Concentrations Of Major Basic Protein Research Articles

INCREASED EOSINOPHIL GRANULE PROTEIN PRODUCTION IN CHRONIC SPONTANEOUS URTICARIA

<h3>Introduction</h3> Chronic spontaneous urticaria (CSU) lesional and non-lesional skin reveals increased numbers of eosinophils, but the exact role of eosinophils in CSU pathogenesis remains unknown. A pathogenic role for eosinophils in CSU was supported by the results of a recent clinical trial demonstrating the efficacy of benralizumab in CSU. Previous studies have identified increased levels of major basic protein (MBP) and eosinophilic cationic protein (ECP) in the sera and skin of CSU subjects; however, eosinophil peroxidase (EPO), the granule protein uniquely expressed by eosinophils, and eosinophil-derived neurotoxin (EDN) were not examined. Therefore, we sought to determine whether eosinophil granule proteins (EGPs) are increased in CSU. <h3>Methods</h3> Sera from CSU subjects (n=11) and healthy controls (n=1) were collected and sent to the NIH Human Eosinophil Section for analysis with their novel multiplex immunoassay which simultaneously measures concentrations of MBP, ECP, EDN, and EPO using Luminex xMAP technology. CSU subjects completed disease activity surveys and complete blood count differentials were analyzed. <h3>Results</h3> Mean concentrations of MBP (1329.23 vs 399.55ng/ml), ECP (285.87 vs 45.31ng/ml), EDN (349.43 vs 38.53ng/ml), and EPO (54.96ng/ml vs undetectable) were markedly increased in CSU subjects compared to the healthy control. We found no correlation between eosinophil counts and granule protein levels, or with Urticaria Activity Scores. <h3>Conclusions</h3> Our results suggest that unique pathways may be activated in a subset of patients with CSU which led to the production of large quantities of EGPs. Additional research is needed to understand the pathways responsible for producing these granule proteins in CSU and their clinical impact.

Association of eosinophil-mediated inflammatory biomarkers with the presence of the Schatzki ring

PurposeThe study aimed to assess the level of inflammatory biomarkers related to eosinophilia: interleukins 5 (IL-5) and 13 (IL-13), eotaxin 3, major basic protein (MBP) and transforming growth factor β1 (TGF-β1) in patients with dysphagia and Schatzki ring (SR), as well as the characteristics of this group of patients in terms of the features differentiating gastroesophageal reflux disease (GERD) and eosinophilic esophagitis (EoE). Patients and methodsWe analyzed 42 patients with dysphagia, each of whom underwent panendoscopy with an assessment of the occurrence of SR, retrospectively assessed EoE Endoscopic Reference Score (EREFS) total, inflammatory and fibrostenotic and serum concentrations of IL-5 and 13, TGF-β1, eotaxin 3 and MBP. All of them completed a symptom and comorbid questionnaire. Patients diagnosed with SR constituted the SR group (n ​= ​8), the rest – the non-SR group. The quantification of the biomarkers was performed by enzyme immunoassay (ELISA). In the data analysis, p ​≤ ​0.05 was considered statistically significant. ResultsWe demonstrated a significant increase in terms of exceeding the reference values of TGF-β1 (37.5% vs 8.8%) and MBP (75% vs 35.3%) in patients with SR compared to the non-SR group (qualitative analysis). There was also a statistically significant increase in the concentration of each of the determined biomarkers (quantitative analysis) in the SR group. ConclusionsThe increase in TGF-β1 and MBP concentrations indicates the inflammatory and probably fibrostenotic pathogenesis of SR. Obtained results do not allow for an unequivocal classification of SR as a complication typical only for GERD or EoE.

Eosinophilic biomarkers for detection of acute exacerbation of chronic obstructive pulmonary disease with or without pulmonary embolism.

