kinases, phosphoinositide-dependent kinase 1 (PDK1), protein kinase B (PKB) and members of the protein kinase C family, tyrosine kinases from the Tec family such as Btk, phospholipase C and GDP-GTP exchange factors for small GTPases, Rac and ARF6 9, 10. A potential mechanism by which differential cellular functions can be elicited by class IA and IB PI3K-induced PI(3,4,5)P3 has recently been identified in neutrophils. PI(3,4,5)P3 and Gβγ-regulated guanine nucleotide exchange factor for Rac (P-Rex1) have been found to activate Rac only in the presence of both PI(3,4,5)P3 and Gβγ, as a consequence of class IB-mediated GPCR stimulation. However, class IA/tyrosine kinase-mediated PI(3,4,5)P3 accumulation cannot stimulate P-Rex1 and therefore does not lead to Rac activation 11, 12.

The roles of p110γ and p110δ in immune cell function have been further defined by gene targeting in mice 13. Targeting of p110δ, by either the direct removal of p110δ or the generation of a catalytically dead p110δ knock-in (KI), leads to aberrant B cell development and signaling from the B cell receptor 1416. Mice lacking p110γ are healthy and viable, but display impaired neutrophil and macrophage chemotaxis in vitro and in vivo17, 18. In addition, mast cells derived from p110γ-deficient mice fail to degranulate fully in response to GPCR agonists such as adenosine, resulting in reduced edema formation in a model of passive systemic anaphylaxis 19. P110γ-deficient mice are also protected from ADP-induced platelet-dependent thromboembolic vascular occlusion. However, bleeding time in the mice was unaffected 20. Finally, p110γ negatively modulates cardiac contractility, supporting the potential of PI3Kγ inhibition in the treatment of heart failure 21.

Control of cell polarity is essential for neutrophil chemotaxis and is dependent on GPCR-mediated myosin assembly at the back edge and F-actin polymerization and phospho PKB/Akt colocalization at the leading edge of the cell 22. Further examination of neutrophil migration in p110γ knockout (KO) mice suggests that this isoform is essential for the direction of cell movement along a chemoattractant gradient by the central role it plays in mediating mechanisms at the leading edge 7. In addition, a recent in vitro study on chemotaxis by Heit et al. highlighted a PI3K versus p38 MAPK signaling hierarchy when comparing chemokine stimulation with formyl-Met-Leu-Phe (fMLP) 23. The movement of neutrophils into sites of inflammation is central to the pathology of several disease conditions, including chronic obstructive pulmonary disorder (COPD) 24 and acute respiratory distress syndrome (ARDS) 25. Indeed, a central role for PI3Kγ has been suggested by data from an intraperitoneal LPS model of lung injury, in which activation of the transcriptional regulatory factor NF-κB was reduced in PI3Kγ KO neutrophils 26.

However, the relative contributions of different PI3K isoforms on neutrophil chemotaxis into the lung have not been adequately investigated. In the present study, we use WT and p110γ KO mice to demonstrate the major role of p110γ within in vitro models of chemotaxis, and confirm these observations in vivo using a chemokine-induced model of airway inflammation. The potential contributory roles of other PI3K isoforms are addressed using inhibitors as well as PI3Kδ kinase-dead KI mice. These studies are the first to address the role of p110γ in chemokine-driven lung inflammation and to compare the relative contributions of p110γ and p110δ via mice gene-targeted for each isoform.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

In vitro analysis of PI3Kγ during early chemotactic events

To thoroughly investigate the extent of PI3Kγ KO neutrophil dysfunction in response to chemokines, in vitro analysis of a range of PI3K-dependent events was undertaken. The chemokines macrophage inflammatory protein-2 (MIP-2) and keratinocyte-derived chemokine (KC) were used as stimuli, as they represent the murine equivalents of IL-8 and GROα, both of which have long been established as neutrophil chemoattractants 27, 28. One of the earliest signaling events following activation of PI3K is the phosphorylation of PKB/Akt. Following a 1-min stimulation with either MIP-2 or KC, phosphorylation of PKB/Akt was diminished in KO neutrophils by between 70% and 80%, respectively (Fig. 1a).

