Airway hypersensitivity induced by eosinophil granule-derived cationic proteins.

Vagal bronchopulmonary C-fiber sensory nerves play an important role in the manifestation of airway hypersensitivity, a common and prominent pathophysiological feature of airway inflammatory diseases. Eosinophil granule-derived cationic proteins are known to be involved in the mucosal damage and development of bronchial hyperresponsiveness during allergic airway inflammation. In view of these background information, we have carried out a series of studies to investigate the effect of cationic proteins on these C-fiber afferents and the mechanism(s) possibly involved; a summary of these studies is presented in this mini-review. Intra-tracheal instillation of either eosinophil granule-derived (e.g., major basic protein, MBP) or synthetic cationic proteins (e.g., poly-l-lysine) induced a sporadic, but intense and lingering discharge of pulmonary C-fibers, and greatly enhanced the chemical and mechanical sensitivities of these afferents in anesthetized rats. The stimulatory and sensitizing effects of these proteins were completely nullified when their cationic charges were neutralized or removed. Furthermore, in isolated rat bronchopulmonary capsaicin-sensitive neurons, eosinophil granule cationic proteins induced a direct and long-lasting (>60 min) but reversible sensitizing effect on their responses to chemical and electrical stimulations. More importantly, our study showed that these cationic proteins exerted an inhibitory effect on the sustained delayed-rectifier voltage-gated K+ current and the A-type, fast-inactivating K+ current; these actions were at least in part responsible for the sensitizing effect in these neurons. In awake mice, intra-tracheal instillation of MBP also induced a slowly developing (peaking in 2-3 days), progressive and sustained (lasting for 3-7 days) elevation of the cough responses to inhaled irritant gases. Taken together, these findings suggest that the enhanced sensitivity of bronchopulmonary C-fibers induced by the eosinophil granule cationic proteins may be a contributing factor in the pathogenesis of bronchial hyperresponsiveness and chronic cough associated with eosinophilic infiltration of the airways.

[The significance and application of serum eosinophil cationic proteinand IgG4 antibody in patients with allergic rhinitistreated by allergic specific immunotherapy].

Objective:To study the significance and application of serum eosinophil cationic protein(ECP) and IgG4 antibody in patients with allergic rhinitis treated by allergic specific immunotherapy.Method:The level of IgG4 antibody and eosinophil cationic protein in serum was measured in 33 cases of allergic rhinitis before treatment and half a year and one year after treatment.The change of ECP level was observed in different age groups,and the relationship between serum IgG4 and ECP after treatment was analyzed.Result:IgG4 antibody in the serum significantly increased after treatment,and the difference was statistically significant(P<0.05).In the serum ECP content gradually reduced after treatment,and the difference was statistically significant(P<0.05).No obvious difference in ECP level was observed among dfferent age groups after treatment (P<0.05).The level of serum IgG4 was negatively correlated with serum ECP level despite statistical insignificance(r=-0.138,P>0.05).Conclusion:ECP is a sign of eosinophil activation,which is an important factor leading to the nasal inflammation.The content of serum ECP can be used as an indicator for patients with allergic rhinitis recieving nonspecific immune treatment .IgG4 antibody is a relatively reliable indicator to evaluate the treatment effect of specific immunotherapy,and may be negatively related to the serum ECP levels.

Eosinophil Cationic Protein Shows Survival Effect on H9c2 Cardiac Myoblast Cells with Enhanced Phosphorylation of ERK and Akt/GSK-3ß under Oxidative Stress.

Eosinophil cationic protein (ECP) is well known as a cationic protein contained in the basic granules of activated eosinophils. Recent studies have reported that ECP exhibits novel activities on various types of cells, including rat neonatal cardiomyocytes. Here we evaluated the effects of ECP on rat cardiac myoblast H9c2 cells. Our results showed that ECP enhanced the survival of the cells, in part by promoting the ERK and Akt/GSK-3ß signaling pathways. ECP attenuated the cytotoxic effects of H2O2 on H9c2 cells as well as the production of reactive oxygen species, the number of apoptotic cells and caspase 3/7 activity in the cells. In conclusion, ECP activated the ERK and Akt/GSK-3ß pathways, resulting in anti-oxidative effects on H9c2 cells that attenuated apoptosis.