Eosinophilia has been implicated in the pathophysiology of acute exacerbation of chronic obstructive pulmonary disease (AECOPD). However, the role of eosinophil activation in the development of AECOPD remains unclear. In the present study, the reliability of plasma levels of eosinophil activation markers, including eosinophil cationic protein (ECP), major basic protein (MBP), eosinophil-derived neurotoxin (EDN) and eosinophil peroxidase (EPX), were measured and used as diagnostic biomarkers of AECOPD with or without pulmonary embolism (PE). A total of 47 patients with AECOPD, 30 patients with AECOPD/PE and 35 healthy adults were enrolled in the present study. Plasma levels of ECP, EDN, EPX and MBP were measured using commercial ELISA kits. The mean concentrations of plasma ECP, EDN, EPX and MBP in the patients with AECOPD was significantly 2.87-, 3.06-, 1.60- and 1.92-fold higher, respectively, compared with the control group (P<0.05). Similar results were obtained in patients with AECOPD/PE, for whom plasma levels of ECP, EDN, EPX and MBP were significantly 2.06-, 2.21-, 1.42- and 2.42-fold higher, respectively, compared with the controls (P<0.05). No significant differences were observed in the levels of these proteins between patients with AECOPD or AECOPD/PE. Among the four potential markers, ECP was determined to be the optimal marker for distinguishing patients with AECOPD or AECOPD/PE from the controls. No significant correlation was observed between marker concentrations and gender, age or disease severity. The results of the present study may have clinical applications in the diagnosis of AECOPD using these novel biomarkers.

Eosinophil Cationic Protein (ECP), a predictive marker of bullous pemphigoid severity and outcome

Bullous Pemphigoid (BP) is an inflammatory rare autoimmune bullous dermatosis, which outcome cannot be predicted through clinical investigations. Eosinophils are the main immune infiltrated cells in BP. However, the release of Major Basic Protein (MBP), Eosinophil Derived Neurotoxin (EDN), and Eosinophil Cationic Protein (ECP) upon eosinophil activation has still not been evaluated with respect to BP development. MBP, EDN and ECP were measured by ELISA in serum (n = 61) and blister fluid (n = 20) of patients with BP at baseline, and in serum after 2 months of treatment (n = 41). Eosinophil activation in BP patients was illustrated at baseline by significantly higher MBP, EDN and ECP serum concentrations as compared with control subjects (n = 20), but without distinction according to disease severity or outcome. EDN and ECP values were even higher in the blister fluids (P < 0.01 and P < 0.05, respectively), whereas MBP values were lower (P < 0.001). ECP serum concentration decreased after 60 days of treatment in BP patients with ongoing remission but not in patients who later relapsed (P < 0.05). A reduction of at least 12.8 ng/mL in ECP concentrations provided a positive predictive value for remission of 81%, showing that ECP serum variation could be a useful biomarker stratifying BP patients at risk of relapse.

Development of a suspension array assay in multiplex for the simultaneous measurement of serum levels of four eosinophil granule proteins

The concentrations of major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil derived neurotoxin (EDN) and eosinophil peroxidase (EPO) have been associated with eosinophilic disease severity. Whereas a variety of techniques have been used to measure individual eosinophil granule protein concentration, none of these methods efficiently measures MBP, ECP, EDN and EPO simultaneously. A multiplex suspension array system was developed to simultaneously measure the concentrations of MBP, ECP, EDN and EPO in serum. The assay showed excellent inter- and intra-assay reliability, and serum levels of MBP, ECP and EDN from eosinophilic subjects analyzed by ELISA and multiplex were highly correlated (r=0.8579; P<0.0001, r=0.6356; P=0.0006 and r=0.8600; P<0.0001, respectively, Spearman rank correlation). Moreover, the multiplex assay required 500-fold less serum than a single ELISA to achieve comparable sensitivity. Absolute eosinophil count and eosinophil surface expression of the activation marker, CD69, were significantly correlated with concentrations of MBP, EDN and EPO, but not ECP, in serum from eosinophilic subjects. Furthermore, subjects with eosinophilic gastrointestinal disorder and normal peripheral absolute eosinophil counts (<0.5×109/l) had significantly increased concentrations of MBP (P<0.0001), ECP (P<0.0001), EDN (P=0.0001) and EPO (P<0.0001) compared to normal donors. In summary, the eosinophil granule protein multiplex assay provides a rapid and reliable way to measure eosinophil granule protein levels and should prove useful in assessing patterns of degranulation in patients with eosinophilic disorders.

