Eosinophil peroxidase induces the expression and function of acid-sensing ion channel-3 in allergic rhinitis:in vitro evidence in cultured epithelial cells

BACKGROUND:
Acid-sensing ion channels (ASIC) are a family of acid-activated ligand-gated cation channels. As tissue acidosis is a feature of inflammatory conditions, such as allergic rhinitis (AR), we investigated the expression and function of these channels in AR.
OBJECTIVES:
The aim of the study was to assess expression and function of ASIC channels in the nasal mucosa of control and AR subjects.
METHODS:
Immunohistochemical localization of ASIC receptors and functional responses to lactic acid application were investigated. In vitro studies on cultured epithelial cells were performed to assess underlying mechanisms of ASIC function.
RESULTS:
Lactic acid at pH 7.03 induced a significant rise in nasal fluid secretion that was inhibited by pre-treatment with the ASIC inhibitor amiloride in AR subjects (n = 19). Quantitative PCR on cDNA isolated from nasal biopsies from control and AR subjects demonstrated that ASIC-1 was equally expressed in both populations, but ASIC-3 was significantly more highly expressed in AR (P < 0.02). Immunohistochemistry confirmed significantly higher ASIC-3 protein expression on nasal epithelial cells in AR patients than controls (P < 0.01). Immunoreactivity for EPO+ eosinophils in both nasal epithelium and submucosa was more prominent in AR compared with controls. A mechanism of induction of ASIC-3 expression relevant to AR was suggested by the finding that eosinophil peroxidase (EPO), acting via ERK1/2, induced the expression of ASIC-3 in epithelial cells. Furthermore, using a quantitative functional measure of epithelial cell secretory function in vitro, EPO increased the air-surface liquid depth via an ASIC-dependent chloride secretory pathway.
CONCLUSIONS:
This data suggests a possible mechanism for the observed association of eosinophils and rhinorrhoea in AR and is manifested through enhanced ASIC-3 expression.

HIF-2alpha Associated Familial Erythrocytosis Supports the PHD2-HIF2-alpha-VHL Axis as the Major Regulator of Erythropoietin Production

Transcriptionally erythropoietin (Epo) synthesis is tightly regulated by the hypoxia inducible factor (HIF), which is composed of one alpha and one beta subunit that are constitutively expressed. The beta subunit is non-variable, but three different alpha subunits give rise to three isoforms of HIF. The alpha subunit is proteasomally regulated in the presence of oxygen by hydroxylation of the proline in the LXXLAP motif of the oxygen dependent degradation (ODD) domain of HIFalpha, catalysed by members of the prolyl hydroxylase domain (PHD) family of enzymes. This allows the von Hippel Lindau (VHL) protein to associate with the alpha subunit, which is subsequently tagged with ubiquitin and degraded by the proteasome. Any defect in the oxygen sensing pathway that allows the alpha subunit to escape proteasomal regulation leads to elevated expression of HIF target genes.

Recently mutations in both VHL and PHD2 have been identified in a cohort of patients with erythrocytosis, but no mutations were found in the ODD domain of HIF1alpha. Instead, investigation of the homologous region in HIF-2alpha revealed four different mutations, Pro534Leu, Met535Val, Gly537Arg and Gly537Trp in seven individuals/families. Affected individuals presented at a young age with elevated serum Epo. Several individuals have a clinical history of thrombosis, but no evidence of a von Hippel Lindau-like syndrome.

To define how the four mutations relate to the erythrocytosis phenotype functional assays were performed in vitro. Binding of PHD2 to the four HIF-2alpha mutants was impaired to varying degrees, with both the Gly537 mutants showing the greatest reduction. The association of VHL with the hydroxylated Met535Val mutant peptide was similar to wild type HIF- 2alpha, but was decreased in the other three HIF-2alpha mutants. Expression of three HIF- 2alpha target genes, adrenomedullin, NDRG1 and VEGF, was significantly up-regulated in cells stably transfected with the mutants under normoxia compared to wild type HIF-2alpha. Mutations in the ODD domain of HIF-2alpha disrupt proteasomal regulation by reducing the association with PHD2 and hence hydroxylation. Furthermore the binding of VHL is also impaired, even when HIF-2alpha is hydroxylated. Examination of the three-dimensional structure of hydroxylated HIF-1alpha bound to VHL confirms that amino acids close to site of hydroxylation (Pro-531 in isoform 2) are important for this association. These observations, together with recent studies utilising murine models of erythrocytosis, support the PHD2-HIF-2alpha-VHL axis as the major regulator of erythropoietin.