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Figure 1. Early chemotactic events are impaired in KO neutrophils in vitro. (a) PKB/Akt phosphorylation (Ser473) in WT (solid bars) and KO (hatched bars) neutrophils stimulated with 3 nM MIP-2 or KC for 1 min. Representative phosho- and total Akt gels are also shown. (b) Shape change analysis of WT and KO neutrophils in response to 3 nM MIP-2 or KC stimulation for 5 min, measured by mean fluorescent density. Significant differences (Student's t-test) between WT and KO responses are shown (*, p<0.05; **, p<0.005).

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Membrane ruffling is another cellular event that precedes cell movement and can be observed via FACS analysis as a shift along the forward scatter axis, indicating a change in cell size. Taking the dramatic changes seen in WT neutrophil mean fluorescent density as 100%, KO neutrophils were compared upon stimulation with either MIP-2 or KC. Shape change was reduced by 90% and 80%, respectively, in KO cells (Fig. 1b).

Chemotaxis across a 3-µm membrane in response to chemokines was also measured. Migration of KO neutrophils was reduced by up to 55% or 40% in response to MIP-2 or KC, respectively, when compared to WT cells (Fig. 2a). Chemokinesis was also measured by addition of increasing concentrations of chemokine stimuli to the upper side of the membrane, thus removing the gradient. Chemokinesis was found to be unaffected in neutrophils lacking PI3Kγ, with approximately one third of WT and KO cells passing through the membrane in response to undirected chemokine activation (Fig. 2b). No addition of chemokine stimuli to either side of the membrane results in near zero cell movement (Fig. 2b). When chemokinesis was taken into account, the chemotactic component is reduced in KO neutrophils by up to 75% and 60% in response to MIP-2 and KC, respectively (Fig. 2a).

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Figure 2. KO neutrophil chemotaxis, but not chemokinesis, is impaired in vitro. (a) Chemotaxis of WT and KO neutrophils in response to 3 nM MIP-2 or KC stimulation for 90 min, measured as a percentage of the total number of neutrophils present in unfractionated bone marrow. (b) Chemotaxis versus chemokinesis analyzed by addition of increasing concentrations of MIP-2 to the above filter insert until an equimolar concentration was reached, also shown (dotted line) in Fig. 2a. Levels of chemotaxis plus chemokinesis are also shown in response to no stimuli. Significant differences (Student's t-test) between WT and KO responses are shown (*, p<0.05; **, p<0.005).

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Role of PI3Kγ during single-phase neutrophil influx into the lung in response to chemokine

To confirm in vitro observations of greatly reduced, though not completely inhibited, neutrophil migration, MIP-2 or KC was directly instilled into WT and KO mice intra-nasally. WT mice responded comparatively to either MIP-2 or KC, generating early peaks of neutrophil influx at 5 h, which by 14 and 32 h were approaching background levels in both WT and KO mice (Fig. 3a, b). BAL neutrophil levels in KO mice were reduced by up to 65% and 75% in MIP-2- and KC-treated animals, respectively. The KO response was similarly inhibited at later time points, although in comparatively low numbers (Fig. 3a, b). No IL-1β or TNF-α, cytokines also associated with neutrophilia 29, could be detected in BAL supernatants (data not shown).

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Figure 3. Chemokine-induced neutrophil influx into the lung is impaired in vivo. (a) Neutrophils migrated into the BAL fluid of WT (solid bars) and KO (hatched bars) mice in response to saline or 30 µg/kg MIP-2 administered intra-nasally and measured at 5, 14 and 32 h. (b) As above with KC as stimulus. Significant differences (Student's t-test) between WT and KO responses are shown (*, p<0.05; **, p<0.005).