Eosinophil Granular Proteins Damage Bronchial Epithelial Cells Infected with Respiratory Syncytial Virus

Background: Respiratory syncytial (RS) virus infection is an important exacerbating factor in acute bronchial asthma. However, the precise mechanisms responsible for viral infection-induced exacerbations of asthma are uncertain. To elucidate the role of eosinophilic inflammation in the pathogenesis of virus-induced asthma, we investigated the effects of eosinophil granule proteins on bronchial epithelial cell infected with RS virus. Methods: Morphological changes and cytopathic effects in human type II pulmonary alveolar epithelial cells (A549) infected with RS virus and/or eosinophil granule proteins such as major basic protein (MBP), eosinophil peroxidase (EPO), eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) were observed by microscopy. Apoptosis/necrosis was evaluated by trypan blue exclusion test. We also measured 8 types of phosphorylated proteins in MBP-treated A549 cells infected with RS virus. Results: Although RS virus alone did not affect the cytopathic effects of A549 cells, high concentrations of MBP or a combination of 4 granule proteins resulted in cytopathic effects. MBP or EPO, but not ECP or EDN, induced cytotoxicity and necrosis of the infected A549 cells. Furthermore, MBP induced the phosphorylation of the extracellular signal-regulated kinase (ERK) 1/2, p38 mitogen-activated protein kinase (MAPK), Jun-N-terminal protein kinase (JNK), and signal transducer and activator of transcription (STAT)3 in the infected cells. Conclusions: These results suggest that eosinophil granule proteins, specifically MBP, damage bronchial epithelial cells infected with RS virus and that the MAPK family are involved in these responses, indicating that eosinophilic inflammation might be associated with the pathophysiology of RS virus-induced acute exacerbations of asthma.

Effects of respiratory syncytial virus infection and major basic protein derived from eosinophils in pulmonary alveolar epithelial cells (A549)

RSV (respiratory syncytial virus)-induced pneumonia and bronchiolitis may be associated with hyperresponsive conditions, including asthma. Eosinophilic proteins such as MBP (major basic protein) may also be associated with the pathophysiology of asthma. To elucidate the roles of RSV infection and MBP in the pathogenesis of pneumonia with hyperresponsiveness, we investigated the effects of RSV infection and MBP on A549 (alveolar epithelial) cells. CPE (cytopathic effects) in A549 cells were observed by microscopy. Apoptosis and cell death was evaluated by flow cytometric analysis and modified MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. We also measured 15 types of cytokines and chemokines in A549 cell supernatants. Although RSV alone did not affect the CPE of A549, high concentrations of MBP resulted in cell death within 24 h. Combinations of RSV and MBP synergistically induced cell death. In A549 cells infected with RSV alone, the release of GM-CSF (granulocyte-macrophage colony-stimulating factor) was significantly enhanced compared with control cells (no infection). In the cells treated with MBP alone, the production of IL (interleukin)-2, 4, 5, 7, 10, 12, 13, 17, IFN (interferon)-γ, GM-CSF, G-CSF (granulocyte colony-stimulating factor) and MIP (macrophage inflammatory protein)-1β was significantly increased compared with control cells. Notably, the levels of GM-CSF and IL-17 in RSV/MBP-treated cells were significantly higher than those treated with MBP alone. These results suggest that MBP synergistically enhanced the release of various cytokines/chemokines and the cell death of RSV-infected A549 cells, indicating that MBP may be closely associated with the pathophysiology of allergic reactions in bronchiolitis/pneumonia due to RSV.

The role of eosinophil major basic protein in angiogenesis

Eosinophil-derived major basic protein (MBP) plays an active role in allergic inflammation and tissue remodelling. However, its role in angiogenesis has not been established as yet. Therefore our objective was to investigate whether MBP exhibits any direct pro-angiogenic effects. Rat aortic endothelial cells and human umbilical vascular endothelial cells were cultured with different concentrations of MBP and their viability (Trypan blue exclusion test), proliferation (thymidine incorporation) and capillary-like structure formation (matrigel assay) were investigated in vitro. The angiogenic activity of MBP was then tested in vivo using the chick chorio allantoic membrane (CAM) assay. Subcytotoxic concentrations of MBP induce endothelial cell proliferation and enhance the pro-mitogenic effect of vascular endothelial growth factor (VEGF), but do not affect their VEGF release. MBP promotes capillarogenesis by endothelial cells seeded on matrigel and sprouting formation in the CAM assay. Furthermore, we have shown that the pro-angiogenic effect of MBP is not due to its cationic charge since stimulation of the CAMs with the synthetic polycation, poly-L-arginine does not induce any angiogenic effects. These data demonstrate that MBP has pro-angiogenic effects in vitro and in vivo, providing a novel mechanism whereby MBP can participate in tissue inflammation and remodelling in atopic diseases.

