Phosphoinositide 3-OH kinase regulates integrin-dependent processes in neutrophils by signaling through its effector ARAP3 - PubMed (original) (raw)

Phosphoinositide 3-OH kinase regulates integrin-dependent processes in neutrophils by signaling through its effector ARAP3

Laure Gambardella et al. J Immunol. 2013.

Abstract

ARAP3, a GTPase activating protein for Rho and Arf family GTPases, is one of many phosphoinositide 3-OH kinase (PI3K) effectors. In this study, we investigate the regulatory input of PI3K upstream of ARAP3 by analyzing neutrophils from an ARAP3 pleckstrin homology (PH) domain point mutation knock-in mouse (R302, 303A), in which ARAP3 is uncoupled from activation by PI3K. ARAP3 PH domain point mutant neutrophils are characterized by disturbed responses linked to stimulation by either integrin ligands or immobilized immune complexes. These cells exhibit increased β2 integrin inside-out signaling (binding affinity and avidity), and our work suggests the disturbed responses to immobilized immune complexes are secondary to this. In vitro, neutrophil chemotaxis is affected in the mutant. In vivo, ARAP3 PH domain point mutant bone marrow chimeras exhibit reduced neutrophil recruitment to the peritoneum on induction of sterile peritonitis and also reduced inflammation in a model for rheumatoid arthritis. The current work suggests a dramatic regulatory input of PI3K into the regulation of β2 integrin activity, and processes dependent on this, by signaling through its effector ARAP3.

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Figures

Figure 1. Incorporating a PH domain point mutation does not affect ARAP3 expression

(A) Protein from 1×106 bone marrow derived neutrophils from control (Arap3+/+), heterozygous (Arap3+/PH*) or homozygous mutant (Arap3PH*/PH*) mice was immunoblotted for ARAP3 (top panel) or β-COP (bottom panel) as a loading control. Representative experiment shown from four independent experiments. (B) Peripheral blood cells from bone marrow chimeras reconstituted with Arap3PH*/PH* (PH*) or matched control (WT) hematopoietic stem cells were analysed using a Vet ABC animal blood cell counter. Data shown (means ±SEM) were obtained with 14 wild-type and 12 knock-in chimeras generated with three individual bone marrow donors per genotype.

Figure 2. Integrin-dependent events are upregulated in Arap3PH*/PH* neutrophils

(A-F) Bone marrow-derived Arap3PH*/PH* (PH*) and matched wild-type control (WT) neutrophils were prepared, primed with TNFα and GM-CSF or mock primed (A,D) and (all) pre-incubated with luminol as described in materials and methods. 5×105 cells were plated into 96 well plates containing fMLF as a soluble stimulus (A,D) or that had been coated with fibrinogen (B,E) or polyRGD (C,F). Light emission was measured in a Berthold Microluminat Plus luminometer. Data were recorded in duplicate. Data (means ± range) from a representative experiment are shown in panels A-C whilst panels D-F represent accumulated light emissions (means ± SEM) from four separate, pooled experiments expressed as a percentage of the responses obtained with control neutrophils. (G-I) Neutrophils were allowed to adhere to heat inactivated serum-blocked (hiFCS) or polyRGD-coated plastic. Lysates were prepared and immunoblotted with antibodies specific for phospho-PKB (Ser 473) or phospho-p38 (T180, Y182) or β-COP as a loading control. A representative example is shown (G). Blots were quantitated using ImageJ software; integrated data obtained from five independent experiments are shown (H,I). (J,K) Gelatinase granule release was measured by zymography of supernatants of neutrophils that were allowed to adhere to hiFCS-blocked or fibrinogen-coated dishes. As a control, neutrophils were stimulated with fMLF in the presence of cytochalasin B (CB). A representative experiment is shown (J; the samples were run on two separate gels and are pasted next to one another for ease of viewing here, this is indicated by a dotted line). Integrated, quantitated data obtained from four independent experiments are plotted (K). (L-N) Neutrophils were allowed to adhere to pRGD-coated tissue culture dishes, washed and fixed. Numbers of attached cells (phase dark) per field of view, and the percentage of spread cells (phase light) were counted. Integrated data obtained from three separate experiments (L,M) and representative examples (N) are shown. (All) Raw data were analysed by T-test (Mann Whitney); * p<0.05; ** p<0.01; ***p<0.001.