Helicobacter pylori outer membrane vesicle proteins induce human eosinophil degranulation via a ß2 Integrin CD11/CD18- and ICAM-1-dependent mechanism.

Eosinophil cationic protein (ECP), a cytotoxic protein contained in eosinophils granules, can contribute to various inflammatory responses. Although Helicobacter pylori infection increases infiltration of eosinophils, the mechanisms of eosinophil degranulation by H. pylori infection are largely unknown. The goal of this study was to investigate the role of H. pylori outer membrane vesicles (OMVs) in modulating eosinophil degranulation. We found that eosinophils treated with H. pylori OMVs released significantly more ECP compared with untreated controls. In addition, eosinophils cocultured with OMV-preexposed primary gastric epithelial cells exhibited significantly increased ECP release. Similarly, eosinophils cocultured with culture supernatant (CM) from primary gastric epithelial cells exposed to OMVs (OMV-CM) released significantly higher amounts of ECP compared with eosinophils cocultured with CM from unexposed control cells. Furthermore, OMVs and OMV-CM both induced the upregulation of ICAM-1 on gastric epithelial cells and ß2 integrin CD11b on eosinophils. In addition, both transduction of ICAM-1 shRNA into gastric epithelial cells and treatment with neutralizing mAbs to CD18 significantly decreased OMV-mediated or OMV-CM-mediated release of ECP. These results suggest that the eosinophil degranulation response to H. pylori OMVs occurs via a mechanism that is dependent on both ß2 integrin CD11/CD18 and ICAM-1.

A biologically inspired lung-on-a-chip device for the study of protein-induced lung inflammation.

This study reports a biomimetic microsystem that reconstitutes the lung microenvironment for monitoring the role of eosinophil cationic protein (ECP) in lung inflammation. ECP induces the airway epithelial cell expression of CXCL-12, which in turn stimulates the migration of fibrocytes towards the epithelium. This two-layered microfluidic system provides a feasible platform for perfusion culture, and was used in this study to reveal that the CXCL12-CXCR4 axis mediates ECP induced fibrocyte extravasation in lung inflammation. This 'lung-on-a-chip' microdevice serves as a dynamic transwell system by introducing a flow that can reconstitute the blood vessel-tissue interface for in vitro assays, enhancing pre-clinical studies. We made an attempt to develop a new microfluidic model which could not only simulate the transwell for studying cell migration, but could also study the migration in the presence of a flow mimicking the physiological conditions in the body. As blood vessels are the integral part of our body, this model gives an opportunity to study more realistic in vitro models of organs where the blood vessel i.e. flow based migration is involved.

Protein post-translational modification in host defense: the antimicrobial mechanism of action of human eosinophil cationic protein native forms.

Knowledge on the contribution of protein glycosylation in host defense antimicrobial peptides is still scarce. We have studied here how the post-translational modification pattern modulates the antimicrobial activity of one of the best characterized leukocyte granule proteins. The human eosinophil cationic protein (ECP), an eosinophil specific granule protein secreted during inflammation and infection, can target a wide variety of pathogens. Previous work in human eosinophil extracts identified several ECP native forms and glycosylation heterogeneity was found to contribute to the protein biological properties. In this study we analyze for the first time the antimicrobial activity of the distinct native proteins purified from healthy donor blood. Low and heavy molecular weight forms were tested on Escherichia coli cell cultures and compared with the recombinant non-glycosylated protein. Further analysis on model membranes provided an insight towards an understanding of the protein behavior at the cytoplasmic membrane level. The results highlight the significant reduction in protein toxicity and bacteria agglutination activity for heavy glycosylated fractions. Notwithstanding, the lower glycosylated fraction mostly retains the lipopolysaccharide binding affinity together with the cytoplasmic membrane depolarization and membrane leakage activities. From structural analysis we propose that heavy glycosylation interferes with the protein self-aggregation, hindering the cell agglutination and membrane disruption processes. The results suggest the contribution of post-translational modifications to the antimicrobial role of ECP in host defense.

