Interaction and complex-formation between the eosinophil cationic protein and alpha 2-macroglobulin.
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ABSTRACT:
SUBMITTER: Peterson CG
PROVIDER: S-EPMC1148198 | biostudies-other | 1987 Aug
REPOSITORIES: biostudies-other
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Project description:On starch-gel or polyacrylamide-gel electrophoresis of human serum, a supernumerary zone of aspartate aminotransferase activity may be demonstrated, migrating with the slow alpha(2) protein zone. This appearance is due only to cationic aspartate aminotransferase, bound by alpha(2)-macroglobulin. The binding is strongly potentiated by dilute borate buffers.
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Project description:BackgroundEosinophilic granulocytes are important for the human immune system. Many cationic proteins with cytotoxic activities, such as eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN), are released from activated eosinophils. ECP, with low RNase activity, is widely used as a biomarker for asthma. ECP inhibits cell viability and induces apoptosis to cells. However, the specific pathway underlying the mechanisms of ECP-induced cytotoxicity remains unclear. This study investigated ECP-induced apoptosis in bronchial epithelial BEAS-2B cells and elucidated the specific pathway during apoptosis.ResultsTo address the mechanisms involved in ECP-induced apoptosis in human BEAS-2B cells, investigation was carried out using chromatin condensation, cleavage of poly (ADP-ribose) polymerase (PARP), sub-G1 distribution in cell cycle, annexin V labeling, and general or specific caspase inhibitors. Caspase-8-dependent apoptosis was demonstrated by cleavage of caspase-8 after recombinant ECP treatment, accompanied with elevated level of tumor necrosis factor alpha (TNF-alpha). Moreover, ECP-induced apoptosis was effectively inhibited in the presence of neutralizing anti-TNF-alpha antibody.ConclusionIn conclusion, our results have demonstrated that ECP increased TNF-alpha production in BEAS-2B cells and triggered apoptosis by caspase-8 activation through mitochondria-independent pathway.
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Project description:Eosinophil granulocytes reside in respiratory mucosa including lungs, in the gastro-intestinal tract, and in lymphocyte associated organs, the thymus, lymph nodes and the spleen. In parasitic infections, atopic diseases such as atopic dermatitis and asthma, the numbers of the circulating eosinophils are frequently elevated. In conditions such as Hypereosinophilic Syndrome (HES) circulating eosinophil levels are even further raised. Although, eosinophils were identified more than hundred years ago, their roles in homeostasis and in disease still remain unclear. The most prominent feature of the eosinophils are their large secondary granules, each containing four basic proteins, the best known being the eosinophil cationic protein (ECP). This protein has been developed as a marker for eosinophilic disease and quantified in biological fluids including serum, bronchoalveolar lavage and nasal secretions. Elevated ECP levels are found in T helper lymphocyte type 2 (atopic) diseases such as allergic asthma and allergic rhinitis but also occasionally in other diseases such as bacterial sinusitis. ECP is a ribonuclease which has been attributed with cytotoxic, neurotoxic, fibrosis promoting and immune-regulatory functions. ECP regulates mucosal and immune cells and may directly act against helminth, bacterial and viral infections. The levels of ECP measured in disease in combination with the catalogue of known functions of the protein and its polymorphisms presented here will build a foundation for further speculations of the role of ECP, and ultimately the role of the eosinophil.
