Monocyte-mediated antibody-dependent cellular cytotoxicity: a clinical test of monocyte function.
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The lack of a simple, rapid, and quantitative test of the functional activity of the monocyte has hampered studies of the contribution of this cell type to host defense and human disease. This report describes an assay of antibody-dependent cellular cytotoxicity, which depends exclusively upon the monocyte as the effector cell and therefore provides a convenient test of monocyte function. In this system, mononuclear leukocytes (MNL) obtained by Ficoll-Hypaque separation of whole blood are cytotoxic for 51Cr-labeled human erythrocyte targets coated with anti-blood group antibody. Removal of phagocytic monocytes from the MNL by iron ingestion, followed by exposure to a magnetic field, completely abolishes all cytotoxic activity from the remaining MNL population. Similarly, in severely mono-cytopenic patients with aplastic anemia, cytotoxic effector activity is absent. In normals and less severely monocytopenic aplastic anemia patients, cytotoxicity correlates significantly (p less than 0.001) with monocyte number. Application of this monocyte-mediated antibody-dependent cellular cytotoxicity assay to the study of patients with the Wiskott-Aldrich syndrome has revealed defective monocyte cytotoxic activity in spite of normal monocyte numbers, suggesting that this test may be useful for the assessment of monocyte function in a variety of clinical situations. (+info)
Actin polymerization: Where the WASP stings.
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How do extracellular signals induce actin polymerization, as required for many cellular responses? Key signal transducers, such as the small GTPases Cdc42 and Rac, have now been shown to link via proteins of the WASP family to the Arp2/3 complex, which nucleates actin polymerization. (+info)
Mutations that cause the Wiskott-Aldrich syndrome impair the interaction of Wiskott-Aldrich syndrome protein (WASP) with WASP interacting protein.
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Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, eczema, immune deficiency, and a proclivity toward lymphoid malignancy. Lymphocytes of affected individuals show defects of activation, motility, and cytoskeletal structure. The disease gene encodes a 502-amino acid protein named the WAS protein (WASP). Studies have identified a number of important interactions that place WASP in a role of integrating signaling pathways with cytoskeletal function. We performed a two-hybrid screen to identify proteins interacting with WASP and cloned a proline-rich protein as a specific WASP interactor. Our clone of this protein, termed WASP interacting protein (WIP) by others, shows a difference in seven amino acid residues, compared with the previously published sequence revealing an additional profilin binding motif. Deletion mutant analysis reveals that WASP residues 101-151 are necessary for WASP-WIP interaction. Point mutant analyses in the two-hybrid system and in vitro show impairment of WASP-WIP interaction with three WASP missense mutants known to cause WAS. We conclude that impaired WASP-WIP interaction may contribute to WAS. (+info)
The Wiskott-Aldrich syndrome protein directs actin-based motility by stimulating actin nucleation with the Arp2/3 complex.
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Actin polymerization at the cell cortex is thought to provide the driving force for aspects of cell-shape change and locomotion. To coordinate cellular movements, the initiation of actin polymerization is tightly regulated, both spatially and temporally. The Wiskott-Aldrich syndrome protein (WASP), encoded by the gene that is mutated in the immunodeficiency disorder Wiskott-Aldrich syndrome [1], has been implicated in the control of actin polymerization in cells [2] [3] [4] [5]. The Arp2/3 complex, an actin-nucleating factor that consists of seven polypeptide subunits [6] [7] [8], was recently shown to physically interact with WASP [9]. We sought to determine whether WASP is a cellular activator of the Arp2/3 complex and found that WASP stimulates the actin nucleation activity of the Arp2/3 complex in vitro. Moreover, WASP-coated microspheres polymerized actin, formed actin tails and exhibited actin-based motility in cell extracts, similar to those behaviors displayed by the pathogenic bacterium Listeria monocytogenes. In extracts depleted of the Arp2/3 complex, WASP-coated microspheres and L. monocytogenes were non-motile and exhibited only residual actin polymerization. These results demonstrate that WASP is sufficient to direct actin-based motility in cell extracts and that this function is mediated by the Arp2/3 complex. WASP interacts with diverse signaling proteins and may therefore function to couple signal transduction pathways to Arp2/3-complex activation and actin polymerization. (+info)
The human WASP-interacting protein, WIP, activates the cell polarity pathway in yeast.
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WIP, the Wiskott-Aldrich syndrome protein-interacting protein, is a human protein involved in actin polymerization and redistribution in lymphoid cells. The mechanism by which WIP reorganizes actin cytoskeleton is unknown. WIP is similar to yeast verprolin, an actin- and myosin-interacting protein required for polarized morphogenesis. To determine whether WIP and verprolin are functional homologues, we analyzed the function of WIP in yeast. WIP suppresses the growth defects of VRP1 missense and null mutations as well as the defects in cytoskeletal organization and endocytosis observed in vrp1-1 cells. The ability of WIP to replace verprolin is dependent on its WH2 actin binding domain and a putative profilin binding domain. Immunofluorescence localization of WIP in yeast cells reveals a pattern consistent with its function at the cortical sites of growth. Thus, like verprolin, WIP functions in yeast to link the polarity development pathway and the actin cytoskeleton to generate cytoskeletal asymmetry. A role for WIP in cell polarity provides a framework for unifying, under a common paradigm, distinct molecular defects associated with immunodeficiencies like Wiskott-Aldrich syndrome. (+info)
The thrombocytopenia of Wiskott Aldrich syndrome is not related to a defect in proplatelet formation.
