Combined immunodeficiency with severe inflammation and allergy caused by ARPC1B deficiency
2016; Elsevier BV; Volume: 140; Issue: 1 Linguagem: Inglês
10.1016/j.jaci.2016.09.061
ISSN1097-6825
AutoresTaco W. Kuijpers, Anton T. J. Tool, Ivo van der Bijl, Martin de Boer, Michel van Houdt, Iris M. De Cuyper, Dirk Roos, Floris van Alphen, Karin van Leeuwen, Emma L. Cambridge, Mark J. Arends, Gordon Dougan, Simon Clare, Ramiro Ramírez‐Solis, Steven T. Pals, David J. Adams, Alexander B. Meijer, Timo K. van den Berg,
Tópico(s)Blood disorders and treatments
ResumoOne of the major protein complexes in actin polymerization and cellular motility is the Arp2/3 complex, consisting of 7 polypeptides.1Campellone K.G. Welch M.D. A nucleator arms race: cellular control of actin assembly.Nat Rev Mol Cell Biol. 2010; 11: 237-251Crossref PubMed Scopus (684) Google Scholar Two of the subunits are actin-related proteins of the Arp2 and Arp3 subfamilies. The remaining 5 regulatory subunits are referred to as ARPC1 (actin-related protein complex-1), ARPC2, ARPC3, ARPC4, and ARPC5. ARPC1 is present in 2 isoforms in humans, ARPC1A and ARPC1B as a WD40 repeat-containing protein, encoded by different genes, whereas the other ARPC subunits do not contain common sequence motifs. Arp2/3 gene deletions result in embryonic lethality in the mouse.2Yae K. Keng V.W. Koike M. Yusa K. Kouno M. Uno Y. et al.Sleeping beauty transposon-based phenotypic analysis of mice: lack of Arpc3 results in defective trophoblast outgrowth.Mol Cell Biol. 2006; 26: 6185-6196Crossref PubMed Scopus (42) Google Scholar The genetic defects in the regulatory proteins for cytoskeletal rearrangements cause different syndromes, mostly dominated by blood and immune phenotypes.3Al-Herz W. Bousfiha A. Casanova J.L. Chatila T. Conley M.E. Cunningham-Rundles C. et al.Primary immunodeficiency diseases: an update on the classification from the International Union of Immunological Societies expert committee for primary immunodeficiency.Front Immunol. 2014; 5: 162Crossref PubMed Scopus (378) Google Scholar The activities of nucleation-promoting factors for actin polymerization are mostly regulated by signal-transduction pathways, one of which involves the activation by the Rho-family guanosine triphosphatases CDC42 and RAC2, and the Wiskott-Aldrich Syndrome protein (WASP) family as the intermediary Arp2/3 activator that can control actin assembly downstream of these small guanosine triphosphatases.4Takenawa T. Suetsugu S. The WASP-WAVE protein network: connecting the membrane to the cytoskeleton.Nat Rev Mol Cell Biol. 2007; 8: 37-48Crossref PubMed Scopus (703) Google Scholar, 5Thrasher A.J. Burns S.O. WASP: a key immunological multitasker.Nat Rev Immunol. 2010; 10: 182-192Crossref PubMed Scopus (296) Google Scholar Here we describe the first human genetic defect in a component of the Arp2/3 complex itself. The ARPC1B mutation results in a combined immunodeficiency with symptoms of immune dysregulation and a mild bleeding tendency, caused by defective actin polymerization in the immune cells, in a 7-year-old male patient born as the first child of consanguineous, healthy Moroccan parents. He has one younger unaffected brother. The first weeks of his postnatal development were uneventful, and his umbilical cord detached spontaneously. At 2 months of age, he was admitted because of gastric bleeding during a febrile period of clinical gastroenteritis. A gastroscopy showed profuse bleeding in the presence of normal coagulation and liver function tests in the presence of a striking leukocytosis (between 25.2 and 55.8 × 106/mL), a normochromic anemia and a mild thrombocytopenia. The white blood cell count slowly improved spontaneously during further follow-up, whereas the thrombocytopenia