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Project description:BackgroundThe eosinophil cationic protein (ECP) is cytotoxic to bacteria, viruses, parasites and mammalian cells. Cells are damaged via processes of pore formation, permeability alteration and membrane leaking. Some clinical studies indicate that ECP gathers in the bronchial tract of asthma sufferers, damages bronchial and airway epithelial cells, and leads to in breathing tract inflammation; therefore, prevention of the cytotoxicity caused by ECP may serve as an approach to treat airway inflammation. To achieve the purpose, reduction of the ECP-cell interactions is rational. In this work, the Chinese herbal combinative network was generated to predict and identify the functional herbs from the pools of prescriptions. It was useful to select the node herbs and to demonstrate the relative binding ability between ECP and Beas-2B cells with or withour herb treatments.ResultsEighty three Chinese herbs and prescriptions were tested and five effective herbs and six prescription candidates were selected. On the basis of effective single-herbal drugs and prescriptions, a combinative network was generated. We found that a single herb, Gan-cao, served as a node connecting five prescriptions. In addition, Sheng-di-huang, Dang-guei and Mu-tong also appeared in five, four and three kinds of prescriptions, respectively. The extracts of these three herbs indeed effectively inhibited the interactions between ECP and Beas-2B cells. According to the Chinese herbal combinative network, eight of the effective herbal extracts showed inhibitory effects for ECP internalizing into Beas-2B cells. The major components of Gang-cao and Sheng-di-huang, glycyrrhizic acid and verbascose, respectively, reduced the binding affinity between ECP and cells effectively.ConclusionsSince these Chinese herbs reduced the binding affinity between ECP and cells and inhibited subsequent ECP entrance into cells, they were potential for mitigating the airway inflammation symptoms. Additionally, we mentioned a new concept to study the Chinese herbs using combinative network in the field of systems biology. The functional single herbs could be identified from the set of prescriptions.
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Project description:BackgroundThe role of eosinophils in cancer is not yet completely understood, but patients with eosinophilia show a trend towards longer survival in several types of cancer, including melanoma. However, eosinophil count at initial diagnosis of metastatic melanoma does not predict survival. Since eosinophil cationic protein (ECP) mediates anticancer effects, such as tissue remodelling and cytotoxic activity, we investigated this marker as an early prognostic marker in metastatic melanoma.MethodsSerum of 56 melanoma patients was collected at the time of diagnosis of metastatic disease. ECP levels as measured by ELISA were correlated with overall survival (OS) in patients before systemic therapy with immunotherapy or chemotherapy. Statistical analyses were performed using the Log-Rank (Mantel-Cox) test.ResultsThe median OS for patients with high serum ECP above 12.2 ng/ml was 12 months (n = 39), compared to 28 months for patients with ECP below this threshold (n = 17; p = 0.0642). In patients with cutaneous melanoma, excluding patients with uveal and mucosal melanoma, the survival difference was even more striking (p = 0.0393). ECP's effect size on OS was observed independently of the consecutive therapy. ECP levels were not correlated with LDH levels.ConclusionECP seems to be a novel prognostic serum marker for the outcome of melanoma patients, which is independent of LDH and easy to perform in clinical practice. The striking negative prognostic value of high ECP level is unanticipated and can guide patient management.
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Project description:Cell-penetrating peptides (CPPs) are short peptides which can carry various types of molecules into cells; however, although most CPPs rapidly penetrate cells in vitro, their in vivo tissue-targeting specificities are low. Herein, we describe cell-binding, internalization, and targeting characteristics of a newly identified 10-residue CPP, denoted ECP(32-41), derived from the core heparin-binding motif of human eosinophil cationic protein (ECP). Besides traditional emphasis on positively charged residues, the presence of cysteine and tryptophan residues was demonstrated to be essential for internalization. ECP(32-41) entered Beas-2B and wild-type CHO-K1 cells, but not CHO cells lacking of cell-surface glycosaminoglycans (GAGs), indicating that binding of ECP(32-41) to cell-surface GAGs was required for internalization. When cells were cultured with GAGs or pre-treated with GAG-digesting enzymes, significant decreases in ECP(32-41) internalization were observed, suggesting that cell-surface GAGs, especially heparan sulfate proteoglycans were necessary for ECP(32-41) attachment and penetration. Furthermore, treatment with pharmacological agents identified two forms of energy-dependent endocytosis, lipid-raft endocytosis and macropinocytosis, as the major ECP(32-41) internalization routes. ECP(32-41) was demonstrated to transport various cargoes including fluorescent chemical, fluorescent protein, and peptidomimetic drug into cultured Beas-2B cells in vitro, and targeted broncho-epithelial and intestinal villi tissues in vivo. Hence this CPP has the potential to serve as a novel vehicle for intracellular delivery of biomolecules or medicines, especially for the treatment of pulmonary or gastrointestinal diseases.
