Mosaicism of NK cells in a patient with Wiskott-Aldrich syndrome

Rare cases of somatic mosaicism resulting from reversion of inherited mutations can lead to the attenuation of blood-cell disorders, including Wiskott-Aldrich syndrome (WAS). The impact of the revertant hematopoietic stem or progenitor cells, particularly their representation in blood-cell populations, is of interest because it predicts the outcome of gene therapy. Here we report an 8-year-old patient with WAS caused by a single nucleotide insertion in the WASP gene that abrogates protein expression. The patient nonetheless had mild disease. We found reversion of the mutation in a fraction of patient lymphocytes. Forty percent of natural killer (NK) cells expressed Wiskott-Aldrich syndrome protein (WASP), and NK cells contained both mutated and revertant (normal) sequences. WASP was not expressed in patient T or B cells; T cells contained only the mutated sequence. The selective advantage of WASP+ NK cells was also demonstrated for carrier females. The enrichment of WASP+-revertant NK cells indicates that WASP provides a selective advantage in this lineage and predicts the success of gene therapy for reconstituting the NK-cell compartment. The importance of reconstituting the NK-cell lineage is discussed.     

Alterations in the renal elastin-elastase system in type 1 diabetic nephropathy identified by proteomic analysis

Diabetes now accounts for >40% of patients with ESRD. Despite significant progress in understanding diabetic nephropathy, the cellular mechanisms that lead to diabetes-induced renal damage are incompletely defined. For defining changes in protein expression that accompany diabetic nephropathy, the renal proteome of 120-d-old OVE26 transgenic mice with hypoinsulinemia, hyperglycemia, hyperlipidemia, and proteinuria were compared with those of background FVB nondiabetic mice (n = 5). Proteins derived from whole-kidney lysate were separated by two-dimensional PAGE and identified by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. Forty-one proteins from 300 visualized protein spots were differentially expressed in diabetic kidneys. Among these altered proteins, expression of monocyte/neutrophil elastase inhibitor was increased, whereas elastase IIIB was decreased, leading to the hypothesis that elastin expression would be increased in diabetic kidneys. Renal immunohistochemistry for elastin of 325-d-old FVB and OVE26 mice demonstrated marked accumulation of elastin in the macula densa, collecting ducts, and pelvicalyceal epithelia of diabetic kidneys. Elastin immunohistochemistry of human renal biopsies from patients with type 1 diabetes (n = 3) showed increased elastin expression in renal tubular cells and the interstitium but not glomeruli. These results suggest that coordinated changes in elastase inhibitor and elastase expression result in increased tubulointerstitial deposition of elastin in diabetic nephropathy. The identification of these coordinated changes in protein expression in diabetic nephropathy indicates the potential value of proteomic analysis in defining pathophysiology.     

WASP and N-WASP in human platelets differ in sensitivity to protease calpain.

Mutations of Wiskott-Aldrich syndrome protein (WASP) underlie the severe thrombocytopenia and immunodeficiency of the Wiskott-Aldrich syndrome. WASP, a specific blood cell protein, and its close homologue, the broadly distributed N-WASP, function in dynamic actin polymerization processes. Here it is demonstrated that N-WASP is expressed along with WASP, albeit at low levels, in human blood cells. The presence of approximately 160 nmol/L rapidly acting N-WASP molecules may explain the normal capacity of WASP-negative patient platelets for early agonist-induced aggregation and filopodia formation. Ex vivo experiments revealed a significant difference between WASP and N-WASP in sensitivity to calpain, the Ca++-dependent protease activated in agonist-stimulated platelets. Through the use of a series of calpain-containing broken cell systems, it is shown that WASP is cleaved in a Ca++-dependent reaction inhibitable by calpeptin and E64d and that N-WASP is not cleaved, suggesting that the cleavage of WASP by calpain functions in normal platelets as part of a Ca++-dependent switch mechanism that terminates the surface projection phase of blood cell activation processes.     

Molecular characterization of sialophorin (CD43), the lymphocyte surface sialoglycoprotein defective in Wiskott-Aldrich syndrome.

Sialophorin (CD43) of leukocytes and platelets is a surface sialoglycoprotein that is phenotypically defective on lymphocytes of patients with the X chromosome-linked immunodeficiency Wiskott-Aldrich syndrome. Previous studies with monoclonal antibodies indicate that sialophorin is a component of a T-lymphocyte activation pathway. Here we describe the cDNA cloning and derived amino acid sequence of human sialophorin. The sequence predicts an integral membrane polypeptide with an N-terminal hydrophobic signal region followed by a mucin-like 235-residue extracellular region with a uniform distribution of 46 serine, 47 threonine, and 24 proline residues. This is followed by a 23-residue transmembrane region and a 123-residue C-terminal intracellular region. These latter regions have been highly conserved during evolution; the intracellular region contains a number of potential phosphorylation sites that might mediate transduction of activation signals. The chromosomal location of the sialophorin gene was determined and the implications of this assignment for the pathogenesis of the Wiskott-Aldrich syndrome are discussed.     

