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Blood, Vol. 113, Issue 21, 5266-5276, May 21, 2009
Wound healing defect of Vav3–/– mice due to impaired β2-integrin–dependent macrophage phagocytosis of apoptotic neutrophils
Blood Sindrilaru et al.
113: 5266
Supplemental materials for: Sindrilaru et al
Immunofluorescence staining
Cryosections of wound tissue were fixed in ice-cold 3.7% paraformaldehyde. Infiltrating PMN and Mφ were detected using antibodies (Ab) against Ly-6G (GR-1) (clone RB6-8C5) and F4/80 (clone BM8), respectively (eBioscience). α-Smooth-Muscle-Actin (αSMA) expression was detected with anti-αSMA mAb (clone 1A4, DakoCytomation, Hamburg, Germany) and endothelial cells were identified with anti-CD31 (PECAM-1) mAb (clone MEC13.3, BD Pharmingen). TGF-β1 staining was performed with anti-LAP (TGF-β1) mAb (R&D Systems), keratinocytes were stained with anti-keratin14 (K14) (clone AF64, Covance, Münster, Germany). Alexa®488 and Alexa®555 Ab (Invitrogen) were used as secondary antibodies. Nuclei were counterstained with DAPI (Fluka, Münich, Germany). For confocal microscopy, fixed cells were permeabilized with 0.5% Triton-X. Anti-CD29 (β1-integrin, clone 18/CD29, BD Transduction Laboratories), anti-CD18 (β2-integrin, clone GAME-46, BD Pharmingen), and anti-Vav3 (Abcam, Cambridge, UK) antibodies were used for double staining. Pictures were acquired using a Zeiss Axiophot microscope (Carl Zeiss Inc, Jena, Germany), digital camera, and corresponding software (Axiocam®, Zeiss). Confocal microscopy was performed using Leica TCS SP% 63×/1.2 oil objective (Leica Microsystems), Wetzlar, Germany) and LSM 410/Axiovert 135M microscope (Zeiss). Western blot analysis
Frozen crushed tissue was dissolved in ice-cold RIPA buffer (Sigma-Aldrich) and cleared by centrifugation. Protein content was determined by Bradford test; equal amounts of protein were resolved by SDS-PAGE (Bio-Rad Laboratories, Inc., Munich, Germany) and transferred to nitrocellulose membrane (Protran®, Schleicher & Schuell, Dassel, Germany). Membranes were blocked with 5% BSA/TBS and incubated with anti-αSMA (Progen, Heidelberg, Germany), anti–TGF-βRII (Santa Cruz, Heidelberg, Germany) and anti-CD31 antibodies and re-probed for total actin (Calbiochem, Bad Soden, Germany) and vimentin (Abcam) followed by HRP-conjugated Ab (Dianova, Hamburg, Germany). Immunoreactions were detected by ECL chemiluminescence (Amersham Biosciences). Bands were digitised with a scanner (ArcusII; Agfa) and the ratio between all band densities of one blot was calculated (ImageQuantV3.3; Molecular Dynamics). Relative protein expression was normalised to the respective values for vimentin as earlier described.1 For Western blot analysis of Vav3 in bone marrow chimeras peripheral blood was collected on heparin from irradiated mice after reconstitution with bone marrow from WT and Vav3−∕− mice. After osmotic erythrocyte lysis, leukocyte pellets were dissolved in ice-cold lysis buffer (50mM Tris-Cl pH 7.6, 150mM NaCl, 1% NP-40, and a protease inhibitor cocktail) for 10min and cleared by centrifugation. Protein content was determined by the Bradford test. Equal amounts of protein were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% BSA/TBS and incubated with anti-Vav3 (Abcam) at a dilution of 1:500 and anti-actin (Calbiochem) at a dilution of 1:10000 antibodes followed by HRP-conjugated antibodies (Dianova). Immunoreactions were detected by ECL chemiluminescence. Generation of murine bone marrow-derived macrophages
Bone marrow cells were flushed from femurs and grown in DMEM supplemented with 10% heat-inactivated FCS, 10% conditioned supernatant from L929 cells, 2% L-Glutamine, Penicillin/Streptomycin 100U/100 µg/ml and 1% non-essential amino acids at 5% CO2 and 37°C.1 Six days-old mature Mφ were used in in vitro assays and for transfer experiments. Generation of murine apoptotic PMN
Mone-marrow cells were flushed from femurs and neutrophils were separated by Percoll (Sigma Aldrich, München, Germany) density gradient centrifugation. PMN collected between the 81% and 62% Percoll columns were rendered apoptotic by ageing by 22h incubation in Iscove’s medium (Sigma Aldrich) at 37°C and 5% CO2 as described.1 For adhesion and phagocytosis assays, leukocytes were incubated with 1 µM CellTracker Orange (CMRA) or 2 µM CellTracker Green (CMFDA) (Invitrogen, Karlsruhe, Germany) in serum-free medium at 37°C for 30min. Immunoprecipitation