Striking deposition of toxic eosinophil major basic protein in mucus: Implications for chronic rhinosinusitis

The mechanisms by which eosinophilic inflammation damages the epithelium and contributes to recurrent acute exacerbations in chronic rhinosinusitis (CRS) have not been fully elucidated. We tested the hypotheses that eosinophils deposit toxic major basic protein (MBP) in the mucus and that MBP reaches concentrations able to damage the sinonasal epithelium. Tissue specimens with mucus attached to the tissue were carefully collected from 22 patients with CRS and examined by using immunofluorescence staining for MBP. This immunofluorescence was digitally analyzed to determine the area covered by MBP and the intensity of the staining (estimating MBP concentration). Levels of MBP in extracts from nasal mucus were quantitated by means of RIA. Heterogeneous eosinophilia was evident within tissue and mucus specimens. All tissue specimens showed intact eosinophils, but diffuse extracellular MBP deposition, as a marker of eosinophil degranulation, was rare. In contrast, all mucus specimens showed diffuse MBP throughout and abundant diffuse extracellular MBP deposition within clusters of eosinophils. Digitized analyses of MBP immunofluorescence revealed increased area coverage (P < .0001) in mucus compared with that seen in tissue. Estimated concentrations of MBP within the clusters suggested toxic levels. MBP concentrations in mucus extract reached 11.7 microg/mL; MBP was not detectable in healthy control subjects. In patients with CRS, eosinophils form clusters in the mucus where they release MBP, which is diffusely deposited on the epithelium, a process not observed in the tissue. Estimated MBP levels far exceed those needed to damage epithelium from the luminal side and could predispose patients with CRS to secondary bacterial infections.

Cationic Proteins and Bronchial Hyperresponsiveness

Chronic airway inflammation in asthma is characterized by the preponderance of eosinophils in the airway walls and lumens. Eosinophils are unique among granulocytes in that they contain intracytoplasmic granules, which stain with eosin. These granules contain a number of preformed enzymes and other proteins, including cationic proteins such as major basic protein (MBP), eosinophil cationic protein, eosinophil-derived neurotoxin, and eosinophil peroxidase. Other granule enzymes include lysophospholipase, collagenase, b -glucuronidase, and arylsulfatase B (1). The principal granule protein in eosinophils is MBP, a highly positively charged, 117–amino acid polypeptide with a molecular weight of 14 kD. It is named for its highly basic isoelectric point (calculated to be 10.9) that is due to its large number of arginine residues (2). It is encoded from a 3.3-kb, five-intron gene located on chromosome 11 (3). MBP is synthesized in the cell as a pre-proprotein and is then processed and transported as a relatively neutral pH, 33-kD proprotein, presumably to protect the eosinophil from the toxic effects of MBP (4). Pro-MBP is converted into mature MBP after its sequestration in the granule crystalloid cores. Upon release, physiologic concentrations of MBP upregulate expression of intercellular adhesion molecule-1 on cultured human nasal epithelial cells (5). Moreover, MBP can induce eosinophil degranulation and the production of interleukin 8 by these cells (6). Thus, MBP can act as an autocrine factor to potentiate further eosinophil recruitment and activation in the airways. In addition, the highly cationic MBP reacts readily with acidic lipids, disordering lipid membranes and resulting in fusion and lysis (4). Accordingly, MBP is a potent bacteriocidal and helminthotoxic agent that is also toxic to tumor cells and other mammalian cells, including the airway epithelium. Naturally occurring cationic polypeptides such as MBP have diverse cytotoxic and noncytotoxic effects on many different cell types due to interactions with cell-surface anion charges. The importance of the cationic charge is supported by the observation that synthetic polycations of similar size and charge, such as polyL -arginine and polyL lysine, mimic many of the effects of MBP. Specific mechanisms are not known, but interactions of polycations with cell-surface anionic charges could interfere with normal cell function in a variety of ways, including the blockade of ion channels, inhibition of transporters, compromise of membrane integrity, and binding to or aggregation of integral membrane proteins (7–11). However, cationic charge alone may not be the only mechanism of action for naturally occurring cationic polypeptides. For example, contractions of guinea pig tracheal smooth muscle by MBP are inhibited by atropine, whereas the contractions due to synthetic polycations of similar charge are not (12). Thus, all of the effects of MBP may not be attributed to cationic charge. Interest in eosinophil-derived cationic proteins, especially MBP, was stimulated by evidence implicating them in the pathogenesis of bronchial hyperresponsiveness (13, 14) (Table 1). In ovalbumin-sensitized guinea pigs and in patients with asthma, a significant correlation exists between the concentrations of MBP and methacholine hyperresponsiveness (15, 16). Additional evidence suggests that neutralization of endogenously secreted MBP, either with a polyanionic peptide or with antibodies to MBP, can prevent antigen-induced bronchial hyperreactivity in sensitized animals. Specifically, the polyanion polyL -glutamic acid inhibits the bronchial hyperresponsiveness induced by MBP in primates (17) as well as the increase in vagal responsiveness induced by antigen provocation in guinea pigs (18). Similarly, anti-MBP antibodies suppress the development of bronchial hyperreactivity after antigen challenge in sensitized guinea pigs without affecting airway eosinophilia (19). Such studies suggest that MBP plays a key role in the alterations of airway function that accompany eosinophilic airway inflammation. Initial in vivo (20) and in vitro studies (21, 22, 23) suggested that the effects of MBP on airway smooth-muscle contractility occurred indirectly, via effects on the airway epithelium. Exposure to MBP or synthetic polycations damages the airway epithelial barrier (24, 25) and increases epithelial permeability to hydrophilic tracers (26). Such actions would expose the underlying airway smooth muscle to higher concentrations of bronchoactive substances. Nevertheless, MBP also exerts important nontoxic effects on the airway epithelium. It has been shown that MBP affects epithelial eicosanoid metabolism, altering the balance between constricting and dilating prostaglandins (27, 28). Furthermore, recent evidence shows that polycations inhibit L -arginine uptake by airway epithelial cells (8) and that a deficiency in nitric oxide production contributes to polycation-induced airway hyperreactivity (9). ( Received in original form February 14, 2000 )

