Identification of a major basic protein in guinea pig eosinophil granules.

Elucidation of the functions of the eosinophil might be accomplished by analysis of the granule constituents. We have purified eosinophils (93% or greater) from the peritoneal cavity of the guinea pig and have investigated a variety of methods to disrupt cells and liberate intact granules. Lysis in 0.34 M sucrose gave the best yield of granules and these had the characteristic morphology of eosinophil granules when examined by electron microscopy. Granules were solubilized by a variety of treatments and the solutions analyzed by polyacrylamide electrophoresis at pH 3 in 6 M urea. Comparison of the electrophoretic patterns of solubilized eosinophil and neutrophil granules revealed a difference: a major portion (53+/-3%; x +/-1 SE) of the protein from the eosinophil granule migrated as a single component. This major band protein has a molecular weight between 6,000 and 12,000 daltons and a pI of 10 or greater. Analysis of eosinophil granule constituents on Sephadex G-50 revealed two main peaks; peak 1 possessed peroxidase activity and peak 2 contained the major band protein. These studies indicate that eosinophil granules contain a cationic protein of low molecular weight which lacks peroxidase activity and which accounts for greater than 50% of granule protein.

Physiochemical and biological properties of the major basic protein from guinea pig eosinophil granules.

Guinea pig eosinophil granules are characterized by the presence of a basic protein of low molecular weight which accounts for greater than 50% of granule protein. This protein, termed the major basic protein (MBP), readily aggregates and becomes insoluble, and the formation of aggregates is dependent on the establishment of disulfide bonds. Analysis of concentrated preparations of MBP often revealed a series of bands which were multiples of a monomeric unit with a mol wt of approximately 11,000. Analysis of reduced and alkylated MBP on a 10% agarose column equilibrated with 6 M guanidinium chloride revealed a single polypeptide chain with a mol wt of 10,800. Amino acid analysis of the protein revealed the presence of 13% arginine, consistent with the basic character of the molecule. Four residues of tryptophan, were present, indicating that MBP is not a histone. The MBP did not increase vascular permeability when injected into the skin of guinea pigs, nor did it antagonize the effect of histamine and bradykinin in the skin. MBP also did not contract the isolated guinea pig ileum and when mixed with histamine or bradykinin did not inhibit their activity on the gut. MBP had only weak, if any, antihistaminic activity. MBP possessed weak bactericidal activity when compared to histone and then only with one strain of E. coli. MBP precipitated DNA, neutralized heparin, and activated papain. On a molar basis MBP was more active than cysteine in activating papain. These results do not point to any unique biological activity associated with MBP other than those expected of a protein as basic as it is and one which possesses reactive sulfhydryl groups. Possible functions of eosinophils based on the properties of the MBP are discussed.

Localization of the guinea pig eosinophil major basic protein to the core of the granule.

The localization of the guinea pig eosinophil major basic protein (MBP) within the cell was investigated by the use of immunoelectron microscopy and by isolation of the granule crystalloids. First, by immunoperoxidase electron microscopy, we found that the MBP of eosinophil granules is contained within the crystalloid core of the granule. Specific staining of cores was present when rabbit antiserum to MBP was used as the first stage antibody in a double antibody staining procedure, whereas staining was not seen when normal rabbit serum was used as the first stage antibody. Second, crystalloids were isolated from eosinophil granules by disruption in 0.1% Triton X-100 and centrifugation through a cushion of 50% sucrose. Highly purified core preparations yielded essentially a single band when analyzed by electrophoresis on polyacrylamide gels containing 1% sodium dodecyl sulfate (SDS). The E1%1cm of the core protein was 26.8 +/- 1.0 (X +/- SEM); the E1%1cm for the MBP was 26.3. The core protein could not be distinguished from the MBP by radioimmunoassay (RIA) and essentially all of the protein in the core preparations could be accounted for as MBP. The results indicate that the MBP is contained in the core of the guinea pig eosinophil granule and that it is probably the only protein present in the core.