persisted (see Table E1 in this article's Online Repository at www.jacionline.org). The mean platelet volume was repeatedly found to be normal (mean of 8.8 fL, n = 9 samples; normal range 7.4-11.7 fL). His second admission was due to an auricular infection (perichondritis by Staphylococcus aureus), which was treated appropriately, but resulted in scarring of his right pinnacle (Fig 1, A). At the age of 5 months, he presented again with a clinical picture of fever and purpuric lesions on his legs, arms (Fig 1, B and C), and scrotum, and poor wound healing (data not shown). Skin biopsy showed a clear leukocytoclastic vasculitis with multiple microthrombi in the vascular lesions (Fig 1, D). We measured autoantibodies against nuclear antigens (ANA, ENA), neutrophil cytoplasmic proteins (ANCA-IFT, MPO, and PR3), platelet antigens, and lupus anticoagulants (LAC), and found all tests to be negative (data not shown). He suffered from 2 similar episodes of vasculitis in the presence of a high number of eosinophils (peaking at 3350 cells/μL at 25 months of age during an infection) that were treated with corticosteroids for 3 months each. His eosinophil counts remained within the normal range thereafter (Table E1). At the age of 4 years, he suffered from a period of prolonged bloody diarrhea from which Salmonella typhimurium was cultured due to a serious pan-colitis with neutrophil and eosinophil infiltration in the biopsies of his colon (Fig 1, E). Recurrent pneumonias that responded to antibiotics have led to mild bronchiectasis. Apart from infections, he developed serious eczema and showed anaphylaxis after ingestion of nuts. The immunoglobulin spectrum shows increased IgA and IgE (Table E1). Because of the early leukocytosis and initial bleeding tendency, we excluded leukocyte adhesion defects, including LAD-III.6Kuijpers T.W. van de Vijver E. Weterman M.A. de Boer M. Tool A.T. van den Berg T.K. et al.LAD-1/variant syndrome is caused by mutations in FERMT3.Blood. 2009; 113: 4740-4746Crossref PubMed Scopus (176) Google Scholar The most eminent in vitro findings consisted of the neutrophil defect in motility and directed movement (chemotaxis) due to an F-actin polymerization defect (Fig 1, F and G); a result supported by confocal analysis (see Fig E1, A, in this article's Online Repository at www.jacionline.org), adhesion was unimpaired (data not shown). Enhanced azurophil granule release on cell activation (see Fig E2 in this article's Online Repository at www.jacionline.org) was noted by the release of proteolytic activity and the upregulation of CD63 as an integral membrane marker for azurophil granules, but normal activation of nicotinamide adenine dinucleotide phosphate oxidase, phagocytosis, and killing of S aureus and Escherichia coli (Fig E2; data not shown). The initial bleeding tendency seemed to be associated with a very mild platelet dysfunction in a double-colored aggregation assay designed previously to determine platelet function under conditions of reduced platelet counts when standard aggregometry tests fail to be accurate (see Fig E3, A, in this article's Online Repository at www.jacionline.org).7De Cuyper I.M. Meinders M. van de Vijver E. de Korte D. Porcelijn L. de Haas M. et al.A novel flow cytometry-based platelet aggregation assay.Blood. 