Figure 3. β2 integrins on _Arap3_PH*/PH*neutrophils have higher affinity and avidity

(A) Wild-type control and _Arap3_PH*/PH* bone marrow cells were or were not pre-incubated with 20ng/ml TNFα at 37°C before being labelled with PE-conjugated anti GR1, APC-conjugated anti Mac1 and FITC-conjugated anti LFA1. For FACS analysis, GR1-positive cells were gated and Mac1 and LFA1 staining was measured. Results were analysed with FlowJo V6.4.7 software. Cells from 15 knock-in and 11 control chimeras obtained from three separate reconstitutions were analysed in three experiments. Representative traces are shown. black lines, wild-type; grey lines, _Arap3_PH*/PH* ; broken lines, no TNFα, full lines, with TNFα. (B) Unstimulated, bone marrow derived control (WT) and _Arap3_PH*/PH* (PH*) neutrophils were allowed to bind to ICAM1-Fc in solution in the absence of any stimulation. Cells were washed, fixed and bound ICAM1 was stained with a fluorescently conjugated secondary antibody. Washed, stained cells were allowed to settle on electrostatically coated slides and signal strength was measured by quantitative immunofluorescence. Mean fluorescence intensity obtained from 64 knock-in and 50 control cells in three independent experiments is plotted (mean ± SEM) and representative examples are shown. (C) Integrin clustering was analysed in unstimulated control and _Arap3_PH*/PH* neutrophils in solution. Bone marrow derived neutrophils were prepared and incubated at 37C in the presence of anti LFA1 or anti Mac1, washed, fixed, stained with a fluorescently conjugated secondary antibody and allowed to settle on electrostatically labelled slides. Distribution of fluorescence was assessed by confocal microscopy. Representative examples (stained for LFA1) are shown together with their corresponding heatmaps (obtained by analysis with ImageJ). Pooled results stem from four independent experiments, in each of which 25 cells per genotype were analysed are plotted (mean ± SEM). (B,C). Results obtained were analyed by paired T-tests. * p<0.05; ** p<0.01.

Figure 4. Immune complex-induced events are upregulated in Arap3PH*/PH* neutrophils

(A,B) Bone marrow-derived Arap3PH*/PH* (PH*) and matched wild-type control (WT) neutrophils were prepared and pre-incubated with luminol as described in materials and methods. 5×105 cells were plated into 96 well plates that had been coated with an immune-complex (BSA anti-BSA). Light emission was measured in a Berthold Microluminat Plus luminometer. Data were recorded in duplicate. Data (means ± range) from a representative experiment are shown in panel A whilst panel B represents accumulated light emissions (means ± SEM) from four independent experiments expressed as a percentage of the responses obtained with wild-type control neutrophils. (C-E) Neutrophils were allowed to adhere to heat inactivated BSA-blocked (BSA) or immune complex-coated (BSA-αBSA) plastic. Lysates were prepared and subjected to immunoblotting with antibodies specific for phospho-PKB (Ser 473) or phospho-p38 (T180, Y182) or β-COP as a loading control. A representative example is shown (C). Blots were quantitated using ImageJ software; integrated data obtained from five independent experiments are shown (D,E). (F,G) Gelatinase granule release was measured by zymography of supernatants of neutrophils that were allowed to adhere to BSA-blocked or immune complex-coated dishes. A representative experiment is shown (F; the samples were not in this order on the original gel and have been pasted next to one another for ease of viewing, this is indicated by a dotted line). Integrated, quantified data obtained from four independent experiments are plotted (G). (H-J) Neutrophils were allowed to adhere to immune complex-coated tissue culture dishes, washed and fixed. Numbers of attached cells (phase dark) per field of view, and the percentage of spread cells (phase light) were counted. Integrated data obtained from three separate experiments are plotted (I,J) and representative examples are shown (H). (All) Raw data were analysed by T-test (Mann Whitney). * p<0.05; ** p<0.01; ***p<0.001.

Figure 5. FcγR are not affected in _Arap3_PH*/PH* neutrophils

(A) Surface distribution of FcγRII/III and FcγRIV was analysed by FACS analysis of bone marrow cell populations. Seven wild-type and seven knock-in chimeras obtained from three different pairs of donors were analysed in two independent experiments. A representative example is shown. Black lines represent control cells and grey lines _Arap3_PH*/PH*. FcγRII/III FACS: broken lines represent unstimulated and full lines TNFα-stimulated samples; FcγRIV FACS: dotted lines represent isotype control. (B) Immune complex induced ROS formation depends on signalling through FcγRs. Neutrophils were or were not pre-incubating with FcγR blocking antibodies and/or isotype controls as indicated, before being plated onto immobilised immune complexes for ROS assays. Pooled results from three independent experiments are plotted (mean ± SEM). Differences between wild-type and _Arap3_PH*/PH* were highly significant (p<0.001) for all conditions except anti-FcγRII/III + anti-FcγRIV (p>0.05), two-way ANOVA with Bonferroni post-test. (C) Cross-linking FcγRII/III does not cause increased ROS production in _Arap3_PH*/PH* neutrophils. Bone marrow derived neutrophils were prepared from control and knock-in mice and incubated with anti FcγRII/III with or without priming. After washing, ROS production was measured on cross-linking the FcγR with a F(ab’)2 fragment. A representative example is shown (left) and integrated results from five independent experiments are plotted (mean ± SEM). Differences did not reach statistical significance (p>0.1; Mann Whitney T-test). (D) Control and _Arap3_PH*/PH* neutrophils were incubated with FcγRII/III antibody, washed, and stimulated by F(ab’)2 fragment-mediated cross-linking for the indicated amounts of time. Time 0, no cross-linking. Lysates were prepared and protein subjected to immunoblotting. PKB and p38 phosphorylation was analysed using phospho-specific antibodies; β-COP served as loading control. A representative experiment from four independent experiments is shown.