Characterization of "peak E," a novel amino acid associated with eosinophilia-myalgia syndrome.

Epidemiologic studies strongly associate eosinophilia-myalgia syndrome (EMS) with ingestion of tryptophan containing a contaminant ("peak E"). Prior reports have suggested that peak E is the di-tryptophan N alpha-animal of acetaldehyde. Spectral and chemical studies now demonstrate that peak E is 1,1'-ethylidenebis[tryptophan]. This novel amino acid may be the etiological agent responsible for EMS, or it may be a marker of a still unidentified causal agent.

Comparative properties of the Charcot-Leyden crystal protein and the major basic protein from human eosinophils.

Guinea pig eosinophil granules contain a protein, the major basic protein (MBP), which accounts for more than half of the total granule protein, has a high content of arginine, and displays a remarkable tendency to form disulfide-linked aggregates. In this study we have purified a similar protein from human eosinophil granules and have compared the human MBP to the protein comprising the Charcot-Leyden crystal (CLC). Eosinophils from patients with various diseases were purified and disrupted, and the granule fraction was obtained. Examination of the granule fraction by transmission electron microscopy showed numerous typical eosinophil granules. Analyses of granule lysates by gel filtration and by polyacrylamide gel electrophoresis revealed the presence of peroxidase and MBP with properties similar to that previously found in guinea pig eosinophil granules. The human MBP had a molecular weight of 9,200, contained less than 1% carbohydrate, was rich in arginine, and readily formed disulfide-bonded aggregates. CLC were prepared from eosinophil-rich cell suspensions by homogenization in hypotonic saline. The supernates following centrifugation of cell debris spontaneously formed CLC. Analysis of CLC revealed the presence of a protein with a molecular weight of 13,000 containing 1.2% carbohydrate. The protein displayed a remarkable tendency to aggregate even in the presence of 0.2 M acetic acid. Human MBP and CLC protein differed in their molecular weights, carbohydrate compositions, and amino acid analyses. Mixtures of the MBP and the CLC protein yielded two bands in polyacrylamide gel electrophoresis. Neither eosinophil protein increased vascular permeability in the guinea pig skin or contracted the guinea pig ileum. The results indicate that the human MBP and the CLC are distinct substances with properties such that one cannot be derived from the other.

Acidic polyamino acids inhibit human eosinophil granule major basic protein toxicity. Evidence of a functional role for ProMBP.

Eosinophil granule major basic protein (MBP), a potent toxin for helminths and mammalian cells in vitro, is a single polypeptide chain rich in arginine. MBP has been localized on damaged helminths and tissues in hypersensitivity diseases including bronchial asthma. The MBP cDNA indicates that MBP is translated as a slightly acidic preproprotein with an acidic propart. To test the hypothesis that the acidic pro-part of proMBP inhibits the toxicity of mature MBP, acidic polyamino acids (aa) were used as antagonists of MBP toxicity to K562 cells and guinea pig tracheal epithelium and used as antagonists of MBP airway hyperresponsiveness in primates. The acidic poly aa inhibited MBP toxicity and MBP airway hyperresposiveness. The acidic poly aa inhibited MBP toxicity in a charge-dependent manner similar to that proposed for proMBP, suggesting that the acidic pro-part of proMBP functions to mask mature MBP toxicity. This inhibition was not limited to MBP, but also applied to polyarginine and eosinophil cationic protein. These acidic poly aa may be useful to inhibit the actions of a number of cationic toxins released by the eosinophil in numerous hypersensitivity diseases.

Elevated serum levels of the eosinophil granule major basic protein in patients with eosinophilia.