2013; 121: e70-e80Crossref PubMed Scopus (40) Google Scholar Also, detection of glycoprotein IIb/IIIa integrin activation and upregulation of CD62P and CD63 from the granules were tested and compared with control platelets similarly activated (Fig E3, B). Although spreading of patient platelets was different from control platelets (Fig E1, B) and CD62P and CD63 upregulation was slightly reduced, glycoprotein IIb/IIIa activation seemed intact. Clinically, the bleeding tendency was not apparent anymore after these initial bleeding events during further follow-up in the presence of a mild thrombocytopenia. Exome sequencing failed to pick up a mutation (because of the default “variant caller” parameter settings in this complex genetic defect), but subsequent proteomics analysis of the platelets and neutrophils indicated a complete lack of ARPC1B (Fig 2, A-D). ARPC1B is an essential hematopoietic component of the Arp2/3 complex for F-actin polymerization. The ARPC1B deficiency was caused by a homozygous complex frameshift mutation in the ARPC1B gene (c.491_495TCAAGdelCCTGCCCins), as confirmed by additional targeted sequencing approaches with improved, customized “variant caller” parameter settings for the detection of complex mutations (see Fig E4 in this article's Online Repository at www.jacionline.org). The complex nature of the mutation in this family might be the consequence of a double strand break repaired by nonhomologous end joining, or by a microhomology-mediated end-joining mechanism.8Kent T. Chandramouly G. McDevitt S.M. Ozdemir A.Y. Pomerantz R.T. Mechanism of microhomology-mediated end-joining promoted by human DNA polymerase θ.Nat Struct Mol Biol. 2015; 22: 230-237Crossref PubMed Scopus (195) Google Scholar, 9Rodgers K. McVey M. Error prone repair of DNA double-strand breaks.J Cell Physiol. 2016; 231: 15-24Crossref PubMed Scopus (217) Google Scholar Proteomics analysis demonstrated the presence of the Arp2/3 complex in normal neutrophils, T cells, and platelets to consist of ARPC1B, ARPC2, ARPC3, ARPC4, ARPC5, and ARPC5L, but there was an absence of ARPC1A. Because ARPC1B was also expressed in tissue cells, the migration defect observed in neutrophils was expected to be present in primary fibroblasts as well. However, these cells showed normal migratory behavior, and we may conclude that the nonhematopoietic expression of ARPC1A is apparently sufficient to rescue the fibroblasts from a detectable defect (see Fig E5 in this article's Online Repository at www.jacionline.org). Expression of ARPC1A and ARPC1B were variably present in additional nonhematopoietic cell lines (HeLa, SKBR3, and HEK273), indicating redundancy of ARPC1 proteins, whereas only ARPC1B protein was detected in hematopoietic cell lines (Daudi, Ramos, Jurkat, NB4, U937), which is similar to the various findings with fibroblasts versus blood cells tested (data not shown). Together, these novel data on ARPC1 protein expression may well explain why this ARPC1B defect manifests primarily as a hematological and immunological disease. When Arpc1b−/− mice (Arpc1btm1a(EUCOMM)Wtsi) were generated and compared with the patient (see Table E2 in this article's Online Repository at www.jacionline.org) as part of a phenotyping screen, no overt changes in whole blood cell counts were found. However, plasma total IgE levels were increased at 16 weeks (0 ± 0 ng/mL in wild-type mice vs 220.6 ± 266.9 ng/mL in Arpc1b−/− mice). In 75% of the Arpc1b-deficient mice, mild inflammation of the blood vessels was observed, targeting the aorta and/or the mesenteric and pancreatic arteries (Fig 2, E). On being challenged with the attenuated S typhimurium M525 and unlike their wild-type equivalents, Arpc1b−/− mice showed signs of moderate salmonellosis by day 3 postinfection, with 8 of 18 mice having to be sacrificed by day 5 (Fig 2, F). In contrast, all wild-type mice survived the challenge. The fact that ARPC1B is the only ARPC1 isoform in hematopoietic cells, whereas both ARPC1A and ARPC1B are present in tissue cells, suggests a differential use of Arp2/3 activities in tissue and blood cells. Severe food allergies, eczema, and autoimmunity are also observed in WASP- and WIP-deficient patients causing Wiskott-Aldrich(-like) syndrome,3Al-Herz W. Bousfiha A. Casanova J.L. Chatila T. Conley M.E. Cunningham-Rundles C. et al.Primary immunodeficiency diseases: an update on the classification from the International Union of Immunological Societies expert committee for primary immunodeficiency.Front Immunol. 