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Project description:BackgroundAlthough blood eosinophils are a frequently used marker of type 2 inflammation in children with asthma, their sensitivity is relatively poor. Additional markers of type 2 inflammation are needed.ObjectiveWe hypothesized that plasma concentrations of eosinophil cationic protein (ECP), a marker of eosinophil activation, would be useful for detection of type 2 inflammation and would predict poorer asthma outcomes over 1 year.MethodsChildren and adolescents 6 through 17 years (N = 256) with confirmed asthma completed a baseline visit and a follow-up visit at 12 months. A subset also underwent systemic corticosteroid responsiveness testing with intramuscular triamcinolone. Outcome measures at 12 months included uncontrolled asthma, lung function, and asthma exacerbations treated with systemic corticosteroids.ResultsPlasma ECP concentrations ranged from 0.03 to 413.61 ng/mL (median, 6.95 ng/mL) and were consistently associated with other markers of type 2 inflammation. At baseline, children in the highest ECP tertile had poorer asthma control, more airflow limitation, and more exacerbations, but also had greater symptom improvement with intramuscular triamcinolone. At 12 months, associations between the highest ECP tertile and exacerbations, but not lung function or asthma control, persisted after covariate adjustment. However, the sensitivity of ECP was modest and was not markedly different from that of blood eosinophil counts.ConclusionPlasma ECP concentrations may be a useful marker of type 2 inflammation in children and may help identify those children at highest risk for recurrent exacerbations who could benefit from corticosteroid treatment. However, additional markers may be needed to improve sensitivity for outcome detection.
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Project description:Eosinophil cationic protein (ECP)/ribonuclease 3 is a member of the RNase A superfamily involved in inflammatory processes mediated by eosinophils. ECP is bactericidal, helminthotoxic, and cytotoxic to tracheal epithelium cells and to several mammalian cell lines although its RNase activity is low. We studied the thermal stability of ECP by fourth-derivative UV absorbance spectra, circular dichroism, differential scanning calorimetry, and Fourier transform infrared spectroscopy. The T (1/2) values obtained with the different techniques were in very good agreement (T (1/2) approximately 72 degrees C), and the stability was maintained in the pH range between 5 and 7. The ECP calorimetric melting curve showed, in addition to the main transition, a pretransitional conformational change with a T (1/2) of 44 degrees C. Both calorimetric transitions disappeared after successive re-heatings, and the ratio DeltaH versus DeltaH (vH) of 2.2 indicated a significant deviation from the two-state model. It was observed that the thermal unfolding was irreversible. The unfolding process gives rise to changes in the environment of aromatic amino acids that are partially maintained in the refolded protein with the loss of secondary structure and the formation of oligomers. From the thermodynamic analysis of ECP variants, the contribution of specific amino acids, such as Trp10 and the region 115-122, to thermal stability was also determined. The high thermal stability of ECP may contribute to its resistance to degradation when the protein is secreted to the extracellular medium during the immune response.
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Project description:The eosinophil granule proteins, major basic protein (MBP) and eosinophil cationic protein (ECP), activate mast cells during inflammation; however the mechanism responsible for this activity is poorly understood. We found that some theoretical tryptase-digested fragments of MBP and ECP induced degranulation of human cord blood-derived mast cells (HCMCs). The spectrum of activities of these peptides in HCMCs coincided with intracellular Ca2+ mobilization activities in Mas-related G-protein coupled receptor family member X2 (MRGPRX2)-expressing HEK293 cells. Two peptides corresponding to MBP residues 99-110 (MBP (99-110)) and ECP residues 29-45 (ECP (29-45)), respectively, induced degranulation of HCMCs and intracellular Ca2+ mobilization in MRGPRX2-expressing HEK293 cells in a concentration-dependent manner. Stimulation with MBP (99-110) or ECP (29-45) induced the production of prostaglandin D2 by HCMCs. The activities of MBP (99-110) and ECP (29-45) in both HCMCs and MRGPRX2-expressing HEK293 cells were inhibited by MRGPRX2-specific antagonists. In conclusion, these results indicated that MBP and ECP fragments activate HCMCs, and it may occur via MRGPRX2. Our findings suggest that tryptase-digested fragments of eosinophil cationic proteins acting via the MRGPRX2 pathway may further our understanding of mast cell/eosinophil communication.
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