A radioimmunoassay was established for the human eosinophil granule major basic protein (MBP). The mean level of MBP in sera from 105 normal control patients was 454 ng/ml, whereas in a sample of 188 patients with various forms of diseases, including the hypereosinophilic syndrome, levels as high as 14,000 ng/ml were measured. Serum levels of MBP did not correlate with eosinophil counts in normal subjects, but a positive correlation was seen in patients with eosinophilia; the patients with eosinophil counts greater than 350/mm3 generally showed increased levels of MBP. Many patients with skin disease and normal eosinophil counts had elevated levels of serum MBP. Monomer MBP has a molecular weight of 9,300, but in sera of patients with eosinophilia, the MBP activity was of high molecular weight, greater than 50,000. Analyses of serum by Sephadex G-200 and by electrofocusing suggest that MBP is not simply polymerized, but rather is bound to a larger carrier molecule. Monomeric MBP can be isolated from serum by reduction of serum with dithiothreitol, alkylation with iodoacetamide, and acidification to pH 2 followed by fractionation on Sephadex G-50 at pH 2. Under these conditions, up to 80% of the MBP emerges in monomeric form. The results indicate that eosinophil granule proteins circulate in blood covalently bound to serum proteins, and that elevated concentrations of serum MBP are present in some diseases associated with eosinophilia.

Reactivity of monoclonal antibodies EG1 and EG2 with eosinophils and their granule proteins.

Use of the murine monoclonal antibodies EG1 and EG2 has been based on the assumption that EG2 recognizes activated eosinophils. We examined the reactivity of EG1 and EG2 with eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), and stimulated and nonstimulated eosinophils from normal donors. By radioimmunoassay, EG1 recognized only ECP, whereas EG2 recognized both ECP and EDN. By Western blot, EG1 reacted with ECP, EG2 reacted with both ECP and EDN, but EG2 could not distinguish between lysates of stimulated and nonstimulated eosinophils. By immunofluorescence, EG1 and EG2 at 20 microg/mL stained 95-100% of nonstimulated eosinophils, regardless of fixative; EG1 and EG2 at 0.1 microg/mL stained 61-90% of acetone- and paraformaldehyde-fixed and only 5-21% of methanol-fixed nonstimulated eosinophils. Thus, the reactivity of EG1 and EG2 with eosinophils depends on the method of fixation and antibody concentration; and EG2, in contrast to previous reports, cannot reliably discriminate between resting and activated eosinophils.

Eosinophil granule proteins in peripheral blood granulocytes.

Eosinophils contain four principal cationic proteins, major basic protein (MBP), eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), and eosinophil peroxidase (EPO). To determine the quantities of these proteins in granulocytes and whether they are specific to eosinophils, their concentrations in lysates of human granulocytes were measured using specific radioimmunoassays. The effect of different methods for eosinophil lysis on the recovery of the proteins was also studied. Maximal recovery occurred at pH 2 for MBP and pH 5.6 for the other granule proteins. The proteins cosedimented with eosinophils and their concentrations (mean +/- SEM) in ng/10(6) eosinophils (and in nM/10(6) eosinophils) were: MBP, 8,982 +/- 611 (641.6); EDN, 3,283 +/- 116 (178.4); ECP, 5,269 +/- 283 (250.9); and EPO, 12,174 +/- 859 (171.5). Basophils from a normal person contained (in ng/10(6) cells) MBP, 2,374; EDN, 214; ECP, 77; and EPO, 17. Highly purified neutrophils contained (in ng/10(6) cells) MBP, 3 +/- 0.5; EDN, 72 +/- 9; and ECP, 50 +/- 12. Therefore we conclude that these proteins are mainly expressed in eosinophils, but that certain ones are present in basophils and neutrophils.

Cloning and sequence analysis of the human gene encoding eosinophil major basic protein.

