Shwachman-Diamond syndrome neutrophils have altered chemoattractant-induced F-actin polymerization and polarization characteristics

Abstract

delayed in SDS neutrophils. This is the first study revealing a molecular defect, namely defective F-actin cytoskeletal remodeling, which may contribute to impaired chemotaxis in SDS patients. Design and Methods Neutrophil isolation and cell cultureBlood was obtained after informed consent of healthy volunteers (26-43 years) and SDS patients (2-29 years; all with mutations 183-184 A>CT [K62X]/IVS2 258+2T>C [C84fs]). The study was approved by the Medical Ethics Committees of the AMC Hospital and the institutional review board and was in accordance with the Declaration of Helsinki. Neutrophils were isolated as described. 8 PLB-985 and GFP-Rac2 PLB-985 17 cells were cultured in RPMI/10% FCS/penicillin (200 µg/mL)/streptomycin (200 µg/mL)/ L-glutamine (4 mM). PLB-985 cells were differentiated with 0.5% dimethylformamide (DMF; Sigma-Aldrich) for 5-7 days. Cellular spreading and immunofluorescenceNeutrophils or PLB-985 cells in Hepes medium (132 mM NaCl, 20 mM Hepes, 6mM KCL, 1 mM MgSO4, 1.2 mM K2HPO4, 1 mM CaCl2, 5 mM Glucose, 2.5% human albumin (Cealb; Sanquin reagents)) were seeded on fibronectin-coated coverslips and allowed to adhere for 30 min. Cells were stimulated with 20nM fMLP, fixed with 4% paraformaldehyde/PBS and processed for immunofluorescence staining as described. 18 Antibodies used: SBDS (rabbit polyclonal), Phalloidin-Alexa488 or BODIPY conjugated. Pictures were taken with a Zeiss LSM510 microscope with a Zeiss 65x oil lens and processed with LSM 510 software. Neutrophils (2×10 6 /mL) in Hepes medium were stimulated with 100nM fMLP or 40nM C5a. At indicated time points 2×10 5 cells were harvested and fixed with buffer 1 (Intraprep Permeabilization Reagent Kit; Immunotech), permeabilized with buffer 2 and stained with Phalloidin-Alexa488 (Molecular Probes, Invitrogen). Cells were analyzed on LSRII (BD Biosciences) flow cytometer for F-actin content. F-actin polymerization and cellular polarizationTo determine F-actin polarization, neutrophils were embedded with Mowiol 4-88 and analyzed by confocal microscopy. For each experiment at least 50 cells per time-point were analyzed in a blind fashion. Results and Discussion SBDS co-localizes with F-actin and Rac2 in human neutrophilsTo investigate a possible link between SBDS and the actin cytoskeleton, we performed immunofluorescence stainings in neutrophils and PLB-985 cells, with an anti-SBDS antibody that we have generated and which specifically recognized SBDS (Online Supplementary Figure S1A/B). In resting human neutrophils we observed that SBDS is localized prominently to the nucleus and to a lesser extent to the cytoplasm (Online Supplementary Figure 1C). However, in these resting cells there is not much polymerized F-actin cytoskeleton detectable (data not shown) and therefore we set out to investigate SBDS in relation to the F-actin cytoskeleton in activated neutrophilic cells. Human peripheral blood neutrophils were allowed to adhere to fibronectin and were subsequently stimulated with fMLP. These activated cells displayed Factin enriched c...

Mutations in signal recognition particle SRP54 cause syndromic neutropenia with Shwachman-Diamond–like features

“…Although tra- patients, including profound neutropenia and defective exocrine pancreas development. Neutrophils of SDS patients have been reported to show impaired chemotaxis and migration deficiency (34,35), which lead to repeated infections, as was also observed in all 3 patients reported here. In accordance with the results of these studies, we observed a decrease in the capacity of neutrophils to migrate toward the injury site in the zebrafish model ( Figure 5).…”

Sbds is required for Rac2-mediated monocyte migration and signaling downstream of RANK during osteoclastogenesis

“…21,22 The SBDS homolog in Dictyostelium discoideum, an amoeba model to study neutrophils, is enriched in pseudopods of migrating cells, 38 further implicating a role for SBDS in chemotaxis. Migration of monocytes/ , WT and Sbds-null nuclear extracts, whereas the mutant consensus oligonucleotide had no effect (n ϭ 4): *P Ͻ .005; **P Ͻ .01.…”

“…20 In addition to concerns of neutropenia and recurrent infections, neutrophils of affected persons have been shown to exhibit impaired chemotaxis and deficiency in migration. 21,22 A lesscharacterized abnormality with SDS patients is their defect in bone homeostasis, a parameter that is normally maintained via the balanced activities of bone-producing osteoblasts and boneresorbing osteoclasts. 23 A unique feature of osteoclast formation, or osteoclastogenesis (OCG), involves multiple fusions of preosteoclasts derived from cells from the monocyte-macrophage lineage, a process requiring a dynamic actin cytoskeleton that is regulated by small GTPases.…”

Marrow failure: a window into ribosome biology

“…The bone marrow is typically mildly dysplastic and hypocellular. Abnormalities of neutrophil chemotaxis have been reported, 55,56 though patients are able to form purulent abscesses and empyemas. 57 Neutrophil respiratory burst activity is normal.…”