Eosinophil Cationic Protein Gene Research Articles

Eosinophil Cationic Protein: Overview of Biological and Genetic Features

The eosinophil cationic protein (ECP) is a small polypeptide that originates from activated eosinophil granulocytes. A wide range of stimuli has been shown to induce the secretion of ECP. The gene that encodes the human ECP is located on chromosome 14, and the protein shares the overall three-dimensional structure and the RNase active-site residues with other proteins in the RNase A superfamily. Several single-nucleotide polymorphisms in the human ECP gene have been currently described. ECP has many biological functions, including an immunoregulatory function, the regulation of fibroblast activity, and the induction of mucus secretion in the airway. Additionally, the protein is a potent cytotoxic molecule and has the capacity to kill mammalian and nonmammalian cells. The purpose of this article was to review the known biological and genetic characteristics of ECP that contribute to the understanding of this protein's role in the development and progression of a wide variety of diseases.

Eosinophil catonic protein – current concepts and controversies

Eosinophil cationic protein (ECP) is a heterogeneous molecule originating from activated eosinophil granulocytes. Biological activity and the cellular content of ECP are determined by genetic and posttranslational factors. Several single nucleotide polymorphisms (SNPs) in human ECP gene (RNASE3) have been described so far. ECP is a mediator in host immune response to parasites, bacteria and viruses. By its cytotoxic and non-cytotoxic activity, ECP may also cause side-effects in the host's own tissues. The largest number of clinical studies is focused on the role of ECP in eosinophil-related disorders, particularly in asthma. Although present in numerous body fluids, difficult bioavailability of biological material, invasive sampling methods and complex sample management prior to ECP level determination are the reasons that serum is most commonly used in routine laboratory practice. As numerous biological and methodological preanalytical factors (the type of collection test-tube, temperature and duration of blood clotting, centrifugation, hemolysis) may affect test result, the sample for serum ECP determination should be collected under standardized conditions. Regarding interpretation of results, it is necessary, along with absolute ECP concentration values, to monitor changes in ECP concentration during the duration of disease or after implemented therapy, and interpret ECP test result in combination with other laboratory and clinical findings. Rational approach to selection of new tests is indeed one of important requirements that medical workers meet today. To enable them to determine the clinical significance of ECP with better certainty, further studies on a large number of specific patient groups are needed.

Associations of ECP (eosinophil cationic protein)-gene polymorphisms to allergy, asthma, smoke habits and lung function in two Estonian and Swedish sub cohorts of the ECRHS II study

BackgroundThe Eosinophil Cationic Protein (ECP) is a potent multifunctional protein. Three common polymorphisms are present in the ECP gene, which determine the function and production of the protein. The aim was to study the relationship of these ECP gene polymorphisms to signs and symptoms of allergy and asthma in a community based cohort (The European Community Respiratory Health Survey (ECRHS)).MethodsSwedish and Estonian subjects (n = 757) were selected from the larger cohort of the ECRHS II study cohort. The prevalence of the gene polymorphisms ECP434(G>C) (rs2073342), ECP562(G>C) (rs2233860) and ECP c.-38(A>C) (rs2233859) were analysed by DNA sequencing and/or real-time PCR and related to questionnaire-based information of allergy, asthma, smoking habits and to lung functions.ResultsGenotype prevalence showed both ethnic and gender differences. Close associations were found between the ECP434(G>C) and ECP562(G>C) genotypes and smoking habits, lung function and expression of allergic symptoms. Non-allergic asthma was associated with an increased prevalence of the ECP434GG genotype. The ECP c.-38(A>C) genotypes were independently associated to the subject being atopic.ConclusionOur results show associations of symptoms of allergy and asthma to ECP-genotypes, but also to smoking habits. ECP may be involved in impairment of lung functions in disease. Gender, ethnicity and smoking habits are major confounders in the evaluations of genetic associations to allergy and asthma.