Human versus mouse eosinophils: "that which we call an eosinophil, by any other name would stain as red".

The respective life histories of human subjects and mice are well defined and describe a unique story of evolutionary conservation extending from sequence identity within the genome to the underpinnings of biochemical, cellular, and physiologic pathways. As a consequence, the hematopoietic lineages of both species are invariantly maintained, each with identifiable eosinophils. This canonical presence nonetheless does not preclude disparities between human and mouse eosinophils, their effector functions, or both. Indeed, many books and reviews dogmatically highlight differences, providing a rationale to discount the use of mouse models of human eosinophilic diseases. We suggest that this perspective is parochial and ignores the wealth of available studies and the consensus of the literature that overwhelming similarities (and not differences) exist between human and mouse eosinophils. The goal of this review is to summarize this literature and in some cases provide experimental details comparing and contrasting eosinophils and eosinophil effector functions in human subjects versus mice. In particular, our review will provide a summation and an easy-to-use reference guide to important studies demonstrating that although differences exist, more often than not, their consequences are unknown and do not necessarily reflect inherent disparities in eosinophil function but instead species-specific variations. The conclusion from this overview is that despite nominal differences, the vast similarities between human and mouse eosinophils provide important insights as to their roles in health and disease and, in turn, demonstrate the unique utility of mouse-based studies with an expectation of valid extrapolation to the understanding and treatment of patients.

Eosinophil-induced neurotoxicity: the role of eosinophil cationic protein/RNase 3.

We analyze the effect of ECP on primary cultures of cerebellar granule cells (CGCs) and astrocytes in an effort to understand the role of ECP in the eosinophil-induced neurotoxicity. We have shown that ECP induces dose-dependent cell death in both CGCs and astrocytes. The effect of ECP action on cell morphology is consistent with apoptosis for both cell types. The apoptotic mechanism involves ECP binding on the cell surface and an increase in the free cytosolic Ca(2+) concentration. It is associated with the activation of caspase-3, -8 and -9, processes that are also involved in the apoptosis induced either by stroke or other neurodegenerative conditions. Our results open new insights to clarify the neurotoxic effects associated to ECP in the hypereosinophilic syndrome.

Human eosinophil cationic protein enhances stress fiber formation in Balb/c 3T3 fibroblasts and differentiation of rat neonatal cardiomyocytes.

We found that eosinophil cationic protein (ECP) stimulated the growth of mouse Balb/c 3T3 fibroblasts. ECP-treated 3T3 cells were more flattened and exhibited enhanced stress fiber formation. The enhancement of cytoskeleton after addition of recombinant ECP appeared stable and was able to inhibit disassembly of actin filaments that was induced by fibroblast growth factor-2. The ROCK inhibitor, Y-27632, abrogated this enhancement on stress fiber formation that was induced by ECP indicating the involvement of Rho/ROCK signaling pathway. The effect of ECP was assessed on the differentiation of primary cardiomyocytes derived from rat neonatal heart since the development of actin filaments is significantly related with organization of stress fibers. As the result, both beating rate and the expression of cardiac muscle specific markers such as atrial natriuretic factor were enhanced in the presence of ECP. Thus ECP may also function as a cardiomyocyte differentiation factor.

The coding ECP 434(G>C) gene polymorphism determines the cytotoxicity of ECP but has minor effects on fibroblast-mediated gel contraction and no effect on RNase activity.