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The role of PI3Kδ and other PI3K isoforms in residual PI3Kγ-independent neutrophil chemotaxis

In all parameters studied thus far, low yet significant levels of residual neutrophil chemotactic function were observed. Recent studies using a selective inhibitor of PI3Kδ have suggested a role for this isoform in neutrophil chemotaxis 30. To determine whether a PI3Kδ-dependent mechanism was required in our experimental systems, neutrophils from PI3Kδ kinase-dead KI mice 14 were compared with PI3Kγ KO neutrophils in vitro. Upon MIP-2 stimulation, no reduction in PKB/Akt phosphorylation was observed between WT and PI3Kδ KI neutrophils (Fig. 4a), whereas a 60% drop was seen in PI3Kγ KO cells (Fig. 4b). A control of isoform-non-specific PI3K inhibition was included using the broad-spectrum PI3K inhibitor wortmannin. At 100 nM, wortmannin was able to ablate PKB/Akt phosphorylation in WT, PI3Kδ KI and PI3Kγ KO cells. These data suggest an involvement of other PI3K isoforms, though not PI3Kδ, in the phosphorylation of PKB/Akt following GPCR stimulation and also demonstrate 100 nM to be an appropriate dose of wortmannin, capable of blocking all PI3K isoform signaling via the PKB/Akt pathway. The absence of PI3Kδ function in neutrophil chemotaxis was further demonstrated upon investigation of chemotaxis, with no significant reduction in cell movement observed (Fig. 4c), compared with a 50% drop in PI3Kγ KO neutrophils (Fig. 4d). These data also demonstrate no effect on chemokinesis in the absence of PI3Kδ. Importantly, 100 nM wortmannin treatment reduced levels of chemotaxis by up to 60%, but was unable to significantly reduce chemotaxis below the level seen with PI3Kγ KO neutrophils. LY294002 was also employed as another broad-spectrum PI3K inhibitor, yielding comparable results in WT, PI3Kδ KI (Fig. 4e) and PI3Kγ KO cells (Fig. 4f).

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Figure 4. Role of PI3Kγ, PI3Kδ and other PI3K isoforms during neutrophil chemokine stimulation in vitro. (a) PKB/Akt phosphorylation (Ser473) in WT (solid bars) and PI3Kδ KI (dotted bars) neutrophils, pretreated with saline or 100 nM wortmannin for 20 min, then stimulated with 3 nM MIP-2 for 1 min. Representative phosho- and total Akt gels are also shown. (b) PKB/Akt phosphorylation of WT (solid bars) and PI3Kγ KO (hatched bars) neutrophils treated as above. (c) Chemotaxis of WT (solid bars) and PI3Kδ KI (dotted bars) neutrophils in response to 3 nM MIP-2 stimulation for 90 min, following pretreatment with saline or 100 nM wortmannin. (d) Chemotaxis of WT (solid bars) and PI3Kγ KO (hatched bars) neutrophils treated as above. (e) Chemotaxis of WT (solid bars) and PI3Kδ KI (dotted bars) neutrophils in response to 3 nM MIP-2 stimulation for 90 min, following pretreatment with saline or 10 µM Ly294002. (f) Chemotaxis of WT (solid bars) and PI3Kγ KO (hatched bars) neutrophils treated as above. Significant differences (Student's t-test) compared to the saline-treated WT response are shown (*, p<0.05; **, p<0.005). Significant differences compared to the saline-treated PI3Kδ KI or PI3Kγ KO response are also shown (, p<0.05; ††, p<0.005).