Natural history of idiopathic erythrocytosis - A retrospective case series review

Idiopathic Erythrocytosis (IE) is a diagnosis given to patients who have an absolute erythrocytosis (red cell mass more than 25% above their mean normal predicted value) but who do not have a known form of primary or secondary erythrocytosis (BCSH guideline, 2005). We report here the results of a follow-up study of 80 patients (44 male and 36 female) diagnosed with IE from the United Kingdom and the Republic of Ireland over a 10 year period. Baseline information was initially collected when investigating for molecular causes of erythrocytosis in this group. The diagnosis of IE was made on the basis of a raised red cell mass >25% above mean normal predicted value, absence of Polycythaemia Vera (PV) based on the criteria of Pearson and Messinezy (1996), and the exclusion of secondary erythrocytosis (oxygen saturation >92% on pulse oximetry, no history of sleep apnoea, no renal or hepatic pathology, and a normal oxygen dissociation curve (if indicated). The average age at diagnosis of erythrocytosis was 34.5 (2–74 years). Erythropoietin levels were available for 77/80 of the patients and were low in 18 (23%) and normal or high in 59 (74%). Ultrasound imaging was carried out in 67 patients (84%) at time of diagnosis and no significant abnormalities found. Fourteen patients had a family history of erythrocytosis. These patients have now been followed up for an average of 9.4 years (range 1–39). Out of 80 patients 56 patients can still be classified as having IE, of whom 52 are living (cause of death in the other 4 - lung cancer, RTA, sepsis, unknown). Thirty-five of these patients are regularly venesected, 3 take hydroxyurea (one also venesected), 11 receive no treatment while treatment is unknown in 2. Twenty take aspirin, 1 warfarin and 31 no thromboprophylaxis. Four of these patients had suffered thromboembolic complications (3 with CVA/TIAs and 1 with recurrent DVT) at or before their original diagnosis. Since diagnosis 8 patients have had 9 thrombotic events of which 7 were arterial (1 CVA, 3 TIAs, 1 MI, 2 PVD) and 2 venous (DVT/PE). Twenty take aspirin, 1 dipyridamole, 1 warfarin and 30 take no thromboprophylaxis. Out of the 24 patients who now have a diagnosis other than IE, 8 have been diagnosed with myelo-proliferative disease. Thirteen patients have a molecular abnormality which is likely to account for their erythrocytosis (11 VHL, 1 PHD-2, 1 EPO-receptor mutations). Three patients have secondary erythrocytosis. Older case studies identified a heterogenous group of patients, some of whom probably had apparent erythrocytosis and some who had either primary polycythaemia or secondary causes later identified (Modan and Modan, Najean et al). More recent reviews have identified a more homogenous group with low rates of transformation to myelofibrosis/acute leukaemia and low rates of thrombosis of around 1% patient-year. Follow up of our initial patient group does indeed reveal a heterogeneous group of patients with 10% now diagnosed with an MPD, although when analysis is confined to those patients who continue to fulfil the criteria for IE, the clinical course has been more stable. There has been no progression to MDS or leukaemia in this group (one patient with PV progressed to AML). The rate of thrombosis is 1.6% patient-years which is lower than the rate seen in PV and is consistent with the rate identified in other series. Molecular defects continue to be identified in this group and future investigation is likely to reveal further abnormalities.