2014; 5: 162Crossref PubMed Scopus (378) Google Scholar, 5Thrasher A.J. Burns S.O. WASP: a key immunological multitasker.Nat Rev Immunol. 2010; 10: 182-192Crossref PubMed Scopus (296) Google Scholar, 10Lanzi G. Moratto D. Vairo D. Masneri S. Delmonte O. Paganini T. et al.A novel primary human immunodeficiency due to deficiency in the WASP-interacting protein WIP.J Exp Med. 2012; 209: 29-34Crossref PubMed Scopus (129) Google Scholar but this was genetically excluded in our patient. These immunodeficiencies tend to have a more severe bleeding tendency and more pronounced thrombocytopenia. We have identified a novel combined immunodeficiency with features of recurrent infections, allergic reactions, vasculitis, and mild bleeding tendency. The biology as well as the supportive evidence from the mouse model suggests a direct causal relationship between the ARPC1B mutation and the clinical manifestations. We are very grateful to the patient, his family, and healthy donors. We also thank Dr Ester van Leeuwen for immunological screening and Hans Janssen for his electron microscopic evaluations of blood cells, Simon Forman for IgE measurements, and Dr Hibret Adissu for histopathology. Heparinized venous blood was collected from healthy donors and the patient after informed consent had been obtained. The study was approved by the Academic Medical Center Institutional Medical Ethics Committee in accordance with the 1964 Declaration of Helsinki. Human neutrophils were isolated with a Percoll solution with a density of 1.076 g/mL. Erythrocytes were lysed with isotonic NH4Cl/KHCO3, washed twice in PBS and resuspended in HEPES buffer for further functional testing.E1Kuijpers T.W. Maianski N.A. Tool A.T. Smit G.P. Rake J.P. Roos D. et al.Apoptotic neutrophils in the circulation of patients with glycogen storage disease type 1b (GSD1b).Blood. 2003; 101: 5021-5024Crossref PubMed Scopus (94) Google Scholar, E2Kuijpers T.W. Maianski N.A. Tool A.T. Becker K. Plecko B. Valianpour F. et al.Neutrophils in Barth syndrome (BTHS) avidly bind annexin-V in the absence of apoptosis.Blood. 2004; 103: 3915-3923Crossref PubMed Scopus (79) Google Scholar, E3Kuijpers T.W. Alders M. Tool A.T. Mellink C. Roos D. Hennekam R.C. Hematologic abnormalities in Shwachman Diamond syndrome: lack of genotype-phenotype relationship.Blood. 2005; 106: 356-361Crossref PubMed Scopus (69) Google Scholar PBMCs were collected from the surface of the Percoll solution, washed twice and resuspended in PBS containing 0.5% (wt/vol) BSA for immunophenotyping or for further functional studies in Iscove modified Dulbecco medium supplemented with 10% (vol/vol) fetal calf serum and antibiotics.E4Kuijpers T.W. Bende R.J. Baars P.A. Grummels A. Derks I.A. Dolman K.M. et al.CD20 deficiency in humans results in impaired T cell-independent antibody responses.J Clin Invest. 2010; 120: 214-222Crossref PubMed Scopus (271) Google Scholar, E5Kuijpers T.W. Baars P.A. Aan de Kerk D.J. Jansen M.H. Derks I.A. Bredius R.G. et al.A novel mutation in CD132 causes X-CID with defective T-cell activation and impaired humoral reactivity.J Allergy Clin Immunol. 2011; 128: 1360-1363.e4Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar Platelet-rich plasma and washed platelets were prepared as previously described.E6De Cuyper I.M. Meinders M. van de Vijver E. de Korte D. Porcelijn L. de Haas M. et al.A novel flow cytometry-based platelet aggregation assay.Blood. 