The neutrophil elastase mutant affects viability and differentiation of the human monocytic THP‐1 cell

Deficiency in neutrophils (neutropenia) caused by mutations in neutrophil elastase (NE, ELA2) has been extensively investigated. Monocytes and neutrophils are derived from a common myeloid progenitor; therefore, ELA2 mutations can also influence monocyte development. These effects have not been well described. In this study, we used the human monocytic THP‐1, to carry the human wild‐type and G185R mutant ELA2 gene. Growth, death, differentiation and BiP expression were evaluated in the two stable cell lines and in the wild‐type THP‐1 cells. Exogenous wild‐type ELA2 markedly increased THP‐1 differentiation, whereas G185R ELA2 was incompetent to promote THP‐1 differentiation in response to all‐trans retinoic acid (ATRA). Indeed, during differentiation induced by ATRA, G185R cell line showed significant cell death. Also, up‐regulated BiP expression accompanied cell death in the G185R cells, suggesting that the overexpression of G185R elastase increases apoptosis through an unfolded protein response. The G185R cells treated with lithium chloride (LiCl; a Wnt signalling activator) displayed higher BiP expression but similar cell viability compared with THP1 and HNEwt/THP1 cells treated with LiCl. This suggested that Wnt signalling might increase cellular tolerance to endoplasmic reticulum stress, enabling mutant monocyte survival. Copyright © 2012 John Wiley & Sons, Ltd.     

Hereditary neutropenia: dogs explain human neutrophil elastase mutations

Mutations in ELA2, the gene encoding neutrophil elastase (NE), cause the human diseases cyclic neutropenia (CN) and severe congenital neutropenia (SCN). Numerous mutations are known, but their lack of consistent biochemical effect has proven puzzling. The recent finding that mutation of AP3B1, which encodes the beta subunit of adaptor protein complex 3 (AP3), is the cause of canine CN suggests a model for the molecular basis of hereditary neutropenias, involving the mistrafficking of NE: AP3 recognizes NE as a cargo protein, and their interaction implies that NE is a transmembrane protein. Computerized algorithms predict two NE transmembrane domains. Most CN mutations fall within predicted transmembrane domains and lead to excessive deposition of NE in granules, whereas SCN mutations usually disrupt the AP3 recognition sequence, resulting in excessive transport to the plasma membrane.     

Characterization of mutant neutrophil elastase in severe congenital neutropenia

Severe congenital neutropenia is a heritable human disorder characterized by neutropenia and acute myelogenous leukemia. We recently determined that the majority of cases result from de novo or autosomal dominantly inherited heterozygous mutations in ELA2, encoding neutrophil elastase. Neutrophil elastase is a chymotryptic serine protease localized in granules of neutrophils and monocytes and is the major target of inhibition of the serpin alpha(1)-antitrypsin. The mutations causing severe congenital neutropenia consist of amino acid missense substitutions, in-frame deletion, splice donor mutation producing a deletion, splice acceptor mutation causing insertion of novel residues, and protein truncating mutations of the carboxyl terminus resulting from nonsense substitutions and deletions leading to frameshifts. We have expressed 14 mutant forms of neutrophil elastase in vitro and have characterized their biochemical properties. The mutations have variable effects on proteolytic activity, eliminating the possibility that the disease results from haploinsufficiency. There is no evidence that the mutant enzymes are cytotoxic. The mutant enzymes retain vulnerability to inhibition by alpha(1)-antitrypsin, but demonstrate variable avidity for interaction with this serpin. Somewhat surprisingly, the mutant enzymes inhibit the wild type enzyme when both are coexpressed within the same cell, suggesting the potential to interfere with normal subcellular trafficking or post-translational processing.     

Progenitor cell self-renewal and cyclic neutropenia

Objectives: Cyclic neutropenia (CN) is a rare genetic disorder where patients experience regular cycling of numbers of neutrophils and various other haematopoietic lineages. The nadir in neutrophil count is the main source of problems due to risk of life-threatening infections. Patients with CN benefit from granulocyte colony stimulating factor therapy, although cycling persists. Mutations in neutrophil elastase gene ( ELA2 ) have been found in more than half of patients with CN. However, neither connection between phenotypic expression of ELA2 and CN nor the mechanism of cycling is known.
Materials and methods:  Recently, a multicompartment model of haematopoiesis that couples stem cell replication with marrow output has been proposed. In the following, we couple this model of haematopoiesis with a linear feedback mechanism via G-CSF.
Results:  We propose that the phenotypic effect of ELA2 mutations leads to reduction in self-renewal of granulocytic progenitors. The body responds by overall relative increase of G-CSF and increasing progenitor cell self-renewal, leading to cell count cycling.
Conclusion:  The model is compatible with available experimental data and makes testable predictions.     