A monoclonal antibody to sialophorin (CD43) induces homotypic adhesion and activation of human monocytes

Treatment of human monocytes for 24-48 h with the anti-CD43 mAb L10 caused five- to sevenfold stimulation of hydrogen peroxide-producing capacity, an established characteristic of activated monocytes. Peroxide-producing capacity induced by L10 antibody (1.6 +/- 0.3 nmol H2O2/micrograms DNA/h) was comparable with that induced by IFN-gamma (1.3 +/- 0.4 nmol H2O2/micrograms DNA/h), but appeared more rapidly (maximal at 24 h) than in the IFN-gamma-treated monocytes (maximal at 48 h). Treatment of monocytes with L10 mAb also caused dramatic increase in aggregation (homotypic adhesion). Induction of monocyte aggregation by L10 mAb required incubation for 1-8 h in the presence of Mg2+ and was abrogated by TA-1, an anti-LFA-1-alpha mAb. Thus, L10-induced monocyte activation proceeds via a Mg2+-requiring aggregation stage involving LFA-1. Whereas the extent of monocyte aggregation induced by L10 mAb and by IFN-gamma were comparable, the L10-induced aggregation occurred more rapidly (maximal at 8 h) than the IFN-gamma-induced aggregation (maximal at 24 h). The more rapid appearance of aggregation and increased hydrogen peroxide capacity in L10-treated monocytes suggests that the L10-induced activation pathway is independent of IFN-gamma-and IFN-gamma-R dependent events. These findings suggest that the surface molecule CD43 is the receptor of an independent activation pathway that leads in lymphocytes to proliferation and in monocytes to activation.     

WASP localizes to the membrane skeleton of platelets

Patients with Wiskott-Aldrich syndrome (WAS), an X-linked blood cell disease, suffer from severe thrombocytopenia due to accelerated loss of defective platelets. The affected gene encodes WASP, an actin regulatory protein thought to reside in the cytoplasm of resting leucocytes. In contrast, this study showed that, for platelets, one-quarter of WASP molecules fractionate in the detergent-insoluble high speed pellet characterized as the membrane skeleton, the scaffold structure that underlies the lipid bilayer and stabilizes the surface membrane. Following treatment of platelets with thrombin and stirring, which induces cytoarchitectural remodelling, WASP and other membrane skeletal components sedimented at lower g force and partitioned in the low-speed pellet. Thrombin and stirring also induced WASP tyrosine phosphorylation, a rapid activating reaction, and proteolytic inactivation by cysteine protease calpain. Both the alteration of the sedimentation profile and the proteolytic inactivation were specific for the membrane skeletal pool of WASP and were abrogated in alphaIIb beta3 integrin-deficient platelets and in normal platelets treated with an integrin antagonist. The findings demonstrate that WASP is a component of the resting platelet membrane skeleton and participates in membrane skeletal rearrangements downstream of integrin outside-in signalling. The possible implications for the platelet defect in WAS are discussed.     

The ovalbumin serpins revisited: perspective from the chicken genome of clade B serpin evolution in vertebrates

Serpin superfamily proteins, most of which are serine protease inhibitors, share an unusual mechanism rooted in their conserved metastable tertiary structure. Although serpins have been identified in isolated members of archea, bacteria, and plants, a remarkable expansion is found in vertebrates. The chicken protein ovalbumin, a storage protein from egg white, lacking protease inhibitory activity, is an historical member of the superfamily and the founding member of the subgroup known as ov-serpins (ovalbumin-related serpins) or clade B serpins. In the human, ov-serpins include 13 proteins involved in the regulation of inflammation, apoptosis, angiogenesis, and embryogenesis. Here, a detailed analysis of the chicken (Gallus gallus) genome identified 10 clade B serpin genes that map to a single approximately 150-kb locus and contain the signature protein sequence of serpins and the gene structure of ov-serpins, with either seven or eight exons. Orthologues of PAI-2 (SERPINB2), MNEI (SERPINB1), PI-6 (SERPINB6), and maspin (SERPINB5) are highly conserved. Comparison with human ov-serpins identified avian-specific and mammal-specific genes. Importantly, a unique model of mammalian ov-serpin evolution is revealed from the comparative analysis of the chicken and human loci. The presence of a subset of ov-serpin genes in zebrafish (Danio rerio) gives insight into the ancestral locus. This comparative genomic study provides a valuable perspective on the evolutionary pathway for the clade B serpins, allowing the identification of genes with functions that may have been conserved since the origin of vertebrates. In addition, it suggests that "newer" serpins, such as ovalbumin, have contributed to vertebrate adaptation.     

The neutrophil serine protease inhibitor serpinb1 preserves lung defense functions in Pseudomonas aeruginosa infection

Neutrophil serine proteases (NSPs; elastase, cathepsin G, and proteinase-3) directly kill invading microbes. However, excess NSPs in the lungs play a central role in the pathology of inflammatory pulmonary disease. We show that serpinb1, an efficient inhibitor of the three NSPs, preserves cell and molecular components responsible for host defense against Pseudomonas aeruginosa. On infection, wild-type (WT) and serpinb1-deficient mice mount similar early responses, including robust production of cytokines and chemokines, recruitment of neutrophils, and initial containment of bacteria. However, serpinb1(-/-) mice have considerably increased mortality relative to WT mice in association with late-onset failed bacterial clearance. We found that serpinb1-deficient neutrophils recruited to the lungs have an intrinsic defect in survival accompanied by release of neutrophil protease activity, sustained inflammatory cytokine production, and proteolysis of the collectin surfactant protein-D (SP-D). Coadministration of recombinant SERPINB1 with the P. aeruginosa inoculum normalized bacterial clearance in serpinb1(-/-) mice. Thus, regulation of pulmonary innate immunity by serpinb1 is nonredundant and is required to protect two key components, the neutrophil and SP-D, from NSP damage during the host response to infection.