Eosinophil major basic protein increases membrane permeability in mammalian urinary bladder epithelium.

The eosinophil granule protein major basic protein (MBP) is toxic to a wide variety of cell types, by a poorly understood mechanism. To determine whether the action of MBP involves an alteration in membrane permeability, we tested purified MBP on rabbit urinary bladder epithelium using transepithelial voltage-clamp techniques. Addition of nanomolar concentrations of MBP to the mucosal solution caused an increase in apical membrane conductance only when the voltage across the apical membrane was cell interior negative. The magnitude of the MBP-induced conductance was a function of MBP concentration, and the rate of the initial increase in conductance was a function of the transepithelial voltage. The MBP-induced conductance was nonselective for K+ and Cl-. Mucosal Ca2+ reversed the induced conductance, whereas mucosal Mg2+ partially blocked the induced conductance and slowed the rate of the increase in conductance. The induced conductance was partially reversed by changing the voltage gradient across the apical membrane to cell interior positive. Prolonged exposure resulted in an irreversible loss of the barrier function of the urinary bladder epithelium. These results suggest that an increase in cell membrane ion permeability is an initial step in MBP-induced loss of barrier function.

Eosinophils, major basic protein, and polycationic peptides augment bovine airway myocyte Ca2+ mobilization.

Previous studies in vivo or in isolated airway preparations have suggested that eosinophil-derived polycationic proteins enhance airway smooth muscle tone in an epithelium-dependent manner. We assessed the direct effects of activated human eosinophil supernatant, major basic protein (MBP), and polycationic polypeptides on basal and agonist-stimulated intracellular Ca2+ concentrations ([Ca2+]i) in cultured bovine tracheal smooth muscle (TSM) cells. A 1-h incubation of myocytes with activated eosinophil buffer resulted in a doubling of basal [Ca2+]i and increased responsivity to histamine compared with myocytes that were exposed to sham-activated eosinophil buffer. In addition, concentration-dependent acute transient increases and subsequent 1-h sustained elevations of basal [Ca2+]i were observed immediately after addition of MBP and model polycationic proteins. Finally, both peak and plateau [Ca2+]i responses to bradykinin addition were augmented significantly in cultured myocytes that had been exposed to low concentrations of MBP or model polycationic proteins but were inhibited at greater concentrations. This elevated [Ca2+]i to polycationic proteins was manifest in epithelium-denuded bovine TSM strips as concentration-dependent increased basal tone. We conclude that activated eosinophil supernatant, MBP, and other polycationic proteins have a direct effect on both basal and subsequent agonist-elicited Ca2+ mobilization in cultured TSM cells; TSM strips in vitro demonstrated, respectively, augmented and diminished responses to the contractile agonist acetylcholine. It is possible that alteration in myocyte Ca2+ mobilization induced by these substances may influence clinical states of altered airway tone, such as asthma.

Time-course of antigen-induced airway inflammation in the guinea-pig and its relationship to airway hyperresponsiveness.