<Emphasis Type="Italic">In vitro</Emphasis> study of HAX1 gene therapy by retro viral transduction as a therapeutic target in severe congenital neutropenia

Severe congenital neutropenia (SCN) is a primary immunodeficiency disease in which a number of underlying gene defects are responsible for abnormalities in neutrophil development. The HCLS1-associated protein X1 (HAX1) mutation is associated with an autosomal-recessive form of SCN. Considering the potential of gene therapy approaches for the treatment of monogenic disorders, in this study we aimed to develop retroviral vectors expressing coding sequences (CDS) to be used for the removal of the genetic blockade in deficient hematopoietic cells. Following amplification of CDS with primers containing appropriate restriction sites, HAX1 CDS was cloned into an intermediate vector using TA-cloning. The sequence was transferred into a retroviral vector, followed by retroviral packaging in Plat-A cells. To show HAX1 protein expression, HEK293T cells were exposed to 10 multiplicity of infection (MOI) of retroviral particles and HAX1 expression was confirmed in these cells, using indirect intracellular flow cytometry. This vector was applied for in vitro transduction of hematopoietic stem cell with HAX1 mutation; after 11 days, cultured cells were analyzed for CD66acde and CD177 (neutrophil surface markers) expression. Increased neutrophil production in HAX1 viral vector-expressing hematopoietic cells was observed as compared to control vector transduced cells. Hence, according to the results, this type of therapy could be considered a potential treatment protocol for the disease.     

The Role of Apoptosis in the Pathophysiology of Chronic Neutropenias Associated with Bone Marrow Failure

Chronic neutropenia syndromes associated with bone marrow (BM) failure comprise distinct congenital and acquired hematologic disorders with varying degree of neutropenia due to decreased or ineffective BM neutrophil production. Recent evidence suggests that defective granulocytopoiesis in these neutropenia states is a consequence of accelerated apoptotic cell death of BM myeloid progenitor cells and/or their differentiated progeny. Inherited or spontaneously appearing mutations in the ELA2 gene encoding for neutrophil elastase have been implicated in the accelerated apoptotic process of the BM myeloid cells in patients with cyclic and severe congenital neutropenia. A disturbed balance between pro-apoptotic and anti-apoptotic intracellular or membrane molecules such as down-regulation of the bcl-2 family members or up-regulation of the death receptor Fas, have been implicated in neutropenia associated with myelokathexis, Shwachman-Diamond syndrome and acquired chronic idiopathic neutropenia of adult. In this review we summarize the available evidence suggesting that abnormally increased apoptosis and impaired proliferative and differentiating properties of neutrophil progenitor and precursor cells represent a common pathogenetic mechanism for impaired granulocytopoiesis in both acquired idiopathic and congenital neutropenia states. The underlying distinct cellular and molecular abnormalities and the role of the BM microenvironment are extensively analysed.     

The role of apoptosis in the pathophysiology of chronic neutropenias associated with bone marrow failure

Chronic neutropenia syndromes associated with bone marrow (BM) failure comprise distinct congenital and acquired hematologic disorders with varying degree of neutropenia due to decreased or ineffective BM neutrophil production. Recent evidence suggests that defective granulocytopoiesis in these neutropenia states is a consequence of accelerated apoptotic cell death of BM myeloid progenitor cells and/or their differentiated progeny. Inherited or spontaneously appearing mutations in the ELA2 gene encoding for neutrophil elastase have been implicated in the accelerated apoptotic process of the BM myeloid cells in patients with cyclic and severe congenital neutropenia. A disturbed balance between pro-apoptotic and anti-apoptotic intracellular or membrane molecules such as downregulation of the bcl-2 family members or upregulation of the death receptor Fas, have been implicated in neutropenia associated with myelokathexis, Shwachman-Diamond syndrome and acquired chronic idiopathic neutropenia of adult. In this review we summarize the available evidence suggesting that abnormally increased apoptosis and impaired proliferative and differentiating properties of neutrophil progenitor and precursor cells represent a common pathogenetic mechanism for impaired granulocytopoiesis in both acquired idiopathic and congenital neutropenia states. The underlying distinct cellular and molecular abnormalities and the role of the BM microenvironment are extensively analysed.     

HAX1: A versatile,intrinsically disordered regulatory protein

HAX1 is a relatively small, ubiquitously expressed, predominantly mitochondrial, intrinsically disordered protein. It has been implicated in the regulation of apoptosis, cell migration, calcium cycling, proteostasis, angiogenesis, autophagy and translation. A wide spectrum of functions, numerous interactions and still elusive molecular mechanisms of action make HAX1 an intriguing subject of research. Moreover, HAX1 is involved in the pathogenesis of diseases; its deficiency leads to neutropenia and its overexpression is associated with cancer. In this review we aim to describe the characteristics of HAX1 gene and protein, and comprehensively discuss its multiple functions, highlighting the emerging role of HAX1 in protection from stress and apoptosis through maintaining cellular proteostasis and homeostasis.     