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To determine whether similar PI3Kγ-dependent and PI3K isoform-independent processes occur in vivo, PI3Kγ KO or PI3Kδ KI and their equivalent WT mice were given a single 50 µg/kg wortmannin i.v. dose, 1 h prior to MIP-2 instillation. As with in vitro chemotaxis, no difference in in vivo neutrophil influx into the lung was observed between saline-treated WT and PI3Kδ KI mice. Wortmannin treatment reduced levels of neutrophil influx by up to 60%, but not any further (Fig. 5a). However, a reduction of neutrophil influx was typically seen in saline-treated PI3Kγ KO mice. Furthermore, wortmannin treatment reduced WT neutrophil levels by a similar extent to that seen in treated PI3Kδ KI mice and, importantly, not significantly below the level seen in PI3Kγ KO mice (Fig. 5b). These data demonstrate that PI3Kδ has no role in murine neutrophil chemotaxis, which is predominantly PI3Kγ mediated. Other PI3K isoforms, though possibly involved in PKB/Akt phosphorylation, do not play a functional role in the chemotactic process. The residual cell movements observed both in vitro and in vivo are mediated via a wortmannin-resistant (or LY294002-resistant) and therefore class I PI3K-independent mechanism.

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Figure 5. Role of PI3Kδ, PI3Kγ and other PI3K isoforms during chemokine-induced neutrophil influx into the lung. (a) Neutrophil migration into the BAL fluid of WT (solid bars) and PI3Kδ KI mice (dotted bars), 5 h following intra-nasal MIP-2 instillation. (b) Neutrophil migration into the BAL fluid of WT (solid bars) and PI3Kγ KO mice (hatched bars), 5 h following intra-nasal MIP-2 instillation. Mice were given saline or 50 µg/kg wortmannin i.v. 1 h prior to MIP-2 instillation. Significant differences (Student's t-test) compared to the saline-treated WT response are shown (*, p<0.05; **, p<0.005). Significant differences compared to the saline-treated PI3Kδ KI or PI3Kγ KO response are also shown (, p<0.05; ††, p<0.005).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

The data presented in this study demonstrate the pivotal role played by PI3Kγ in murine neutrophil chemotaxis during an airway inflammatory response; this is the first investigation to determine the relative contributions of p110γ and p110δ via the direct comparison of gene-targeted mice. The specific and central role for PI3Kγ in chemokine-stimulated GPCR-mediated neutrophil chemotaxis was established both in vitro and in vivo. Following stimulation with either MIP-2 or KC chemokine, neutrophils were analyzed for PKB/Akt phosphorylation (an early consequence of GPCR stimulation), changes in cell morphology associated with early chemotaxis, and cell movement itself. Neutrophils lacking PI3Kγ displayed a large reduction in these parameters, confirming the pivotal role of class IB PI3K in all stages of GPCR-driven chemotactic events. Chemotaxis versus chemokinesis was also investigated, with no role for PI3Kγ in non-directional movement demonstrated, a finding in agreement with several studies proposing PI3Kγ function as a ‘compass’ for the cell, orchestrating F-actin polymerization and phospho-PKB/Akt colocalization at the leading edge of a migrating neutrophil 7, 31. However, in all in vitro systems, residual chemotactic function was still identified in KO neutrophils, over and above that which could be ascribed to chemokinesis. The residual chemotactic function observed is largely in agreement with in vitro studies investigating PI3Kγ and cell movement. Incomplete inhibition of neutrophil chemotaxis toward GPCR agonists 7, 17 and partially inhibited macrophage migration to MCP-1 32 have been observed 1719. Indeed, intra-nasal instillation of either MIP-2 or KC stimulated neutrophil influx into the lung, and although greatly reduced in PI3Kγ KO mice, residual neutrophils were still evident. These experiments confirm the central role played by PI3Kγ in chemotaxis, but also further allude to a GPCR-mediated, γ isoform-independent chemotactic process unattributable to any non-GPCR mechanism investigated (e.g. IL-1β or TNF-α).