Identification of polymorphisms in the RNase3 gene and the association with allergic rhinitis

Eosinophil cationic protein (ECP), a potent cytotoxic molecule, is released by activated eosinophils. ECP has been suggested to be involved in tissue remodeling of allergic diseases. The ECP (RNase3) gene is a candidate gene in atopic diseases. RNase3 polymorphisms have been reported to have an association with atopy. We determined whether polymorphisms in the RNase3 gene are associated with allergic rhinitis in a Korean population. The Taqman assay, restriction fragment length polymorphism (PCR-RFLP), and high-resolution melt (HRM) were used for genotyping. Three single nucleotide polymorphisms (SNPs; g.-550A>G, g.371G>C, and g.499G>C) were identified. The genotype of the SNPs was analyzed in patients with allergic rhinitis and controls without allergic rhinitis. The genotype and allele frequencies were compared between both groups. The genotype frequencies of the g.-550A>G and g.371G>C SNPs were not significantly different between patients with allergic rhinitis and controls (P > 0.05). However, in patients with allergic rhinitis, the genotype and allele frequencies of the g.499G>C SNP of RNase 3 were significantly different from those of the control group (P < 001, P = 0.034, respectively). Haplotype analysis demonstrated the presence of the following five different (-550)-(+371)-(+499) major haplotypes: A-G-G, G-C-C, G-G-G, G-C-G, and A-G-C. The G-C-G haplotype was positively associated with allergic rhinitis (P = 0.048), while the G-G-G haplotype was negatively associated with allergic rhinitis (P = 0.004). Our study suggests that RNase3 polymorphisms are potentially associated with susceptibility to allergic rhinitis.

P3.64. The 434(G > C) polymorphism in the eosinophil cationic protein gene and its association with tissue eosinophilia in oral squamous cell carcinomas

P3.64. The 434(G > C) polymorphism in the eosinophil cationic protein gene and its association with tissue eosinophilia in oral squamous cell carcinomas

The 434(G&gt;C) polymorphism in the eosinophil cationic protein gene and its association with tissue eosinophilia in oral squamous cell carcinomas

The aim of this study was to investigate the prevalence of the Eosinophil cationic protein (ECP)-gene polymorphism 434(G>C) in oral squamous cell carcinoma (OSCC) patients and its association with tumor-associated tissue eosinophilia (TATE), demographic, clinical, and microscopic variables. The ECP genotypes of 165 healthy individuals and 157 OSCC patients were detected by PCR-RFLP analysis after cleavage of the amplified DNA sequence with enzyme PstI. TATE was obtained by morphometric analysis. Chi-square test or Fisher's exact test was used to analyze the association of ECP-gene polymorphism 434(G>C) with TATE, demographic, clinical, and microscopic variables in OSCC patients. Disease-free survival and overall survival were calculated by the Kaplan-Meier product-limit actuarial method and the comparison of the survival curves were performed using log rank test. Most of healthy individuals (53.33%) and OSCC patients (57.97%) were heterozygous for the ECP 434(G>C) polymorphism. Based on numerical differences, our results showed that OSCC patients with intense TATE and at least one C allele had a higher frequency of bilateral neck dissection, local recurrence, vascular embolization, involved resection margins, and postoperative radiotherapy. No statistically significant differences on survival rates were found in OSCC patients presenting different ECP 434(G>C) genotypes. These results suggest a tendency towards a poor clinical outcome in OSCC patients with intense TATE and 434GC/CC genotypes, probably due to an ECP genetic variant with altered cytotoxic activity.

The 434(G>C) polymorphism within the coding sequence of Eosinophil Cationic Protein (ECP) correlates with the natural course of Schistosoma mansoni infection

Schistosomiasis is a chronic parasitic infection with over 200 million people infected worldwide. In Schistosoma mansoni infections, parasite-derived eggs get trapped in the liver, causing the formation of granulomas, which may develop into periportal fibrosis and portal hypertension, and thus severe morbidity. Eosinophil cationic protein (ECP) is a secretory protein of eosinophil granulocytes that efficiently kills the larval stage of S. mansoni, but also affects fibroblast functions. We have investigated the prevalence of the ECP gene polymorphism 434(G>C) in two African populations, from an S. mansoni endemic area in Uganda (n=297) and from a non-endemic area in Sudan (n=78), and also compared these with a Swedish population (n=209). The genotype frequencies in the Ugandan population differed significantly from both the Sudanese and Swedish populations (P<0.001). In the Ugandan population there was a significant association between genotype and prevalence of infection (P=0.03), with lower prevalence in subjects with the GG genotype compared with GC (P=0.02) and CC (P=0.03). There was also a trend towards an association with periportal fibrosis (P=0.08) in the Ugandan population. This suggested association was confirmed when the predominant tribe (n=212) was analysed separately (P=0.004). Our results suggest that ECP may be an important protein, both in the immune response against S. mansoni and in the development of periportal fibrosis. The results also suggest genetic selection towards the ECP 434CC genotype in populations living in S. mansoni endemic areas.