Congenital neutropenia: diagnosis, molecular bases and patient management

The term congenital neutropenia encompasses a family of neutropenic disorders, both permanent and intermittent, severe (<0.5 G/l) or mild (between 0.5-1.5 G/l), which may also affect other organ systems such as the pancreas, central nervous system, heart, muscle and skin. Neutropenia can lead to life-threatening pyogenic infections, acute gingivostomatitis and chronic parodontal disease, and each successive infection may leave permanent sequelae. The risk of infection is roughly inversely proportional to the circulating polymorphonuclear neutrophil count and is particularly high at counts below 0.2 G/l. When neutropenia is detected, an attempt should be made to establish the etiology, distinguishing between acquired forms (the most frequent, including post viral neutropenia and auto immune neutropenia) and congenital forms that may either be isolated or part of a complex genetic disease. Except for ethnic neutropenia, which is a frequent but mild congenital form, probably with polygenic inheritance, all other forms of congenital neutropenia are extremely rare and have monogenic inheritance, which may be X-linked or autosomal, recessive or dominant. About half the forms of congenital neutropenia with no extra-hematopoetic manifestations and normal adaptive immunity are due to neutrophil elastase (ELANE) mutations. Some patients have severe permanent neutropenia and frequent infections early in life, while others have mild intermittent neutropenia. Congenital neutropenia may also be associated with a wide range of organ dysfunctions, as for example in Shwachman-Diamond syndrome (associated with pancreatic insufficiency) and glycogen storage disease type Ib (associated with a glycogen storage syndrome). So far, the molecular bases of 12 neutropenic disorders have been identified. Treatment of severe chronic neutropenia should focus on prevention of infections. It includes antimicrobial prophylaxis, generally with trimethoprim-sulfamethoxazole, and also granulocyte-colony-stimulating factor (G-CSF). G-CSF has considerably improved these patients' outlook. It is usually well tolerated, but potential adverse effects include thrombocytopenia, glomerulonephritis, vasculitis and osteoporosis. Long-term treatment with G-CSF, especially at high doses, augments the spontaneous risk of leukemia in patients with congenital neutropenia.     

Hax1 regulates neutrophil adhesion and motility through RhoA

Kostmann disease is an inherited severe congenital neutropenia syndrome associated with loss-of-function mutations in an adaptor protein HS1-associated protein X-1 (Hax1). How Hax1 regulates neutrophil function remains largely unknown. In this paper, we use ribonucleic acid interference to deplete Hax1 in the neutrophil-like cell line PLB-985 and identify Hax1 as a negative regulator of integrin-mediated adhesion and chemotaxis. Using microfluidics, we show that depletion of Hax1 impairs neutrophil uropod detachment and directed migration. Hax1-deficient cells also display increased integrin-mediated adhesion and reduced RhoA activity. Moreover, depletion of RhoA induces increased neutrophil adhesion and impaired migration, suggesting that Hax1 regulates neutrophil adhesion and chemotaxis through RhoA. Accordingly, activation of RhoA is sufficient to rescue adhesion of Hax1-deficient neutrophils. Together, our findings identify Hax1 as a novel regulator of neutrophil uropod detachment and chemotaxis through RhoA.     

G6PC3 mutations are associated with a major defect of glycosylation: a novel mechanism for neutrophil dysfunction

Glucose-6-phosphatase, an enzyme localized in the endoplasmic reticulum (ER), catalyzes the hydrolysis of glucose-6-phosphate (G6P) to glucose and inorganic phosphate. In humans, there are three differentially expressed glucose-6-phosphatase catabolic genes (G6PC1-3). Recently, it has been shown that mutations in the G6PC3 gene result in a syndrome associating congenital neutropenia and various organ malformations. The enzymatic function of G6PC3 is dependent on G6P transport into the ER, mediated by G6P translocase (G6PT). Mutations in the gene encoding G6PT result in glycogen storage disease type-1b (GSD-1b). Interestingly, GSD-1b patients exhibit a similar neutrophil dysfunction to that observed in G6PC3-deficient patients. To better understand the causes of neutrophil dysfunction in both diseases, we have studied the neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase of patients with G6PC3 and G6PT syndromes. Unexpectedly, sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments indicated hypo-glycosylation of gp91(phox), the electron-transporting component of the NADPH oxidase, in all of these patients. Rigorous mass spectrometric glycomic profiling showed that most of the complex-type antennae which characterize the neutrophil N-glycome of healthy individuals were severely truncated in the patients' neutrophils. A comparable truncation of the core 2 antenna of the O-glycans was also observed. This aberrant neutrophil glycosylation is predicted to have profound effects on the neutrophil function and merit designation of both syndromes as a new class of congenital disorders of glycosylation.     