Erythropoietin receptor and hematological disease

This review will discuss evidence for the role of the erythropoietin (Epo) receptor in the development of erythrocytosis and other hematological disorders, The possible causative role of mutations of other genes in the pathogenesis of idiopathic erythrocytosis will be considered, Polycythemia vera (PV) is a myeloproliferative disorder that is caused by an undefined stem cell abnormality, characterized by a significant erythrocytosis, leukocytosis, and thrombocytosis. However, erythrocytosis may arise from apparent (or relative) polycythemia in which the hematocrit is raised due to a low plasma volume. In such cases the red cell mass is normal. A group of disorders with increased red cell mass caused by stimulation of erythrocyte production is known as secondary polycythemia, Investigation of such patients may reveal a congenital abnormality such as high affinity hemoglobin or an acquired abnormality caused, for example, by smoking, renal Vascular impairment, or an Epo-producing tumor. Even after thorough examination there remains a cohort of patients for whom no definite cause for the erythrocytosis can be established, A careful clinical history may reveal whether this idiopathic erythrocytosis is likely to be congenital and/or familial, in which case the term

Erythrocytosis due to a mutation in the erythropoietin receptor gene

Familial erythrocytosis, associated with high haemoglobin levels and low serum erythropoietin (Epo), has been shown to co-segregate with a sequence repeat polymorphism at the 5' region of the erythropoietin receptor (EpoR) in a large Finnish family. We have investigated the cause of erythrocytosis in an English boy. Sequencing of the cytoplasmic region of the EpoR detected a de novo transition mutation of G to A at nucleotide 6002. This mutation resulted in the formation of a stop codon at amino acid 439 with the loss of 70 amino acids from the carboxy terminus. The mutation (G6002A) has arisen independently in a Finnish family and de novo in this English boy. Patients with unexplained erythrocytosis and low serum Epo levels should be investigated for EpoR mutations.

Sequence analysis of the 3' hypoxia-responsive element of the human erythropoietin gene in patients with erythrocytosis

Erythrocytosis arises from a variety of pathogenic mechanisms. We sequenced a 256-bp region 3' to the erythropoietin (Epo) gene which included a 24- to 50-bp minimal hypoxia-responsive element spanning HIF-1- and HNF-4-binding sites in 12 patients with erythrocytosis and 4 normal subjects. Four polymorphisms were found, none of which affected the HIF-1-binding site, although one polymorphism was present in the HNF-4 consensus region. The data indicate that none of these polymorphisms cause erythrocytosis.

Selective block in erythropoietin-induced differentiation of growth factor-independent retrovirally-infected TF-1 cells

The erythroleukaemic cell line TF-1, infected with either the pBabe neo retrovirus or the retrovirus bearing the human erythropoietin (hEpo) gene, developed three growth factor-independent clones. Erythropoietin (Epo), interleukin-3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) accelerated the proliferation of these clones. Autonomous growth of the clones was independent of Epo because it was not altered by Epo anti-sense oligonucleotides, nor was Epo detectable in culture supernatants. Cells from the mutant clones could not be induced by Epo to express glycophorin A and haemoglobin synthesis was markedly reduced. Haemin reversed the block in Epo-induced haemoglobin synthesis. Acquisition of growth factor-independence appears to be linked with the selective loss of differentiation capacity. These cells may provide a useful model for the study of the mechanisms involved in leukaemic transformation.

Serum erythropoietin levels in paroxysmal nocturnal haemoglobinuria: Implications for therapy

In order to assess the rationale and possible indications for the use of recombinant erythropoietin in paroxysmal nocturnal haemoglobinuria (PNH), we have measured endogenous erythropoietin (Epo) levels in 18 patients with PNH and in 44 patients with iron deficiency anaemia (IDA), In both groups of patients we found a significant inverse correlation between Epo and haemoglobin (Hb). However, the mean Epo level was significantly higher in the PNH group (385 mU/ml) than in the IDA group (136 mU/ml), The range of Epo levels at any given Hb was greater in the PNH group than in the IDA group. There was a significant positive correlation between Epo and absolute reticulocyte count, Since Epo administration is unlikely to benefit patients with high levels of endogenous Epo, we conclude that in the majority of patients with PNH there is no indication for treatment with Epo.