Eosinophil cationic protein (ECP) is a secretory protein of the eosinophil granulocyte, a cell involved in innate immunity. Functional studies have implicated ECP in numerous processes, such as tissue remodeling in allergic inflammation and cytotoxicity toward a variety of pathogens. Recent genetic studies have suggested that the ECP 434(G>C) polymorphism resulting in an arg97thr substitution would alter the function of ECP in vivo. Functional (in vitro) studies of ECP up until now have either been conducted with native preparations containing an unknown mixture of the ECP(97arg) and ECP(97thr) variants, or with recombinant proteins. Therefore, we have now for the first time extracted the native ECP(97arg) and ECP(97thr) variants from healthy blood donors and tested them functionally in vitro. Our results show that the arg97thr shift dramatically alters the cytotoxic capacity of ECP in vitro; the tested ECP(97arg) variants were cytotoxic toward the small-cell lung cancer cell line NCI-H69, whereas ECP(97thr) was noncytotoxic. RNase activity was unaffected by the arg97thr substitution. Both ECP(97arg) and ECP(97thr) stimulated fibroblast-mediated collagen gel contraction, an experimental model, which depicts wound healing, in a dose-dependent manner. In conclusion, our results demonstrate that the ECP 434(G>C) gene polymorphism affects the functional properties of native ECP, but also that there is a dissociation between different biological activities; the arg97thr substitution impairs the cytotoxic potential of ECP but less the gel contraction and not at all the RNase activity.

The antipathogen activities of eosinophil cationic protein.

The eosinophil cationic protein (ECP) is a secretory ribonuclease, which is found in the eosinophilic leukocyte and involved in the innate immune system. Its cytotoxic activity is effective against a wide range of pathogens, suggesting a relatively non-specific mechanism of action. We review here the specific antipathogen activities that have been characterized for ECP. Although eosinophils and ECP are primarily associated with the host defense against nonphagocytosable pathogens, such as helminthic parasites, ECP has also an antibacterial activity, which is not shared by the other, closely-related eosinophil ribonuclease, the eosinophil derived neurotoxin (EDN). Although there is no evidence for direct involvement in vivo of eosinophils in the host response to bacterial infection, ECP is active against both Gram-negative and Gram-positive bacterial strains and its mechanism depends on its action both at the bacterial cell wall and cytoplasmic membrane levels. Other antipathogen activities, including antihelminthic activity, are also discussed. Modulation of the protein activity by posttranslational modifications and the currently identified polymorphisms are reviewed. Antimicrobial RNases, as innate immune proteins with anti-infective and immunomodulatory properties, present substantial therapeutic potential in the drug development industry, both in the search of alternative antibiotics and for the treatment of inflammatory disorders.

The role of eosinophil cationic protein in patients with Mycoplasma pneumoniae infection.

BACKGROUND: To study the role played by eosinophil cationic protein (ECP) in patients with Mycoplasma pneumonia infection. METHODS: Pediatric patients aged 4 to 14 years old were divided into 3 groups, each consisting of 30 patients. Group 1 comprised patients with known M. pneumoniae infection. Group 2 comprised patients with asthma who were in a stable condition with no infection, acute asthma exacerbation or steroid use in the last 2 months. Group 3 consisted of healthy children and was designated the control group. The level of ECP in patients' serum was measured by an ECP radioimmunoassay kit. RESULTS: There were 90 children enrolled in this study; 59 (65.56%) were boys and 31 (34.44%) were girls. Mean serum ECP levels between males and females was not significantly different (p = 0.544). The variance of serum ECP levels decreased as patient age increased, but there was no relationship between serum ECP level and patient age (gamma = 0.118, p = 0.267). Serum ECP levels were similar in both the M. pneumoniae-infected and asthma groups; serum ECP levels in the control group were less than the levels seen in the other 2 groups. The difference in serum ECP levels among the 3 groups was statistically significant (p < 0.001). CONCLUSION: Both the children who had M. pneumoniae infection and the children with asthma had significantly increased serum ECP levels compared to normal healthy children. The elevated ECP levels found in the serum of patients with M. pneumoniae infection may be associated with damage to the respiratory epithelium and accelerated hypersensitivity in the respiratory system. Decreasing the serum level of ECP may potentially be a method of relieving symptoms in patients with M. pneumoniae infection. Additional studies are warranted to further validate this conclusion.

Sensitization of isolated rat vagal pulmonary sensory neurons by eosinophil-derived cationic proteins.