Identification of WASP mutations,mutation hotspots and genotype-phenotype disparities in 24 patients with the Wiskott-Aldrich syndrome

The Wiskott-Aldrich syndrome (WAS), an X-linked immunodeficiency disease caused by mutation in the recently isolated gene encoding WAS protein (WASP), is known to be associated with extensive clinical heterogeneity. Cumulative mutation data have revealed that WASP genotypes are also highly variable among WAS patients, but the relationship of phenotype with genotype in this disease remains unclear. To address this issue we characterized WASP mutations in 24 unrelated WAS patients, including 18 boys with severe classical WAS and 6 boys expressing mild forms of the disease, and then examined the degree of correlation of these as well as all previously published WASP mutations with disease severity. By analysis of these compiled mutation data, we demonstrated clustering of WASP mutations within the four most N-terminal exons of the gene and also identified several sites within this region as hotspots for WASP mutation. These characteristics were observed, however, in both severe and mild cases of the disease. Similarly, while the cumulative data revealed a predominance of missense mutations among the WASP gene lesions observed in boys with isolated thrombocytopenia, missense mutations were not exclusively associated with milder WAS phenotypes, but also comprised a substantial portion (38%) of the WASP gene defects found in patients with severe disease. These findings, as well as the detection of identical WASP mutations in patients with disparate phenotypes, reveal a lack of phenotype concordance with genotype in WAS and thus imply that phenotypic outcome in this disease cannot be reliably predicted solely on the basis of WASP genotypes. Received: 30 May 1996 / Revised: 16 July 1996     

Increased Nucleosomes and Neutrophil Activation Link to Disease Progression in Patients with Scrub Typhus but Not Murine Typhus in Laos

Cell-mediated immunity is essential in protection against rickettsial illnesses, but the role of neutrophils in these intracellular vasculotropic infections remains unclear. This study analyzed the plasma levels of nucleosomes, FSAP-activation (nucleosome-releasing factor), and neutrophil activation, as evidenced by neutrophil-elastase (ELA) complexes, in sympatric Lao patients with scrub typhus and murine typhus. In acute scrub typhus elevated nucleosome levels correlated with lower GCS scores, raised respiratory rate, jaundice and impaired liver function, whereas neutrophil activation correlated with fibrinolysis and high IL-8 plasma levels, a recently identified predictor of severe disease and mortality. Nucleosome and ELA complex levels were associated with a 4.8-fold and 4-fold increased risk of developing severe scrub typhus, beyond cut off values of 1,040 U/ml for nucleosomes and 275 U/ml for ELA complexes respectively. In murine typhus, nucleosome levels associated with pro-inflammatory cytokines and the duration of illness, while ELA complexes correlated strongly with inflammation markers, jaundice and increased respiratory rates. This study found strong correlations between circulating nucleosomes and neutrophil activation in patients with scrub typhus, but not murine typhus, providing indirect evidence that nucleosomes could originate from neutrophil extracellular trap (NET) degradation. High circulating plasma nucleosomes and ELA complexes represent independent risk factors for developing severe complications in scrub typhus. As nucleosomes and histones exposed on NETs are highly cytotoxic to endothelial cells and are strongly pro-coagulant, neutrophil-derived nucleosomes could contribute to vascular damage, the pro-coagulant state and exacerbation of disease in scrub typhus, thus indicating a detrimental role of neutrophil activation. The data suggest that increased neutrophil activation relates to disease progression and severe complications, and increased plasma levels of nucleosomes and ELA complexes represent independent risk factors for developing severe scrub typhus.     

Congenital neutropenia: Abnormal neutrophil differentiation associated withHLA

Congenital neutropenia (CN), a usually fatal autosomal recessive disease characterized by a maturation arrest of neutrophil differentiation at the promyelocyte stage, is shown to be significantly associated with HLA-B12. A gene-dose effect for the association with B12 has been observed. The genetic determinant responsible for CN is in apparent linkage disequilibrium with the antigen B12. This disease association suggests that the gene (or genes) controlling neutrophilic granulocyte differentiation is closely linked to the HLA complex. This relationship may reflect a basic function of the histocompatibility system, namely the coding for cell-surface determinants fundamental to cell-cell recognition and to control of cellular differentiation.Deceased