2013; 121: e70-e80Crossref PubMed Scopus (110) Google Scholar Isolated platelets were washed twice with sequestrene buffer followed by centrifuging for 5 minutes at 2310g. Platelets were resuspended in HEPES medium supplemented with 0.02 U/mL apyrase. Studies were performed within 6 hours after blood collection. Nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase activity was assessed as the release of hydrogen peroxide determined with an Amplex Red kit (Molecular Probes, Eugene, Ore). Neutrophils (0.25 × 106/mL) were stimulated with 1 mg/mL unopsonized zymosan (MP Biomedicals, Solon, Ohio), serum-treated zymosan (previously describedE4Kuijpers T.W. Bende R.J. Baars P.A. Grummels A. Derks I.A. Dolman K.M. et al.CD20 deficiency in humans results in impaired T cell-independent antibody responses.J Clin Invest. 2010; 120: 214-222Crossref PubMed Scopus (271) Google Scholar), phorbol 12-myrisatate 13-acetate ([PMA]; 100 ng/mL) or platelet-activation factor (PAF)/N-formylmethionine-leucyl-phenylalanine (fMLP) (both 1 μmol/L) in the presence of Amplex Red (0.5 μmol/L) and horseradish peroxidase (1 U/mL). Fluorescence was measured at 30-second intervals for 30 minutes with an Infinite F200-pro plate reader (Tecan, Männedorf, Switzerland) at an excitation wavelength of 535 nm and an emission wavelength of 595 nm. Maximal slope of H2O2 release was assessed per 2-minute interval. Neutrophils (5 × 106/mL) were incubated with calcein-AM (1 μmol/L final concentration; Molecular Probes) for 30 minutes at 37°C, washed twice, and resuspended in HEPES buffer at a concentration of 2 × 106/mL. Adhesion was determined in an uncoated 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Calcein-labeled cells (2 × 105/well, final volume 100 μL) were stimulated with one of the following: granulocyte-colony stimulating factor (G-CSF) (20 ng/mL), 10 mmol/L dithiothreitol ([DTT]; Sigma Aldrich, St Louis, Mo), 20 μg/mL Pam3Cys (EMC Microcollections, Tübingen, Germany), 20 ng/mL bacterial Toll-like receptor-4 ligand LPS (isolated from E coli strain 055:B5; Sigma Aldrich) in the presence of 50 ng/mL LPS-binding protein (R&D Systems, Minneapolis, Minn), 1 μmol/L PAF (Sigma Aldrich), 10 nmol/L C5a (Sigma Aldrich), 1 μmol/L fMLP, C5a (10−8 mol/L), TNF-α (10 ng/mL), or 100 ng/mL PMA. Plates were incubated for 30 minutes at 37°C and washed 3 times with PBS. Adherent cells were lysed in 0.5% (wt/vol) Triton X-100 in H2O for 5 minutes at room temperature. Fluorescence was measured with a Tecan Infinite F200-pro plate reader at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Adhesion was determined as percentage of total input of calcein-labeled cells. Chemotaxis was measured with 3-μm pore-size Fluoroblock inserts (Corning Inc, Corning, NY), in a Falcon 24-well plate. Calcein-labelEd PMNs (2 × 106/mL, 0.3 mL) were pipetted in the insert (upper compartment), and placed in the lower compartment containing 0.8 mL of C5a (10−8 mol/L), IL-8 (10−8 mol/L), or PAF (10−7 mol/L). Fluorescence was measured underneath the filter every 2.5 minutes for 45 minutes with a Tecan Infinite F200-pro plate reader at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Alternatively, TaxiScan chemotaxis was assessed with an EZ-TAXIScan (ECI Inc, Tokyo, Japan) using a 5-μm chip (260 μm length) at 37°C. Chemotaxis was induced by 2 μL of C5a (stock concentration 10−7 mol/L) or 2 μL of fMLP (stock concentration 10−7 mol/L). Serial images were taken every 30 seconds for 60 minutes per experiment. Neutrophils (2 × 106/mL) were stimulated with fMLP (1 μmol/L) or C5a (10−8 mol/L) for the indicated times. Reaction was stopped by adding 2 × 105 cells to ice-cold PBS with 0.5% (wt/vol) paraformaldehyde (PFA), 20 m mol/L NaF and 1% (wt/vol) BSA for 10 minutes. Thereafter, cells were washed and incubated for 30 minutes at 4°C with