The functional heterogeneity of eosinophil cationic protein is determined by a gene polymorphism and post‐translational modifications

The eosinophil is a cytotoxic cell and takes part in parasite killing and tissue-destructive processes by secretion of proteins such as eosinophil cationic protein (ECP). A polymorphism was demonstrated in the ECP gene, giving rise to a substitution of arginine at position 97 with threonine. This polymorphism is related to disease development. To investigate the functional and molecular heterogeneity of native ECP and the functional consequences of the replacement of arginine with a threonine. ECP was purified from healthy blood donors by gel filtration, ion-exchange chromatography and reversed-phase chromatography. Recombinant ECPs i.e. rECP 97(arg) and rECP 97(thr) were produced by the pFASTBAC baculovirus expression system. The cytotoxic activity was determined against an erythroleukaemia or a small cell lung cancer cell line. Native ECP was purified to apparent homogeneity and showed a considerable molecular heterogeneity and a corresponding functional heterogeneity with respect to cytotoxic activity. After reduction, the native cytotoxic ECP showed three bands on sodium dodecylsulphate polyacrylamide gel electrophoresis : one major band at 18-20 kDa and two minor bands at about 10 and 5 kDa, respectively. The 5 kDa contained two masses differing with 56.2 Da, which corresponds to the difference in molecular masses of arginine and threonine. rECP 97(arg) was cytotoxic in contrast to rECP97(thr). Deglycosylation with N-glycosidase F did not affect the cytotoxic activity of native ECP to any measurable extent nor the activity of rECP 97(arg), whereas rECP 97(thr) achieved cytotoxic activity. The RNase activities of the recombinant and native ECPs were similar. We conclude that ECP is present in several molecular forms with varying biological activities. Some of this functional heterogeneity is based on the genetic polymorphism of the ECP gene and some on post-translational modifications. In subjects carrying the ECP 97(thr) variant, the cytotoxic activity may be disguised by N-linked glycosylation of the active site.

A (G→C) transversion in the 3′ UTR of the human ECP (eosinophil cationic protein) gene correlates to the cellular content of ECP

Eosinophil cationic protein (ECP) is a cytotoxic protein produced by and secreted from human eosinophil granulocytes. ECP may be involved in the injury of epithelial cells in allergic diseases such as asthma. The objectives were to determine the prevalence of the ECP gene polymorphism 562(G > C) in apparently healthy subjects and subjects with allergy and relate the prevalence to clinical disease and to serum and cellular levels of ECP. The 562(G > C) ECP gene polymorphism was determined by gene sequencing of the ECP gene from DNA prepared from 163 apparently healthy subjects and 151 subjects with allergic and nonallergic asthma or other diseases. ECP was measured by a sensitive radioimmunoassay. A polymorphism was detected at position 562, which mapped to the 3' untranslated region (UTR) of the gene encoding the ECP (RNase 3). Sixty-nine percent of the population had the 562GG genotype and 4%, the 562CC genotype. The cellular content of ECP in peripheral blood eosinophil granulocytes was significantly lower in cells from subjects with the 562GC (4.6+/-1.5 microg/10(6) eosinophils) and 562CC (3.2+/-0.7 microg/10(6) eosinophils) genotypes as compared with those with the 562GG genotype (6.0+/-1.9 microg/10(6) eosinophils; P < 0.001). A close link was found to the 434(G > C) ECP gene polymorphism. Associations between the 562(G > C) polymorphism or haplotypes of the two polymorphisms to allergy were not found. The 562(G > C) polymorphism in the 3'-end of the UTR of the ECP gene may determine the ECP content in human eosinophils, but unlike the 434(G > C) polymorphism, the 562(G > C) polymorphism is not related to allergy.