Eosinophil granule major basic protein (MBP), a potent toxin for helminths and mammalian cells, is a single polypeptide rich in arginine. The gene, mbp, was cloned and its nucleotide sequence determined. The 3.3-kb gene consists of six exons and five introns, one of which contains an Alu family repeat. The combined exon sequence is similar to previously reported mbp cDNA sequences. The gene is immediately preceded by a putative promoter containing typical TATA and CCAAT boxes. Southern blots indicate that mbp exhibits limited polymorphism.

Sequence of human eosinophil-derived neurotoxin cDNA: identity of deduced amino acid sequence with human nonsecretory ribonucleases.

Several clones of human eosinophil-derived neurotoxin (EDN) cDNA have been isolated from a lambda gt10 cDNA library prepared from mRNA derived from noninduced HL-60 cells. The amino acid (aa) sequence deduced from the coding sequence of the EDN cDNA is identical to the aa sequence of urinary nonsecretory RNase. Comparison of the aa and/or nucleotide (nt) sequences of EDN and other proteins possessing ribonucleolytic activity, namely bovine seminal RNase, human and rat pancreatic RNases, eosinophil cationic protein (ECP), and human angiogenin, shows extensive identity at half-cystine residues and at aa of active sites. Differences in aa sequences at the active sites are often the result of single nt changes in the codons. The data presented here support the concept of a RNase gene superfamily containing secretory and nonsecretory RNases, angiogenin, EDN and ECP.

Recovery of eosinophils from the peritoneal cavity of the guinea pig.

We studied the effects of various conditions on the recovery of eosinophils from the peritoneal cavity of guinea pigs repeatedly lavaged with saline. We compared the effects of ether and halothane on eosinophil production in guinea pigs either lavaged with saline alone or receiving an injection of polymyxin B before saline lavage. With both anesthetics polymyxin B caused a rapid and consistent increase in eosinophil production, although neutrophils were present. With halothane anesthesia, saline lavage alone yielded mean eosinophil values near those found in the polymyxin group. In contrast, saline lavage alone with ether anesthesia yielded a significantly lower mean eosinophil value than in the polymyxin groups with either anesthetic and the saline lavage alone with halothane anesthesia (P less than 0.05). Additional studies showed that females guinea pigs produced greater numbers of peritoneal eosinophils than male guinea pigs and that peritoneal eosinophilia was maintained for up to 20 weeks by weekly peritoneal lavage with saline alone. Castration of male guinea pigs did not result in eosinophil production comparable to female guinea pigs. Infection with Trichinella spiralis did not enhance peritoneal eosinophilia commensurate with that seen in the peripheral blood. These results indicate that saline lavage alone is a sufficient stimulus for eosinophil production in guinea pigs anesthetized with halothane, that greater numbers of eosinophils are produced in females than males and, finally, that eosinophil production continues at high levels for more than 20 weeks.

Discordant and anomalous results among cytotoxicity assays: the confounding properties of eosinophil granule major basic protein on cell viability assays.

When five cytotoxicity methods compared the toxicity of eosinophil granule major basic protein (MBP) and melittin to K562 and HL-60 cells, strikingly discrepant results were noted. Trypan blue staining, propidium iodide/CellTrackerGreen staining and incorporation of 14C-leucine assays indicated MBP damages > 75% of cells by 1 h. In contrast, 51Cr and lactic dehydrogenase (LDH) release assays indicated MBP damages most cells only at 20 h. All methods indicated melittin damages nearly all cells by 1 h. Further studies showed that without cell transfer, dye staining methods indicated MBP produces < 10% cytotoxicity after 4 h. A modified 14C-leucine assay, employing sodium dodecyl sulfate solubilization and trichloroacetic acid precipitation, showed lower cytotoxicity, 48%, at 4 h. Modified 51Cr and LDH assays showed increased cytotoxicities at 4 h, 34% and 58%, respectively. Overall, MBP's ability to cause molecular and cellular adhesion systematically confounds standard cytotoxicity measurements. However, the modified 14C-leucine assay provides a valid measure of MBP's cytotoxicity and may be useful for analyses of 'sticky' cytotoxins.