Mφ were starved for 48h in 0.5% FCS-containing DMEM, plated on ICAM-1 or fibronectin-coated plates and incubated to adhere. At indicated time-points non-adherent cells were washed and adherent macrophages were lysed in ice-cold lysis buffer (50mM Tris-Cl pH 7.6, 150mM NaCl, 10mM NaF, 2mM Na3VO4, 10% glycerol, 1% NP-40, 1mM EDTA, and protease inhibitor cocktail (Roche, Grenzach, Germany)) for 10min. Crude lysates were cleared by centrifugation. 1mg protein lysates were incubated with 3 µg anti-Vav3 pAb (Upstate, USA) and 50:50 slurry of protein A-conjugated Sepharose beads (Amersham Biosciences, Umea, Sweden) with rotation at 4°C for 4h. Immunoprecipitates were washed 4 times, resuspended in SDS sample buffer, resolved by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were probed with anti-Phosphotyrosine mAb (4G10, Upstate), washed, and reprobed with anti-Vav3 pAb. Cytokine ELISA
To quantify TGF-β1 release during in vitro phagocytosis, WT or Vav3−∕− Mφ were co-cultured with WT or Vav3−∕− apoptotic PMN in all combinations at 37°C, and supernatants were harvested after 24h. For TGF-β1 quantification in tissue lysates snap-frozen wound tissue was ground and resuspended in RIPA buffer at 100mg/ml. Supernatants and wound lysates were assessed for TGF-β1 by specific ELISA according to the manufacturer’s protocols (R&D Systems). Flow-cytometry analysis
Successful bone marrow reconstitution was assessed on peripheral blood leukocytes from recipient and donor mice before irradiation and from irradiated mice after reconstitution with bone marrow derived from WT and Vav3−∕− mice using anti–CD45.1-FITC and anti–CD45.2-PE antibodies (all BioLegend, San Diego, California). Allele expression was assessed by flow-cytometry on a Beckton Dickinson FACScan. For analysis of integrin expression levels, bone marrow-derived Mφ and PMN derived from WT and Vav3−∕− mice were labelled with anti–F4/80-PE antibody together with either anti–CD18-FITC (clone C71/16, BD Pharmingen), anti–ICAM-1-Alexa488, or anti–CD61-Alexa488 (BioLegend). Adhesion and phagocytosis experiments with neutralizing antibodies To assess the contribution of different integrins to the activation of Mφ and subsequent release of active TGFβ1 upon phagocytosis of apoptotic PMN, WT Mφ were incubated with saturating concentrations of neutralizing antibodies against β2-integrins (anti-CD18) at 15 µg/ml, or β3-integrins (anti-CD61) at 15 µg/ml or against ICAM-1 (anti-CD54) at 15 µg/ml or with the corresponding isotype control antibodies at the same concentration (all from BioLegend) and were co-cultured with CMRA-labelled apoptotic WT PMN. After 15min of co-culture for adhesion and 45min for phagocytosis, non-adherent/ingested PMN were removed with cold PBS. Remained Mφ and Mφ/PMN complexes were collected by gentle scrapping, stained for the Mφ marker F4/80 and adherent to/PMN phagocytosing CMRA+F4/80+ Mφ were quantified by flow-cytometry. DISCUSSION Several mechanisms may contribute to the impaired recruitment of Vav3−∕− Mφ to wound sites of Vav3−∕− mice. While the transmigration of classically activated monocyte/macrophages subsets occurs independently of β2-integrins,1,2 the recently described Ly6ClowLFA-1+ subpopulation of mature monocytes depends on β2-integrins for emigration.3 Here, we did not further differentiate the recruitment of Mφ subpopulations. Therefore, we cannot reliably discriminate between the possibility that the lack of Ly6ClowLFA-1+ monocytes emigration, which may trigger the recruitment of other Mφ subsets,3 is responsible for the deficient macrophage recruitment, or alternatively, that Vav3 is required for the β1-integrin–dependent emigration of macrophages. In contrast, Vav3 and its combination with Vav1 are dispensable for PMN recruitment to wound sites, substantiating previous findings that combined Vav1/Vav3-deficiency does not affect neutrophil chemotaxis4 and accumulation at sites of immune complex deposition in a model of immune complex-mediated vasculitis.5 As neutrophil migration is essentially mediated by β2-integrins signalling via Src/Syk-kinases in a number of inflammation models6 including wound repair,1,7 our data suggests that Vav3 is not involved in the β2-integrin − Src/Syk-dependent PMN recruitment. In contrast to CD18−∕− mice with severely hampered PMN recruitment, the influx of PMN is not disturbed in Vav3−∕− mice, resulting — due to reduced numbers of Mφ, severely reduced phagocytosis of PMN and to enhanced spillage of PMN-derived proteases and ROS — to a more extended tissue damage when compared to CD18−∕− mice. In CD18−∕− mice, significantly reduced PMN numbers at wound site and a defective phagocytosis of apoptotic PMN contribute to the healing defect due to impaired phagocytic cup formation between Mφ and PMN and reduced release of active TGF-β1, similar to Vav3−∕− mice. However, in contrast to Vav3−∕− mice, in CD18−∕− mice early tissue damage by activated PMN is most likely reduced. REFERENCES 1. Peters T, Sindrilaru A, Hinz B, et al. Wound-healing defect of CD18(−∕−) mice due to a decrease in TGF-beta1 and myofibroblast differentiation. Embo J. 2005;24: 3400–3410.