Alexa Fluor 488 phalloidin (Invitrogen, Carlsbad, Calif), 1:1000 in IntraPrep Permeabilization Reagent (Beckman Coulter, Indianapolis, Ind), washed twice, and analyzed on an FACSCanto-II flow cytometer equipped with FACSDiva software (BD, San Jose, Calif). Data are expressed as mean fluorescence intensity (MFI). Neutrophils (2 × 106/mL) were preincubated with the (priming) agents PAF (1 μmol/L) or cytochalasin B (5 μg/mL) for 5 minutes and were subsequently stimulated with fMLP (1 μmol/L) for 10 minutes. Thereafter, cells were put on ice, washed with HEPES buffer once, and subsequently stained with an antibody against neutrophil granule markers: CD63-APC (IgG1, clone 9A152; USBiological, Salem, Mass). The cells were analyzed on a FACSCanto-II flow cytometer with FACSDiva software. Data are expressed as MFI. Protease release after degranulation was measured with DQ-green BSA (Molecular Probes), which becomes fluorescent on cleavage by proteases. Neutrophils (2.5 × 106/mL) were preincubated with PAF (1 μmol/L) or cytochalasin B (5 μg/mL) for 5 minutes at 37°C in the presence of DQ-BSA (10 μg/mL) and were then stimulated with fMLP (1 μmol/L). Also, a 100% content value with Triton X-100 0.5% (wt/vol) was determined. Fluorescence was measured with an Infinite F200-pro plate reader at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Data are expressed as relative fluorescence units/minute. Fluorescein isothiocyanate (FITC)-labeled zymosan was opsonized with 10% (vol/vol) human pool serum for 10 minutes at 37°C (serum treated zymosan), and then added in a concentration of 1 mg/mL to neutrophils at a final concentration of 5 × 106 PMN/mL. At the indicated time points, the cellular uptake was instantly stopped by adding 2 × 105 cells to ice-cold PBS with 0.5% (wt/vol) PFA, 20 mmol/L NaF, and 1% (vol/vol) BSA. The PMNs were analyzed on a FACSCanto-II flow cytometer equipped with FACSDiva software. The “phagocytosis index” was defined as the percentage of FITC-positive PMNs multiplied by the MFI. Granulocyte bactericidal activity was determined with E coli, strain ML-35, and S. aureus, strain 502A. Bacterial survival was measured by assaying bacterial colony formation as previously described.E7van Bruggen R. Drewniak A. Tool A.T. Jansen M. van Houdt M. Geissler J. et al.Toll-like receptor responses in IRAK-4-deficient neutrophils.J Innate Immun. 2010; 2: 280-287Crossref PubMed Scopus (39) Google Scholar Platelet aggregation tests employing flow cytometry were performed as previously described.E6De Cuyper I.M. Meinders M. van de Vijver E. de Korte D. Porcelijn L. de Haas M. et al.A novel flow cytometry-based platelet aggregation assay.Blood. 2013; 121: e70-e80Crossref PubMed Scopus (110) Google Scholar, E8van de Vijver E. De Cuyper I.M. Gerrits A.J. Verhoeven A.J. Seeger K. Gutiérrez L. et al.Defects in Glanzmann thrombasthenia and LAD-III (LAD-1/v) syndrome: the role of integrin β1 and β3 in platelet adhesion to collagen.Blood. 2012; 119: 583-586Crossref PubMed Scopus (28) Google Scholar Briefly, platelets were washed with sequestrene buffer. Carboxyfluorescein diacetate succinimidyl ester (Molecular Probes) and PKH26-labeled platelets were mixed 1:1 and preincubated with agonists: 20 μmol/L thrombin-related activation peptide-6 (TRAP6; Bachem, Bubendorf, Switzerland); 100 ng/mL PMA (Sigma Aldrich). Samples were taken in time and were fixed in 0.5% (wt/vol) formaldehyde, PBS. For antigen upregulation of CD62P and CD63 (FITC-labeled; Sanquin Reagents, Amsterdam, The Netherlands) platelets were stimulated 5 minutes with 100 ng/mL PMA, 10 μg/mL collagen, or 20 μg/mL TRAP6. Total expression of the β3 integrin (glycoprotein IIb/IIIa) was measured with C17-FITC (Sanquin Reagents), whereas the high-affinity conformation of integrin β3 with first procaspase activating compound (PAC-1)-FITC (BD Biosciences, San Jose, Calif). The extent of activated integrin was determined relative to total integrin expression, that is, after background correction with the isotype control and normalizing ratios of unstimulated platelets to 1.E8van de Vijver E. De Cuyper I.M. Gerrits A.J. Verhoeven A.J. Seeger K. Gutiérrez L. et al.Defects in Glanzmann thrombasthenia and LAD-III (LAD-1/v) syndrome: the role of integrin β1 and β3 in platelet adhesion to collagen.Blood. 