Bystander Cells and Prognosis in Hodgkin Lymphoma

Hodgkin lymphoma (HL) is characterised histologically by a minority of malignant Hodgkin and Reed-Sternberg (HRS) cells surrounded by benign cells, and clinically by a relatively good prognosis. The treatment, however, leads to a risk of serious side effects. Knowledge about the biology of the disease, particularly the interaction between the HRS cells and the surrounding cells, is essential in order to improve diagnosis and treatment.HL patients with abundant eosinophils in the tumours have a poor prognosis, therefore the eosinophil derived protein eosinophil cationic protein (ECP) was studied. Serum-ECP (S-ECP) was elevated in most HL patients. It correlated to number of tumour eosinophils, nodular sclerosis (NS) histology, and the negative prognostic factors high erythrocyte sedimentation rate (ESR) and blood leukocyte count (WBC). A polymorphism in the ECP gene (434(G>C)) was identified and the 434GG genotype correlated to NS histology and high ESR.The poor prognosis in patients with abundant eosinophils in the tumours has been proposed to depend on HRS cell stimulation by the eosinophils via a CD30 ligand (CD30L)-CD30 interaction. However, CD30L mRNA and protein were detected in mast cells and the predominant CD30L expressing cell in HL is the mast cell. Mast cells were shown to stimulate HRS cell lines via CD30L-CD30 interaction. The number of mast cells in HL tumours correlated to worse relapse-free survival, NS histology, high WBC, and low blood haemoglobin.Survival in patients with early and intermediate stage HL, diagnosed between 1985 and 1992, was generally favourable and comparatively limited treatment was sufficient to produce acceptable results for most stages. The majority of relapses could be salvaged. Patients treated with a short course of chemotherapy and radiotherapy had an excellent outcome.In conclusion prognosis is favourable in early and intermediate stages and there are possibilities for further improvements based on the fact that mast cells and eosinophils affect the biology and prognosis of HL.

Return of the eosinophil question in asthma

Return of the eosinophil question in asthma

STAT6-independent regulation of TH2 cell development

STAT6-independent regulation of TH2 cell development

Monocytes, but not macrophages, produce the eosinophil cationic protein.

The eosinophil cationic protein (ECP) is a cytotoxic protein with ribonuclease activity, produced and stored in bone marrow eosinophil myelocytes. Mature circulating eosinophils contain about 10 pg ECP per cell. The aim of this study was to investigate the possibility that monocytes produce and store ECP. By results from flow cytometry and specific protein measurement it is shown that human monocytes contain ECP (monocytes about 10 fg ECP per cell). RT-PCR analysis indicated the presence of mRNA coding for ECP in blood monocytes but not in alveolar macrophages. Furthermore, mRNA coding for ECP and low amounts of the protein were found in three myeloid cell lines representing different stages of monocytic differentiation. Differentiation of U-937 cells to macrophages induced lowered transcription of the ECP gene and reduced protein production. Immunohistochemical staining of lung tissue indicated that lung macrophages do not contain ECP. It is concluded that ECP is produced to a low extent by human monocytes and that the production is shut down during macrophage differentiation. This might indicate an alternative transcriptional regulation of the ECP gene in the monocytic lineage compared to the eosinophil lineage.

Monocytes, but not macrophages, produce the eosinophil cationic protein

The eosinophil cationic protein (ECP) is a cytotoxic protein with ribonuclease activity, produced and stored in bone marrow eosinophil myelocytes. Mature circulating eosinophils contain about 10 pg ECP per cell. The aim of this study was to investigate the possibility that monocytes produce and store ECP. By results from flow cytometry and specific protein measurement it is shown that human monocytes contain ECP (monocytes about 10 fg ECP per cell). RT‐PCR analysis indicated the presence of mRNA coding for ECP in blood monocytes but not in alveolar macrophages. Furthermore, mRNA coding for ECP and low amounts of the protein were found in three myeloid cell lines representing different stages of monocytic differentiation. Differentiation of U‐937 cells to macrophages induced lowered transcription of the ECP gene and reduced protein production. Immunohistochemical staining of lung tissue indicated that lung macrophages do not contain ECP. It is concluded that ECP is produced to a low extent by human monocytes and that the production is shut down during macrophage differentiation. This might indicate an alternative transcriptional regulation of the ECP gene in the monocytic lineage compared to the eosinophil lineage.