2. Henderson RB, Hobbs JA, Mathies M, Hogg N. Rapid recruitment of inflammatory monocytes is independent of neutrophil migration. Blood. 2003;102: 328–335.
3. Auffray C, Fogg D, Garfa M, et al. Monitoring of blood vessels and tissues by a population of monocytes with patrolling behavior. Science. 2007;317: 666–670.
4. Gakidis MA, Cullere X, Olson T, et al. Vav GEFs are required for beta2 integrin-dependent functions of neutrophils. J Cell Biol. 2004;166: 273–282.
5. Utomo A, Hirahashi J, Mekala D, et al. Requirement for Vav Proteins in Post-Recruitment Neutrophil Cytotoxicity in IgG but Not Complement C3-Dependent Injury. J Immunol. 2008;180: 6279–6287.
6. Ley K, Laudanna C, Cybulsky MI, Nourshargh S. Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat Rev Immunol. 2007;7: 678–689.
7. Schymeinsky J, Then C, Sindrilaru A, et al. Syk-Mediated Translocation of PI3Kdelta to the Leading Edge Controls Lamellipodium Formation and Migration of Leukocytes. PLoS ONE. 2007;2: e1132.
Files in this Data Supplement:
- Figure S1. Transfer of WT or Vav3−∕− bone marrow cells in lethally irradiated WT mice resulted in complete reconstitution of the recipient mice (chimeric mice) with the donor bone marrow cells (JPG, 45.8 KB)
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(A) Flow cytometry analysis of the CD45.1 (recipient cells) and CD45.2 (donor cells) alleles on blood leukocytes from bone marrow chimeras showing complete reconstitution with CD45.2 donor cells (red line). CD45.1+ cells from congenic recipients (grey line) and CD45.2+ cells from C57BL/6 (B6) Vav3−∕− donor mice (blue line) before transplantation served as controls. (B) Western blot analysis of Vav3 in lysates of peripheral blood leukocytes isolated from bone marrow chimeras reconstituted with WT and Vav3−∕− bone marrow.
- Figure S2. β2 integrins and to a lesser extent, β3 integrins mediate macrophage adhesion to, phagocytosis of apoptotic neutrophils in vitro (JPG, 129 KB)
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(A) WT Mφ were incubated with saturating concentrations of neutralizing antibodies against CD18 (anti-CD18), ICAM-1 (anti-CD54), or CD61 (anti-CD61) or with the appropriate isotype IgG which served as negative controls (Co) and co-cultured with CMRA-labelled apoptotic WT PMN. In vitro adhesion (left panel) and phagocytosis (right panel) were assessed by flow cytometry as CMRA+F4/80+ conjugates. Results are expressed as percentages of PMN-binding or PMN-phagocytosing Mφ of the total Mφ counts (B) Active TGF-β1 concentrations measured by ELISA in supernatants of co-cultures between Mφ and apoptotic PMN which were incubated with neutralizing antibodies against either CD18 or CD54 or CD61 or with isotype control IgG. Each symbol indicates the median of triplicates. *P <0.05, **P <0.005, **P <0.001. (C) Flow cytometry analysis of WT and Vav3−∕− Mφ gated on F4/80 and PMN gated on GR-1 expression. Diagrams indicate similar expression levels of CD18, CD61, and CD54 for WT Mφ or PMN (blue line) and Vav3−∕− Mφ and PMN (red line), excluding that the defective formation of the phagocytic synapse between apoptotic PMN and Vav3−∕− Mφ is due to decreased β2-integrin (CD18) or ICAM-1 (CD54) expression levels on PMN or Mφ surface. The filled histogram shows isotype IgG, which served as a negative control.
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