Association Testing of Previously Reported Variants in a Large Case-Control Meta-analysis of Diabetic Nephropathy

We formed the GEnetics of Nephropathy–an International Effort (GENIE) consortium to examine previously reported genetic associations with diabetic nephropathy (DN) in type 1 diabetes. GENIE consists of 6,366 similarly ascertained participants of European ancestry with type 1 diabetes, with and without DN, from the All Ireland-Warren 3-Genetics of Kidneys in Diabetes U.K. and Republic of Ireland (U.K.-R.O.I.) collection and the Finnish Diabetic Nephropathy Study (FinnDiane), combined with reanalyzed data from the Genetics of Kidneys in Diabetes U.S. Study (U.S. GoKinD). We found little evidence for the association of the EPO promoter polymorphism, rs161740, with the combined phenotype of proliferative retinopathy and end-stage renal disease in U.K.-R.O.I. (odds ratio [OR] 1.14, P = 0.19) or FinnDiane (OR 1.06, P = 0.60). However, a fixed-effects meta-analysis that included the previously reported cohorts retained a genome-wide significant association with that phenotype (OR 1.31, P = 2 × 10-9). An expanded investigation of the ELMO1 locus and genetic regions reported to be associated with DN in the U.S. GoKinD yielded only nominal statistical significance for these loci. Finally, top candidates identified in a recent meta-analysis failed to reach genome-wide significance. In conclusion, we were unable to replicate most of the previously reported genetic associations for DN, and significance for the EPO promoter association was attenuated.

Regulation of erythropoietin gene expression depends on two different oxygen-sensing mechanisms

Erythropoietin (Epo), a glycoprotein hormone produced principally in the fetal kidney and in the adult liver in response to hypoxia, is the prime regulator of growth and differentiation in erythroid progenitor cells. The regulation of Epo gene expression is not fully understood, but two mechanisms have been proposed. One involves the participation of a heme protein capable of reversible oxygenation and the other depends on the intracellular concentration of reactive oxygen species (ROS), assumed to be a function of pO2. We have investigated the production of Epo in response to three stimuli, hypoxia, cobalt chloride, and the iron chelator desferrioxamine, in Hep3B cells. As expected, hypoxia caused a marked rise in Epo production. When the cells were exposed to the paired stimuli of hypoxia and cobalt no further increase was found. In contrast, chelation of iron under hypoxic conditions markedly enhanced Epo production, suggesting that the two stimuli act by separate pathways. The addition of carbon monoxide inhibited hypoxia-induced Epo production, independent of desferrioxamine concentration. Taken together these data support the concept that pO2 and ROS are sensed independently.

Erythropoietin production:evidence for multiple oxygen sensing pathways

The production of erythropoietin (Epo), the glycoprotein hormone which controls red blood cell formation, is regulated by feedback mechanisms sensing tissue oxygenation. The mechanism of the putative oxygen sensor has yet to be elucidated. There is evidence that at least two pathways participate in hypoxia signal transduction. One appears to involve a specific haem protein, and a second implicates reactive oxygen species (ROS). Iron catalyses the generation of intracellular ROS and therefore alters the cellular redox state. We have investigated the effect of modulating intracellular iron content on Epo production in Hep 3B cells. Iron chelation stimulates Epo production at 20% O2 and enhances Epo production at 1% O2, but it has no additive effect on cobalt-induced Epo production. Excess molar iron inhibited Epo production in response to hypoxia, desferrioxamine (DFO) and cobalt chloride and inhibited the DFO-enhancing effect of hypoxia-induced Epo production. We found that sulphydryl oxidising agents exert a differential inhibitory effect on hypoxia-induced versus DFO-induced Epo production, providing further evidence that multiple pathways of oxygen sensing exist.

Localization of erythropoietin gene expression in proximal renal tubular cells detected by digoxigenin-labelled oligonucleotide probes

Erythropoietin (EPO) is the main humoral stimulus of erythropoiesis. In adult mammals, the kidney releases EPO in response to hypoxic stress. Conflicting data have suggested either renal tubular or peritubular cell origins of EPO synthesis in vivo. In situ hybridization studies were performed to define further the kidney cell type(s) capable of increasing EPO gene expression during hypoxic stimulation. EPO gene expression was stimulated in mice exposed to acute hypobaric hypoxia. Kidneys from hypoxic and control normoxic mice were obtained. Six digoxigenin-labelled oligonucleotide probes complementary to EPO exon sequences were utilized for in situ hybridization for EPO messenger RNA. Positive hybridization signals were identified in some proximal tubular cells, confined to the inner third of the renal cortex of hypoxic mouse kidney.