Studies addressing the question of whether the mechanisms responsible for the residual chemotaxis observed are dependent on other PI3K isoforms remain inconclusive. Here, we employ PI3Kγ KO mice, PI3Kδ enzyme-dead KI mice and broad-spectrum inhibitors of all PI3K to determine the role of PI3K isoforms in neutrophil chemotaxis. A recent study used an inhibitor with 40-fold selectivity for PI3Kδ over PI3Kγ to analyze the role of the δ isoform in neutrophil chemotaxis. Blockade of PI3Kδ did not affect F-actin synthesis, although morphological changes were seen, which were interpreted as PI3Kδ acting as an amplifier of the PI(3,4,5)P3 levels initiated by PI3Kγ activation 30, 33. A further study hinted at a role for PI3Kδ in neutrophil adhesion, although this was restricted to effects on TNF-α-primed epithelium rather than the function of the neutrophil itself 34. Our studies demonstrate that neutrophils from PI3Kδ KI mice show no reduction in PKB/Akt phosphorylation or chemotaxis (including chemokinesis) when compared to WT cells. These data were confirmed in vivo, demonstrating no involvement for the δ isoform following GPCR signaling and chemotaxis. Wortmannin is a potent inhibitor of PI3K, forming a covalent link with a conserved lysine residue in the ATP-binding pocket of the enzyme 35. The extensive use of wortmannin has helped to establish roles for PI3K in phagocytosis-induced respiratory burst 36, neutrophil responses 6, mast cell function 37 and T cell chemotaxis in response to RANTES stimulation 38. Wortmannin has also been demonstrated as a useful tool for PI3K inhibition within longer-term in vivo studies of eosinophil migration and granule release 8. As PI3Kδ proved unlikely to be involved in neutrophil chemotaxis during an inflammatory response, wortmannin was used to determine whether other PI3K isoforms could play a role. The dose of wortmannin used in vitro was able to completely inhibit all Akt/PKB phosphorylation, a signaling element downstream of all PI3K, and therefore an indicator of isoform non-specific PI3K activation 10. However, despite inhibiting all PI3K signaling, wortmannin was unable to inhibit chemotaxis below the reduced level seen with PI3Kγ KO neutrophils. To demonstrate that the response seen to pharmacological intervention was not peculiar to wortmannin, LY294002 was also employed as another broad-spectrum inhibitor (non-covalent) of PI3K 39, yielding comparable results. These data were confirmed in vivo, demonstrating the action of a PI3K- (and PKB/Akt-) independent process responsible for the residual neutrophil chemotaxis observed in the absence of PI3Kγ.

Studies of chemotaxis in other cell types such as B and T lymphocytes have demonstrated a potential involvement of class IA PI3K, in concert with PI3Kγ 40, 41. Here, we demonstrate that this is not the case for neutrophils and must therefore postulate on what other mechanisms may account for the residual chemotaxis we observe. Potential candidate mechanisms for wortmannin-insensitive/PI3K-independent mechanisms of chemotaxis have previously been proposed. Recent studies identifying a mechanism of F-actin polymerization and pseudopod extension using insulin as a primer proved wortmannin insensitive, but could be blocked by inhibitors of Src kinase and NADPH oxidase 42, 43. This suggests a PI3K-independent mechanism of Rac activation which had previously been identified by inhibitor-based studies of fMLP-stimulated human neutrophils 12, 44. Use of wortmannin at the concentrations detailed in this study inhibits all class I PI3K, but not necessarily C2α, a member of the class II family of PI3K, which may be up to 100-fold less sensitive to wortmannin inhibition 45. When concentrations of wortmannin are greatly increased (>10 µM), chemotaxis is completely ablated (data not shown), due to known non-specific inhibition of several signaling pathways 15, 46. These may include C2α inhibition or effect myosin light chain, which has been identified as the key to contractile movement at the back edge of a migrating cell 22.