The causative relationship between airway inflammation and hyperreactivity is unclear, since inflammatory changes have been examined at one or, at most, a few time-points after antigen challenge in both human asthma and animal models. We have made a detailed investigation of inflammatory and functional changes in the airways up to 8 days after antigen challenge in guinea-pigs. In particular, we examined the hypothesis that eosinophil-derived mediators contribute to tissue damage and the development of airway hyperresponsiveness. Following antigen challenge, the influx of inflammatory cells and mediator release in airway tissue and bronchoalveolar lavage fluid were correlated temporally with histopathological changes in airway tissue and airway responsiveness. Eosinophil influx was demonstrable at 4 h. Eosinophilia peaked after 24 h and persisted for at least 8 days. Parallel increases in the concentrations of major basic protein and eosinophil cationic protein in bronchoalveolar lavage fluid indicated that the eosinophils were activated. Eosinophilia was accompanied by subepithelial oedema and epithelial damage co-localized with major basic protein immunoreactivity. A transient neutrophilia (< 48 h duration) and an increase in neutrophil elastase in bronchoalveolar lavage fluid peaked at 14 h. The proportion of airway macrophages with an activated morphology increased at 8 h and remained markedly elevated until 72 h. Airways were hyperresponsive to histamine at 4 h and for at least 8 days. The antigen-induced airway inflammation resemble in time-course and histopathology that seen in antigen-challenged asthmatics, and indicate that the eosinophil and its cytotoxic proteins may be major mediators of airway mucosal damage and airway hyperresponsiveness.

IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor potentiate basophil mediator release stimulated by eosinophil granule major basic protein.

We have examined the potential of IL-3, IL-5, and granulocyte-macrophage (GM)-CSF to enhance basophil activation by eosinophil granule major basic protein (MBP). Preincubating basophil-containing mononuclear cells with 0.01 to 10 ng/ml IL-3 or IL-5 for 15 min at 37 degrees C caused a concentration-dependent enhancement of histamine release stimulated by 1.5 microM MBP. Statistically significant enhancement was evident at 1 ng/ml and was maximal at 10 ng/ml. Preincubation with GM-CSF similarly enhanced MBP-induced histamine release. A 10- to 15-min preincubation with IL-5 maximally increased the level of MBP-stimulated histamine release. Preincubation of cells with 10 ng/ml IL-3 or IL-5 reduced the MBP concentrations required for histamine release by three- to fourfold and enhanced the rate of MBP-induced histamine release. MBP-stimulated histamine release before or after cytokine priming was independent of cytotoxicity as measured by 51Cr release. Consistent with a direct action of the cytokines on basophils, flow cytometric analysis demonstrated the presence of IL-3 and GM-CSF receptors on basophils. MBP also stimulated low levels of leukotriene C4 (LTC4) release from basophils of 84 to 99% purity, and experiments using enriched (18-63%) basophil preparations demonstrated that preincubation with IL-3, IL-5, and GM-CSF also potentiated MBP-stimulated leukotriene C4 release up to threefold in parallel with histamine release. These results indicate that IL-3, IL-5, and GM-CSF may contribute to the pathogenesis of allergic and other disorders characterized by eosinophilia in part through potentiation of basophil mediator release stimulated by MBP.

Major Basic Protein Regulation of Lung Fibroblast Cytokine Production: Role of Cytokine Synergy and Charge