2012; 119: 583-586Crossref PubMed Scopus (28) Google Scholar Both double-colored events and antigen expression were assessed with a LSR II flow cytometer equipped with High Throughput Sampler (HTS) and data were analyzed by FACSDiva version 6.1 software (both from BD). For confocal microscopy, a total of 250,000 neutrophils were seeded on 12-mm coverslips (Menzel-Gläser, Thermo Fisher Scientific, Braunschwieg, Germany) and stimulated either with fMLP (0.1 μmol/L; Sigma-Aldrich) or C5a (0.1 μmol/L; Sigma-Aldrich). Cells were then fixed in 3.7% paraformaldehyde for 30 minutes at room temperature and permeabilized with 0.1% Triton in PBS. In case of washed platelets, an equal number was seeded and activated by collagen (10 μg/mL; Sigma-Aldrich) or TRAP (20 μmol/L; Bachem). After fixation and permeabilization, cells were stained with Phalloidin Texas Red (Invitrogen), Hoechst (Sigma-Aldrich), anti–alpha tubulin (Sigma-Aldrich) followed by secondary antibody goat antimouse IgG Alexa Fluor 488 (Thermo Fisher Scientific) for neutrophils, or Phalloidin Alexa 488 for platelets. Coverslips were analyzed on a Zeiss LSM 510 confocal microscope (Jena, Germany). Images were deconvoluted with Huygens deconvolution software. Fibroblasts were cultured in 10% (vol/vol) FCS in Dulbecco modified Eagle medium (DMEM) and passaged twice a week. For the migration assays, 6.0 × 104 fibroblasts were seeded in a 24-well plate and grown to confluence. Cells from passages 4 to 6 were used. Cells were starved in DMEM 20 hours prior to scratching. The cells were scratched with a yellow pipette tip and washed with PBS. Cells were stimulated with either 10% (vol/vol) FCS in DMEM or 10 ng/mL platelet-derived growth factor-BB (PDGF-BB) (Peprotech, Oak Park, Calif). The wound area was captured every 20 minutes for 45 hours at 37°C and 5% CO2 on a Zeiss observer Z1 microscope with a 10× objective. Healing was quantified manually by measuring wound size in ImageJ (National Institutes of Health, Bethesda, Md). All chemicals were obtained from Life Technologies (Carlsbad, Calif) unless stated otherwise. To isolate platelets, platelet-rich plasma was collected via centrifugation of whole blood for 20 minutes at 120g. Platelets were spun down by centrifugation for 10 minutes at 2000g, washed and resuspended in a buffer comprising 36 mmol/L citric acid, 103 mmol/L NaCl, 5 mmol/L KCl, 5 mmol/L EDTA, 5.6 mmol/L D-glucose, and 10% (vol/vol) ACD-A (BD, Plymouth, UK) at pH 6.5. About 100 × 106 platelets in 25 μL wash buffer were lysed in 8 mol/L urea in 100 mmol/L Tris-HCl (pH 8). Disulfide bonds were reduced with 10 mmol/L DTT for 60 minutes at 20°C, alkylated with 55 mmol/L iodoacetamide for 45 minutes at 20°C, and samples were digested overnight at 20°C with MS-grade trypsin (Promega Benelux, Leiden, The Netherlands). Peptides were desalted and concentrated with Empore-C18 StageTips prepared in-house and eluted with 0.5% (vol/vol) acetic acid, 80% (vol/vol) acetonitrile as described. Sample volume was reduced by SpeedVac and supplemented with 2% (wt/vol) acetonitrile, 0.1% (wt/vol) trifluoroacetic acid to a final volume of 5 μL. Neutrophils were lysed in 4% (wt/vol) SDS, 100 mmol/L DTT, 100 mmol/L Tris-HCl pH 7.5 supplemented with HALT protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific) and processed into tryptic peptides with the filter aided sample preparation method.E9Rappsilber J. Ishihama Y. Mann M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics.Anal Chem. 2003; 75: 663-670Crossref PubMed Scopus (1804) Google Scholar Peptides derived from neutrophils were desalted as described for the platelets. The tryptic peptides derived from platelet or neutrophil proteins (3 μL sample) were separated by nanoscale C18 reverse chromatography coupled on line to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific) via a nanoelectrospray ion source (Nanospray Flex Ion Source, Thermo Fisher Scientific) as described.E10Gazendam R.P. van de Geer A. van Hamme J.L. Tool A.T. van Rees D.J. Aarts C.E. et al.Impaired killing of Candida albicans by granulocytes mobilized for transfusion purposes: a role for granule components.Haematologica. 2016; 101: 587-596Crossref PubMed Scopus (20) Google Scholar All mass spectrometry data were acquired with Xcalibur software (Thermo Fisher Scientific). The raw mass spectrometry files were processed with Proteome Discoverer 1.4.1.14 (Thermo Fisher Scientific). The SEQUEST-HT search algorithm and a protein database with Homo sapiens proteins from human Uniprot databaseE11UniProt ConsortiumThe Universal Protein Resource (UniProt) in 2010.Nucleic Acids Res. 2010; 38: D142-D148Crossref PubMed Scopus (1006) Google Scholar (downloaded February 2015) were employed to identify the peptides and the number of peptide spectral matches (PSMs) for the individual proteins. The peptide precursor mass tolerance was set to 10 parts per million, and fragment ion mass tolerance was set to 0.6 Da. Carbamidomethylation on cysteine residues was used as fixed modification, and oxidation of methionine was used as a dynamic modification. The percolator node in proteome discoverer was utilized for peptide validation (false discovery rate < 1% based on peptide q value). To compare samples, the obtained number of PSMs for a protein was corrected for the difference in the total number of PSMs for all identified proteins in the individual samples. The correction factor for a sample was calculated by dividing the total number of PSMs of healthy control C1 by the total number of PSMs of the sample. Mass spectrometry raw files obtained from the neutrophil samples of healthy individuals and the patient were also processed with the MaxQuant 1.5.2.8 computational platform as described.E10Gazendam R.P. van de Geer A. van Hamme J.L. Tool A.T. van Rees D.J. Aarts C.E. et al.Impaired killing of Candida albicans by granulocytes mobilized for transfusion purposes: a role for granule components.Haematologica. 2016; 101: 587-596Crossref PubMed Scopus (20) Google Scholar, E12Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9223) Google Scholar Label-free quantification based on precursor intensity was used to evaluate the difference in protein level between control and patient samples. Quantitative significance was assessed with an adapted permutation-based false discovery rate t test from Perseus 1.5.1.6 software. Samples were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. Individual proteins were detected with antibodies against ArpC1b (goat polyclonal antibodies, Thermo Fisher Scientific, Rockford, Ill) and against human Glyceraldehyde-3-Phosphate Dehydrogenase (mouse monoclonal antibody; Merck Millipore, Darmstadt, Germany). Secondary antibodies were either donkey-anti-goat-IgG IRDye 800CW, goat-anti-mouse-IgG IRDye 800CW, or goat-anti-rabbit-IgG IRDye 680CW (LI-COR Biosciences, Lincoln, Neb). Quantification of bound antibodies was performed on an Odyssey Infrared Imaging system (
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