In this study, we employ both KO and kinase-dead KI strategies, as well as pharmacological tools, to resolve the complexities of chemokine-induced neutrophilia and PI3K signaling. Taken together, these data demonstrate the pivotal role played by PI3Kγ in GPCR-driven neutrophil movement into the lung during an airway inflammatory response. We also identify a contributory mechanism of neutrophil chemotaxis, independent of all class I PI3K isoforms, which is evident both in vitro and in vivo. This study confirms PI3Kγ as the key to lung neutrophilia in response to chemokines, which, when combined with the important role of PI3Kγ in mast cell function 19, identifies this isoform as an attractive drug target for the treatment of airway inflammatory diseases.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Mice

129Sv PI3Kγ KO 17 and 129Sv WT mice were bred under specific pathogen-free conditions at Charles River (UK). C57BL/6 PI3Kδ KI 14 and C57BL/6 WT mice were bred under specific pathogen-free conditions at UCL. Animals were housed at 24°C in a 12-h light-dark cycle. Food and water were accessible ad libitum. The studies reported here conformed to the UK Animals (scientific procedures) Act 1986.

Materials

Endotoxin-free FBS and BSA, and wortmannin were obtained from Sigma-Aldrich (Poole, Dorset, UK). LY294002 was obtained from Calbiochem (Nottingham, UK). Anti-phospho-PKB/Akt (Ser473) and anti-PKB/Akt antibodies were obtained from New England Biolabs (Beverly, MA). NuPAGE reagents were from Invitrogen Life Technologies (Carlsbad, CA). Quantikine ELISA kits were obtained from R&D Systems (Abingdon, UK). PBS (w/o Ca2+ and Mg2+), RPMI 1640 (w/o phenol red) were purchased from Invitrogen (Paisley, UK). Percoll was obtained from Amersham Biosciences (Buckinghamshire, UK). All chemokines and cytokines were obtained from Peprotec (London, UK). HTS Multiwell Insert System plates were purchased from BD Biosciences (Oxford, UK).

Models of airway inflammation

Female and male PI3Kγ KO, PI3Kδ KI mice and their WT equivalents (5–7 weeks old, groups of n=12) under halothane/oxygen/nitrous oxide anesthesia, were intra-nasally challenged with a 30 µg/kg dose of murine recombinant MIP-2 or KC. The stimulus was given in 50 µl sterile PBS, with PBS alone used as a control. For inhibition studies, wortmannin was diluted in PBS and a 50 µg/kg dose was given in a single 50-µl i.v. injection into a tail vein 1 h prior to chemokine instillation. BAL with 4×0.3 ml PBS was performed at various time points following challenge. Differential cell counts were determined by cytospin of 200 µl BAL fluid, followed by Diff-quick staining. Total cell numbers were counted by hemocytometer following centrifugation at 300×g for 10 min, removal of BAL supernatant and resuspension of the cell pellet in 400 µl methyl violet stain (0.01% methyl violet in 1.5% acetic acid). BAL supernatant was taken for cytokine measurement.

Isolation of bone marrow neutrophils

Neutrophils were isolated from the femur bone marrow of WT and KO mice for PKB phosphorylation and shape change studies. Following removal of the distal epiphyses, bone marrow was flushed from the femurs using a syringe containing cold PBS (w/o Ca2+ and Mg2+). Cells were then centrifuged at 300×g, resuspended in 5 ml and placed on top of a 20-ml 65%:75% Percoll density gradient diluted in PBS. Following centrifugation at 1,500×g for 30 min at 4°C, the interface was removed. Recovered cells were washed in 50 ml PBS substituted with 2% FBS, then resuspended in preparation for assay. Isolated neutrophils were found to be >90% pure by FACS analysis (data not shown).

Determination of PKB/Akt activation

Bone marrow-derived mouse neutrophils (5×105 cells in 30 µl) were stimulated with chemokine (3 nM in 30 µl) for 1 min at 37°C. Samples were then placed on ice, and 20 µl 4× NuPAGE LDS sample buffer was added to stop the reaction, followed by 10 µl NuPAGE reducing agent. For inhibition studies, wortmannin was added 20 min prior to chemokine stimulation.