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The Wiskott-Aldrich syndrome (WAS) is an X-linked hereditary disease characterized by thrombocytopenia with small platelet size, eczema, and increased susceptibility to infections. The gene responsible for WAS was recently cloned. Although the precise function of WAS protein (WASP) is unknown, it appears to play a critical role in the regulation of cytoskeletal organization. The platelet defect, resulting in thombocytopenia and small platelet size, is a consistent finding in patients with mutations in the WASP gene. However, its exact mechanism is unknown. Regarding WASP function in cytoskeletal organization, we investigated whether these platelet abnormalities could be due to a defect in proplatelet formation or in megakaryocyte (MK) migration. CD34(+) cells were isolated from blood and/or marrow of 14 WAS patients and five patients with hereditary X-linked thrombocytopenia (XLT) and cultured in serum-free liquid medium containing recombinant human Mpl-L (PEG-rHuMGDF) and stem-cell factor (SCF) to study in vitro megakaryocytopoiesis. In all cases, under an inverted microscope, normal MK differentiation and proplatelet formation were observed. At the ultrastructural level, there was also no abnormality in MK maturation, and normal filamentous MK were present. Moreover, the in vitro produced platelets had a normal size, while peripheral blood platelets of the same patients exhibited an abnormally small size. However, despite this normal platelet production, we observed that F-actin distribution was abnormal in MKs from WAS patients. Indeed, F-actin was regularly and linearly distributed under the cytoplasmic membrane in normal MKs, but it was found concentrated in the center of the WAS MKs. After adhesion, normal MKs extended very long filopodia in which WASP could be detected. In contrast, MKs from WAS patients showed shorter and less numerous filopodia. However, despite this abnormal filopodia formation, MKs from WAS patients normally migrated in response to stroma-derived factor-1alpha (SDF-1alpha), and actin normally polymerized after SDF-1alpha or thrombin stimulation. These results suggest that the platelet defect in WAS patients is not due to abnormal platelet production, but instead to cytoskeletal changes occuring in platelets during circulation. (+info)
Wiskott-Aldrich syndrome protein regulates podosomes in primary human macrophages.
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Wiskott-Aldrich syndrome protein (WASp) is a hematopoietic-specific, multidomain protein whose mutation is responsible for the immunodeficiency disorder Wiskott-Aldrich syndrome. WASp contains a binding motif for the Rho GTPase CDC42Hs as well as verprolin/cofilin-like actin-regulatory domains, but no specific actin structure regulated by CDC42Hs-WASp has been identified. We found that WASp colocalizes with CDC42Hs and actin in the core of podosomes, a highly dynamic adhesion structure of human blood-derived macrophages. Microinjection of constitutively active V12CDC42Hs or a constitutively active WASp fragment consisting of the verprolin/cofilin-like domains led to the disassemly of podosomes. Conversely, macrophages from patients expressing truncated forms of WASp completely lacked podosomes. These findings indicate that WASp controls podosome assembly and, in cooperation with CDC42Hs, podosome disassembly in primary human macrophages. (+info)
Retrovirus-mediated WASP gene transfer corrects defective actin polymerization in B cell lines from Wiskott-Aldrich syndrome patients carrying 'null' mutations.
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Boys affected with Wiskott-Aldrich syndrome (WAS) present with variable association of thrombocytopenia, eczema and immune deficiency. If untreated, WAS patients may succumb to intracerebral hemorrhages, severe infections or malignancies. Allogeneic bone marrow transplantation (BMT) can cure all aspects of the disease, but HLA-identical donors are not available to all patients and mismatched BMTs are unfortunately associated with high mortality and morbidity. The good success of HLA-matched BMT, however, makes WAS a potential candidate for hematopoietic stem cell gene therapy. WAS patients carry mutations of the Wiskott-Aldrich syndrome protein gene encoding WASP, a 502-amino acid proline-rich protein with demonstrated involvement in the organization of the actin cytoskeleton. To verify the feasibility of genetic correction for this disease, the WASP cDNA was expressed in EBV-immortalized B cell lines obtained from WAS patients using a retroviral vector. Transduced WAS cells showed levels of WASP expression similar to those found in cells from normal donors, without detectable effects on viability or growth characteristics. In addition, retrovirus-mediated expression of WASP led to improvement of cytoplasmic F-actin expression and formation of F-actin-positive microvilli, a process shown to be defective in untransduced WAS cell lines. These preliminary results indicate a potential use for retrovirus-mediated gene transfer as therapy for WAS. (+info)