Eosinophilic infiltration, elevated levels of eosinophil major basic protein (MBP), and a close approximation of eosinophils and fibroblasts are prominent features of the asthmatic airway. Eosinophil-fibroblast interactions have been shown to regulate fibroblast proliferation and eosinophil survival. The importance of these interactions, however, in generating and/or maintaining local inflammation, is not completely understood. Previous work from our laboratory has demonstrated that human lung fibroblasts are potent sources of immunomodulatory cytokines. Thus, we hypothesized that eosinophils are important regulators of this effector function, in part via the ability of eosinophil-derived granule proteins to regulate fibroblast cytokine elaboration. To test this hypothesis, we used ELISA quantification and Northern blot analysis to characterize the effects of purified MBP and eosinophil-derived neurotoxin (EDN) on cytokine production by both unstimulated and recombinant human interleukin-1 alpha (rIL-1-α)- or transforming growth factor- beta (TGF-β)-stimulated human lung fibroblasts. Unstimulated fibroblasts did not produce significant amounts of IL-6, IL-11, leukemia inhibitory factor (LIF), IL-8, or granulocyte-macrophage colony-stimulating factor (GM-CSF). Concentrations of MBP (>44 μg/mL) stimulated the production of IL-6 and IL-11. Lower doses of MBP interacted with rIL-1-α in a synergistic fashion to augment IL-l-induced IL-6, IL-11, IL-8, and GM-CSF production, and with TGF-β to further augment fibroblast production of IL-11 and LIF. These effects were dose- and time-dependent, and were evident following incubation of fibroblasts with rlL-1-α+/—MBP for as little as 4 h and as much as 48 h. Fibroblasts incubated with rIL-1-α (2.5 ng/mL) plus MBP (44 μg/mL) produced threefold to fivefold more IL-6, threefold to tenfold more IL-11, twofold to fivefold more GM-CSF, and twofold to fivefold more IL-8 than fibroblasts incubated with rIL-1-α alone. Fibroblasts incubated with TGFβ (10 ng/mL) plus MBP (22 μg/mL) produced twofold to fourfold more IL-11 and fourfold to fivefold more LIF than cells incubated with TGF-β alone. The synergy of MBP with rIL-1-α or TGF-β was not generalizable to all fibroblast functions, because MBP did not synergize with IL-1 to induce type I collagen or hepatocyte growth factor (HGF) production, or with TGF-β in the induction of IL-6 or HGF. This effect was also not generalizable to all eosinophil granule-derived molecules since EDN did not synergize with rIL-1-α in the induction of IL-11. The effects of MBP appeared to be pretranslationally mediated, since MBP-induced alterations in cytokine protein production were associated with proportional changes in mRNA accumulation. They also appeared to be at least partially charge-mediated since (1) poly-L-arginine, another highly cationic molecule, also synergized with rIL-1-α and TGF-β to augment fibroblast IL-11 and IL-6 protein production and mRNA accumulation; and (2) the effects of MBP on IL-11 production were abolished when fibroblasts were coincubated with MBP and the anionic molecule heparin. These studies demonstrate that MBP stimulates fibroblast IL-6 and IL-11 production and interacts with IL-1-α and TGF-β to synergistically augment fibroblast cytokine production. These effects of MBP may be mediated, at least in part, by cationic charge. Stimulation of stromal cell cytokine production caused by MBP may serve to augment and/or perpetuate the inflammatory response in eosinophil-associated human diseases such as asthma.

Distinguishing features of basophil and neutrophil activation by major basic protein

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Cytokines and eosinophil-derived cationic proteins upregulate intercellular adhesion molecule-1 on human nasal epithelial cells

Background: Allergic and nonallergic rhinitis with eosinophilia syndrome are characterized by tissue eosinophilia and nasal mucosal injury. Recently, it has been shown that the adherence of eosinophils and other leukocytes to epithelial cells is mediated by intercellular adhesion molecule-1 (ICAM-1) and related adherence-promoting glycoproteins. Methods: In this study we examined the constitutive expression of ICAM-1 on human nasal epithelial cells (HNECs), and the effects of interferon-γ, tumor necrosis factor-γ eosinophil major basic protein, and eosinophil cationic protein on the regulation of ICAM-1 expression on these cells. Similar studies were performed with A549 pneumocytes as comparative epithelial cells. Results: Constitutive expression of ICAM-1 was significantly higher on cultured HNECs than on A549 cells, although nasal epithelial cells in tissue specimens did not demonstrate detectable levels of ICAM-1. This spontaneous expression of ICAM-1 on cultured HNECs may explain the unique susceptibility of the nasal mucosa to rhinovirus infection, because ICAM-1 is the epithelial cell receptor for most rhinoviruses. Physiologic concentrations of major basic protein and eosinophil cationic protein stimulated significant upregulation of ICAM-1 on HNECs, which was comparable to that produced by interferon-γ and tumor necrosis factor-α. In contrast, these eosinophil constituents did not stimulate ICAM-1 upregulation on A549 alveolar epithelial cells, although A549 cells did respond to interferon-γ and tumor necrosis factor-α. Conclusion: The observation that eosinophil products upregulate ICAM-1 on HNECs suggests a positive feedback mechanism, in which the products released from migrating eosinophils might promote additional HNEC-leukocyte adherence by enhancing interactions between leukocyteβ 2 integrins (CDU11/18) and nasal epithelial ICAM-1.