Cord blood erythropoietin, pH, PaO2 and haematocrit following caesarean section before labour

We have investigated the relationship between erythropoietin (Epo) and pH, PaO2 and haematocrit in 100 cord blood samples obtained at Caesarean section prior to labour. Of 82 term (> 37 weeks) infants, 64 were appropriately grown (10th-90th centiles), and their mean cord serum Epo and cord blood Epo was 23 +/- 8 mU/ml (mean +/- SD). Strong inverse correlations were found between cord serum Epo and cord blood pH (r = -0.74; p <0.0001), and between cord serum Epo and cord blood PaO2 (r = -0.55; p <0.0001), but not between cord serum Epo and cord haematocrit (r = 0.02; p <0.9). For the 18 preterm babies (gestation 32.4 +/- 4.1 weeks, birth weight 1,820 +/- 476 g), the Epo level was 36 +/- 8 mU/ml, which was not significantly greater than for the term babies. Strong inverse correlations were again found between Epo and pH (r = -0.87; p <0.0001) and Epo and PaO2 (r = -0.69; p <0.002). Babies from complicated pregnancies (intra-uterine growth retardation, pre-eclampsia, antepartum haemorrhage, diabetes mellitus and fetal distress) tended to have higher Epo levels. Thirteen babies had Epo levels > 40 mU/ml, and 11 (85%) of these required neonatal intensive care. Cord serum Epo correlates better with oxygen tension and pH at birth than with fetal growth and haematocrit, which are measures of chronic stress to the fetus.

Two new mutations in the HIF2A gene associated with erythrocytosis

Congenital or familial erythrocytosis/polycythemia can have many causes, and an emerging cause is genetic disruption of the oxygen-sensing pathway that regulates the Erythropoietin (EPO) gene. More specifically, recent studies have identified erythrocytosis-associated mutations in the HIF2A gene, which encodes for Hypoxia Inducible Factor-2a (HIF-2a), as well as in two genes that encode for proteins that regulate it, Prolyl Hydroxylase Domain protein 2 (PHD2) and the von Hippel Lindau tumor suppressor protein (VHL). We report here the identification of two new heterozygous HIF2A missense mutations, M535T, and F540L, both associated with erythrocytosis. Met-535 has previously been identified as a residue mutated in other patients with erythrocytosis; although, the mutation of this particular residue to Thr has not been reported. In contrast, Phe-540 has not been reported as a residue mutated in erythrocytosis, and we present evidence here that this mutation impairs interaction of HIF-2a with both VHL and PHD2. © 2012 Wiley Periodicals, Inc.

Diagnosis and management of congenital and idiopathic erythrocytosis

An erythrocytosis occurs when there is an increased red-cell mass. The causes of erythrocytosis are divided into primary, when there is an intrinsic defect in the erythroid cell, and secondary, when the cause is extrinsic to the erythroid cell. An idiopathic erythrocytosis occurs when the increased red-cell mass has no identifiable cause. Primary and secondary defects can be further classified as either congenital or acquired causes. The diagnostic pathway starts with a careful history and examination followed by measurement of the erythropoietin (EPO) levels. This allows a division of those patients with a low EPO level, who can then be investigated for primary causes of erythrocytosis, and those with a normal or high EPO level, where the oxygen-sensing pathway needs to be explored further. Physiological studies in those with congenital defects in the oxygen-sensing pathway show many changes in the downstream metabolism adapting to the defect, which has a bearing on the management of the disorders. Low-dose aspirin and venesection to an achievable target are the main therapeutic options that can be considered in the management of erythrocytosis. Specific guidance on venesection options should be considered with certain causes such as high oxygen-affinity hemoglobins.