Positive Darwinian selection after gene duplication in primate ribonuclease genes.

Evolutionary mechanisms of origins of new gene function have been a subject of long-standing debate. Here we report a convincing case in which positive Darwinian selection operated at the molecular level during the evolution of novel function by gene duplication. The genes for eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) in primates belong to the ribonuclease gene family, and the ECP gene, whose product has an anti-pathogen function not displayed by EDN, was generated by duplication of the EDN gene about 31 million years ago. Using inferred nucleotide sequences of ancestral organisms, we showed that the rate of nonsynonymous nucleotide substitution was significantly higher than that of synonymous substitution for the ECP gene. This strongly suggests that positive Darwinian selection operated in the early stage of evolution of the ECP gene. It was also found that the number of arginine residues increased substantially in a short period of evolutionary time after gene duplication, and these amino acid changes probably produced the novel anti-pathogen function of ECP.

Intronic Enhancer Activity of the Eosinophil-derived Neurotoxin (RNS2) and Eosinophil Cationic Protein (RNS3) Genes Is Mediated by an NFAT-1 Consensus Binding Sequence

The eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP) are both small, cationic ribonuclease toxins that are stored in and secreted by activated human eosinophilic leukocytes. We have previously shown that optimal expression of the EDN gene is dependent on an interaction between an intronic enhancer element or elements and the 5' promoter region. Here we present evidence demonstrating that the gene encoding ECP is regulated in an analogous fashion and that an intronic enhancer element functioning in both genes is a consensus binding sequence for the transcription factor NFAT-1. Our initial results demonstrate that one or more nuclear proteins isolated from human promyelocytic leukemia (HL-60) cells bind specifically at this consensus site (5'-GGAGAA-3') within the intron of the EDN gene and that disruption of this sequence reduced the characteristic 20-30-fold increase in reporter gene activity observed with the tandem EDN promoter/exon 1/intron construct to background levels. The NFAT-1 consensus site in the ECP gene differs from that found in the EDN gene by a single nucleotide (5'-GGAGAG-3'); the conversion of the 3' G to an A resulted in a further enhancement of the reporter gene activity supported by the ECP promoter/exon 1/intron construct. Interestingly, no "supershift" was observed in gel shift assays performed in the presence of anti-NFAT-1 antiserum, suggesting that a nuclear protein other than NFAT-1 may be acting at this consensus site.

The molecular biology of eosinophil granule proteins.

Here, we briefly review the molecular biology of the human eosinophil granule proteins, major basic protein (MBP), eosinophil peroxidase (EPO), eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). The nucleotide sequence of MBP cDNA indicates that MBP is translated as a 25.2-kilodalton preproprotein; the mpb gene consists of 6 exons and 5 introns spanning 3.3 kilobases (kb). The approximately 2.1-kb nucleotide sequence of EPO cDNA corresponds to a prosequence, light chain and heavy chain in that order; similarities to other peroxidases suggest the existence of a multigene family. EDN and ECP cDNAs and genes are remarkably similar throughout, suggesting a relatively recent divergence. Promoter regions of the 4 genes show interesting differences and similarities which may be related to differential gene regulation.

Structure and chromosome localization of the human eosinophil-derived neurotoxin and eosinophil cationic protein genes: Evidence for intronless coding sequences in the ribonuclease gene superfamily

Human genomic DNAs for the eosinophil granule proteins, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), were isolated from genomic libraries. Alignment of EDN ( RNS2) and ECP ( RNS3) gene sequences demonstrated remarkable nucleotide similarities in noncoding sequences, introns, and flanking regions, as well as in the previously known coding regions. Detailed examination of the 5′-noncoding regions yielded putative TATA and CAAT boxes, as well as similarities to promoter motifs from unrelated genes. A single intron of 230 bases was found in the 5′ untranslated region and we suggest that a single intron in this region and an intronless coding region are features common to many members of the RNase gene superfamily. The RNS2 and RNS3 genes were localized to the q24–q31 region of human chromosome 14. It is likely that these two genes arose as a consequence of a gene duplication event that took place approximately 25–40 million years ago and that a subset of anthropoid primates possess both of these genes or closely related genes.