Administration of Interleukin-2 (IL-2) Results in Increased Plasma Concentrations of IL-5 and Eosinophilia in Patients With Cancer

Abstract Peripheral eosinophilia is almost invariably observed during the course of interleukin-2 (IL-2) therapy and is frequently accompanied by the development of a capillary leak syndrome characterized by edema, weight gain, and oliguria. We studied five patients with advanced malignancy treated with IL-2. Eosinophilia was not present initially but developed in all patients late in the course of therapy, with counts ranging from 2,328/mm3 to 15,958/mm3. In all patients, there was a temporal relationship between the infusion of IL-2 and the appearance of elevated plasma concentrations of IL-5, a growth factor for eosinophils. Granulocyte-macrophage colony-stimulating factor was not detectable in plasma. IL-4 and gamma-interferon plasma levels were variably elevated. Plasma concentrations of major basic protein, a toxic eosinophil granule protein, began increasing before eosinophil counts increased. By the time of the third IL-2 infusion, high concentrations of major basic protein were present in all five patients (up to 5,600 ng/mL) and skin biopsies showed major basic protein deposition in the dermis. Four patients developed significant capillary leak syndrome and all of these patients showed markedly elevated major basic protein levels. The lowest peak plasma concentration of major basic protein (1,751 ng/mL) was observed in the one patient who did not develop edema and weight gain. These results suggest that IL-2 induces IL-5 leading to marked peripheral eosinophilia and extravascular eosinophil degranulation. The release of toxic eosinophil products at extravascular sites and in the circulation may contribute to the pathogenesis of the capillary leak syndrome complicating IL-2 therapy.

Administration of interleukin-2 (IL-2) results in increased plasma concentrations of IL-5 and eosinophilia in patients with cancer

Peripheral eosinophilia is almost invariably observed during the course of interleukin-2 (IL-2) therapy and is frequently accompanied by the development of a capillary leak syndrome characterized by edema, weight gain, and oliguria. We studied five patients with advanced malignancy treated with IL-2. Eosinophilia was not present initially but developed in all patients late in the course of therapy, with counts ranging from 2,328/mm3 to 15,958/mm3. In all patients, there was a temporal relationship between the infusion of IL-2 and the appearance of elevated plasma concentrations of IL-5, a growth factor for eosinophils. Granulocyte-macrophage colony-stimulating factor was not detectable in plasma. IL-4 and gamma-interferon plasma levels were variably elevated. Plasma concentrations of major basic protein, a toxic eosinophil granule protein, began increasing before eosinophil counts increased. By the time of the third IL-2 infusion, high concentrations of major basic protein were present in all five patients (up to 5,600 ng/mL) and skin biopsies showed major basic protein deposition in the dermis. Four patients developed significant capillary leak syndrome and all of these patients showed markedly elevated major basic protein levels. The lowest peak plasma concentration of major basic protein (1,751 ng/mL) was observed in the one patient who did not develop edema and weight gain. These results suggest that IL-2 induces IL-5 leading to marked peripheral eosinophilia and extravascular eosinophil degranulation. The release of toxic eosinophil products at extravascular sites and in the circulation may contribute to the pathogenesis of the capillary leak syndrome complicating IL-2 therapy.

Cytotoxicity of human eosinophil granule major basic protein to human nasal sinus mucosa in vitro

The toxic effects of the human eosinophil granule major basic protein (MBP), reduced and alkylated, were studied on human nasal mucosa in vitro. With a microscope coupled with a television monitor (magnification x 2500) and videotape recorder, we investigated the effects of MBP on the mucosa and the ciliary activity of single cells. In nasal mucosal specimens from normal individuals, MBP, 5 mumol/L and 10 mumol/L, significantly inhibited (p less than 0.01) ciliary activity by 4 and 1 hours of exposure, respectively. At these same MBP concentrations, the mucosal surface profiles were altered by 4 hours of exposure, and ciliostasis was 75% to 100% complete by 9 and 6 hours, respectively. In a mucosal specimen from a patient with nasal allergy, 1 mumol/L of MBP significantly inhibited (p less than 0.01) ciliary activity by 1 hour; alteration of the mucosal surface profile appeared by 3 hours of exposure, and ciliostasis was 75% to 100% by 13 hours. Similar alterations of the mucosal surface profile were observed with specimens from a second patient with allergies; in contrast, 1 mumol/L of MPB had no effect on specimens from a nonallergic patient. These results indicate that MBP damages human upper respiratory epithelium, causing ciliostasis and alteration of the epithelial surface at concentrations likely achieved in vivo. Furthermore, the mucosal specimens from two allergic patients were damaged by concentrations of MBP that had no effect on mucosal specimens from a normal individual.