It has been shown that airway exposure to eosinophil-derived cationic proteins stimulated vagal pulmonary C fibers and markedly potentiated their responses to lung inflation in anesthetized rats (Lee LY, Gu Q, Gleich GJ, J Appl Physiol 91: 1318-1326, 2001). However, whether the effects resulted from a direct action of these proteins on the sensory nerves was not known. The present study was therefore carried out to determine the effects of these proteins on isolated rat vagal pulmonary sensory neurons. Our results obtained from perforated whole cell patch-clamp recordings showed that pretreatment with eosinophil major basic protein (MBP; 2 microM, 60 s) significantly increased the capsaicin-evoked inward current in these neurons; this effect peaked approximately 10 min after MBP and lasted for >60 min; in current-clamp mode, MBP substantially increased the number of action potentials evoked by both capsaicin and electrical stimulation. Pretreatment with MBP did not significantly alter the input resistance of these sensory neurons. In addition, the sensitizing effect of MBP was completely abolished when its cationic charge was neutralized by mixing with a polyanion, such as low-molecular-weight heparin or poly-L-glutamic or poly-L-aspartic acid, before its delivery to the neurons. Moreover, a similar sensitizing effect was also generated by other eosinophil granule-derived proteins (e.g., eosinophil peroxidase). These results demonstrate a direct, charge-dependent, and long-lasting sensitizing effect of cationic proteins on pulmonary sensory neurons, which may contribute to the airway hyperresponsiveness associated with airway infiltration of eosinophils under pathophysiological conditions.

[Role of eosinophils in allergic inflammation].

Eosinophils are one of the cells that play a critical role in the pathogenesis of allergic diseases. The increase in the number of eosinophils in such diseases is regulated by interleukin-5 (IL-5). The author have prepared recombinant rat IL-5 using a baculovirus expression system and examined its biological activities in rat eosinophils. It was demonstrated that recombinant rat IL-5 prolongs the survival of mature eosinophils and differentiates immature eosinophils into mature eosinophils, suggesting that rat IL-5 is a factor for eosinophilia in rats. Recombinant rat eosinophil-associated ribonuclease (Ear)-1 and Ear-2 were also prepared. Eosinophil granule proteins are thought to cause tissue damage due to their cytotoxic activity, but using recombinant rat Ear-1 and Ear-2, it was found that rat Ear-1 and Ear-2 have strong RNase A activity and bactericidal activity, suggesting that these proteins play critical roles in host defense. Finally, the important role of acetylation of histones was clarified in the differentiation of HL-60 clone 15 cells into eosinophils using the histone deacetylase inhibitors sodium n-butyrate, apicidin, and trichostatin A. These findings would be useful for further investigations of the role of eosinophils in allergic inflammation.

Membrane-bound carboxypeptidase E facilitates the entry of eosinophil cationic protein into neuroendocrine cells.

ECP (eosinophil cationic protein) is a major component of eosinophil granule proteins, and is used as a clinical biomarker for asthma and allergic inflammatory disease. ECP has been implicated in damage to the cell membrane of many tissue types, but the mechanism is not well known. In the present study, mECP-eGFP-6H, a recombinant fusion protein containing mature ECP (mECP), enhanced green fluorescence protein (eGFP) and a His(6) tag (6H), has been expressed, purified and added to GH3 neuroendocrine cells to study the internalization ability of ECP. We found that mECP-eGFP-6H entered into GH3 neuroendocrine cells and inhibited the growth of the cells with an IC(50) of 0.8 microM. By yeast two-hybrid screening and immunoprecipitation, we have identified a specific protein-protein interaction between mECP and CPE (carboxypeptidase E), a well characterized metalloprotease. Further in vivo yeast two-hybrid screening has also revealed that residues 318-387 located in a region of unknown function in mature CPE are indispensable for association with mECP. In addition, the uptake of mECP-eGFP-6H is suppressed by dominant-negative expression of the recycling defect mutant pre-pro-HA-CPE(S471A,E472A) in GH3 cells, suggesting that the entry of mECP-eGFP-6H is associated with the recycling of CPE in GH3 cells. Taken together, we have demonstrated that CPE possesses a novel function to facilitate the entry of ECP to neuroendocrine cells, and such an endocytotic process allows the cytotoxic ECP to inhibit growth of the target cells.