Samples (20 µl) were loaded on a 4–12% Bis-Tris NuPAGE gel. Protein was electrotransferred to a nitrocellulose membrane and then blocked with 1% BSA in 10 mM PBS with 0.1% Tween-20. After blocking, the membrane was incubated overnight at 4°C with rabbit polyclonal phospho-PKB/Akt-specific primary antibody (at 1/1,000 dilution), followed by anti-rabbit IgG alkaline-phosphatase-coupled secondary antibody (at 1/2,000 dilution). After washing five times, the bands were detected using ECF Western blotting detection reagents (Amersham Pharmacia Biotech). The membranes were then stripped using the Reblot kit from Chemicon and reprobed with a rabbit polyclonal antibody specific for total PKB/Akt. Densitometry was performed using a chemiluminescence system and analysis software (Image Quant) to determine the ratio between phosphorylated and total PKB/Akt. Data is presented as phospho-PKB/Akt divided by total PKB/Akt band intensity.

Shape change assay

Isolated WT or KO neutrophils were resuspended at 5×106 cells/ml in PBS (w/o Ca2+, Mg2+) + 0.1% BSA. Cells were rested for 30 min at 18°C, then centrifuged at 300×g for 10 min and resuspended at 5×105 cells/ml. Meanwhile, 10 µl of agonist/chemokine was placed at a 10× concentration in polypropylene FACS tubes, to which 90 µl of cells were added, briefly agitated, then incubated at 37°C for 5 min in a water bath. Ice-cold Cellfix solution (250 µl) was then added to each tube; then incubated on ice for 10 min before FACS analysis (FACSCalibur/CellQuest software; BD Biosciences, Oxford, UK). Changes in mean fluorescent density from saline-stimulated controls were compared, taking the WT shift as 100%.

Chemotaxis assay

WT or KO bone marrow was flushed as described, and any debris removed by centrifugation. Unfractionated cells were resuspended at 2×106 cells/ml in assay buffer [RPMI (w/o phenol red) + 2% FBS]. Chemokine stimulus was diluted in assay buffer to 3 µM, and 1-ml aliquots were placed in the lower chambers of a HTS Multiwell Insert System. Buffer only was included as a negative control, and 500 μl buffer:cell suspension was also included to represent maximal chemotaxis. The 3-µm pore filter insert plate was then placed in the lower chambers, and 500 µl of cell suspension was added (buffer only to maximal wells). For inhibition studies, 100 nM wortmannin or 10 µM LY294002 was added to the cells 20 min prior to loading, remaining present for the duration of the assay. Plates were then incubated at 37°C, 5% CO2 for 90 min. Inserts were then removed, and the number of cells that had moved across the filter membrane was quantified by FACS analysis. Auto-fluorescence was measured, first in a chemokine-responsive sample (i.e. WT), from which a neutrophil population was gated based on size and granularity. This gate was then superimposed onto a maximal sample to ascertain the maximum number of neutrophils that could have moved by chemotaxis across the filter, thus providing a percentage value of neutrophils that did so in the responsive sample. The cell movement measured represents chemotaxis and chemokinesis. Chemokinesis was determined by adding increasing amounts of chemokine to the above filter insert until an equimolar concentration was reached, thus activating the cells but removing the gradient.

Statistical analysis

Results are expressed as mean ± standard error of the mean (SEM). Statistical significance (p<0.05) was determined using a one-tailed, unpaired Student's t-test.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

B.V. was funded by the Ludwig Institute for Cancer Research and the UK Biotechnology and Biological Sciences Research Council. B.V., M.P.W. and E.H. had a grant from the European Union Fifth Framework (Program QLG1–2001–02171/BBW 00.0564–1). M.P.W. was supported by a research grant from Novartis, Horsham, and the Swiss National Science Foundation (3100–064906.01).

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