Figure 6. RhoA activation is affected in _Arap3_PH*/PH* neutrophils

(A-C) Wild-type control (WT) and _Arap3_PH*/PH* (PH*) neutrophils were kept in suspension or plated onto polyRGD-coated tissue culture plastic. Cells were lysed with ice-cold lysis buffer. (A, B) Clarified lysates were subjected to ‘pull-down’ assays using GST-Ral GDS as bait to determine GTP-loaded fractions of Rap (A) and using GST-MT2 bait to determine GTP-loaded Arf6 (B). Results obtained from a minimum of five independent experiments were pooled and plotted (mean ± SEM, left); representative examples are shown (right). L, lysates, PD, pull-downs. Blots were probed with an antibody specific for Rap1 (A) and Arf6 (B). (C) Clarified lysates were employed in RhoA G-LISA assays to determine GTP-loaded RhoA. Results from five pooled, independent experiments are presented (mean ± SEM). (A-C) Raw data were analysed by paired T-tests. * p<0.05; ** p<0.001. (D) Indirect assessment of localisation of RhoA activation. Neutrophils were or were not stimulated for the indicated time with 1μM fMLF in solution, allowed to settle on a glass coverslip for 3 minutes, fixed and stained using phalloidin, to visualise filamentous actin and anti phospho myosin light chain. Representative cells from three independent experiments are shown.

Figure 7. _Arap3_PH*/PH* neutrophils have a chemotaxis defect

(A-F) In vitro chemotaxis assays. Bone marrow derived _Arap3_PH*/PH* and control neutrophils were allowed to chemotax towards 300nM fMLF (A-C) or towards 10nM MIP2 (D-F) in Dunn chambers. Movements were recorded by time-lapse imaging. (A,D) Pooled tracks of individual cells from experiments carried out with three separate cell preparations were plotted using the Ibidi chemotaxis tool plug-in into ImageJ. The source of chemoattractant is at the below. The tracks were analysed using the Ibidi chemotaxis tool’s statistics features. Accumulated and Euclidian distances and directionality are plotted (mean ± SEM; B,C; E,F). (G) Neutrophil recruitment to the peritoneum in a model for sterile peritonitis. Bone marrow chimeras (generated with four bone marrow donors per genotype) were intraperitoneally injected with 0.25ml thioglycollate containing broth. Mice were sacrificed 4.5 hours after injection, their peritonea were flushed and Mac1high GR1-positive neutrophils were counted. Pooled results obtained from two separate experiments are plotted. (B-G) Data were analysed by T-tests (Mann Whitney). * p<0.05; *** p<0.001. (H-I) Serum transfer arthritis. Twelve wild-type and 13 _Arap3_PH*/PH* bone marrow chimeras were injected with 150μl arthritic serum and six wild-type and six _Arap3_PH*/PH* bone marrow chimeras were injected with 150μl control serum in two separate experiments. Joints were scored daily for two weeks. Ankle thickness and clinical score are plotted. Circles, and dotted lines, wild-type bone marrow chimeras; triangles and full lines, _Arap3_PH*/PH* bone marrow chimeras. Blue symbols, control serum; red symbols, arthritogenic serum. The area under the graph was compared by T-test (Mann Whitney); ankle thickness, p=0.053; clinical score, p=0.023. (I) Wax sections of decalcified joints from chimeras reconstituted with control (WT) or _Arap3_PH*/PH* (PH*) bone marrows induced as indicated were H&E stained to visualise leukocyte infiltration on day 4 after serum injection. Representative examples from sections obtained with 6 arthritogenic and 2 control serum-injected mice mice in two independent experiments are shown.

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Phosphoinositide 3-OH kinase regulates integrin-dependent processes in neutrophils by signaling through its effector ARAP3 - PubMed (original) (raw)