Elevated levels of the eosinophil granule major basic protein in the sputum of patients with bronchial asthma.

The eosinophil granule major basic protein (MBP) is toxic to parasites and mammalian cells. Because eosinophilia is characteristic of asthma, we tested the effect of MBP on bronchi and assayed sputa for this protein. We found that MBP damaged bronchial epithelium in vitro and produced changes that mimicked those in asthma. Radioimmunoassay of sputa from 100 consecutive patients with respiratory diseases revealed MBP levels above 0.1 mug/ml in 13 patients, and 11 of these had asthma. In 15 patient hospitalized for asthma, MBP levels of sputum were markedly elevated. Treatment with bronchodilators and glucocorticoids caused an increase peak expiratory flow rate, a reduction in blood eosinophils, and a decrease in the serum and sputum levels of MBP. The results indicate that eosinophil granule constituents are released into the bronchi in asthma and that measurement of sputum MBP may be useful in identifying asthma. The possibility that the eosinophil damages bronchial epithelium in asthma is discussed.

Comparative toxicity of purified human eosinophil granule cationic proteins for schistosomula of Schistosoma mansoni.

The human eosinophil granule contains several distinctive cationic proteins that have been purified to homogeneity, including major basic protein (MBP), eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN). Two earlier studies have shown that MBP and ECP both damage schistosomula of Schistosoma mansoni in vitro in a dose-dependent fashion. The present study expands upon these observations by comparing the toxicity of MBP, ECP, as well as EDN when tested at equimolar concentrations (0.03-2 X 10(-5) M). On a molar basis, ECP was 8 to 10 times more potent than MBP, and the ECP-mediated killing of schistosomula was qualitatively different than that of MBP. Purified ECP produced complete fragmentation and disruption of schistosomula, whereas MBP produced a distinctive ballooning and detachment of the tegumental membrane. In contrast, EDN was only marginally toxic at high concentrations and caused crinkling of the tegumental membrane. Heating MBP and ECP for four hr at 56 degrees C caused precipitation and loss of toxicity for MBP, but not for ECP. Native MBP (with reactive sulfhydryl groups intact) and stabilized, reduced and alkylated MBP had comparable toxicity. To determine the relative contribution of MBP, ECP and other potentially helminthotoxic eosinophil granule constituents to schistosomulum damage, fractions of acid soluble granule extracts prepared by chromatography on Sephadex G-50 columns were analyzed for toxicity to schistosomula and for MBP and ECP levels by radioimmunoassay. Schistosomula were killed by fractions containing MBP, and to a much lesser and more variable extent by fractions containing EDN and a 21,000 dalton protein, but not by fractions coincident with the elution of ECP, which contained concentrations of ECP below that required to produce significant killing of schistosomula by the purified protein. Therefore, although ECP is a more potent helminthotoxin for schistosomula than MBP on a molar basis, MBP, by virtue of its abundance in the granule, accounts for the bulk of the toxicity in fractions of acid solubilized granules obtained from eosinophils of patients with marked eosinophilia.

Comparative toxicity of purified human eosinophil granule proteins for newborn larvae of Trichinella spiralis.

Eosinophils have been implicated in both in vivo and in vitro destruction of helminths. One approach toward elucidating the role of the eosinophil in parasite killing has been to test the toxicity of purified eosinophil granule proteins for parasites in vitro. Previously, major basic protein (MBP) and eosinophil cationic protein (ECP) were shown to be toxic for schistosomules of Schistosoma mansoni, while eosinophil-derived neurotoxin (EDN) was only marginally so. We tested the toxicity of MBP, ECP, and EDN over a range of concentrations (0.006-5 X 10(-4) M) for newborn larvae of Trichinella spiralis. Our observations confirm previous reports of toxicity of mildly reduced and alkylated (R & A) MBP. At concentrations of 5 X 10(-5) M and above, R & A MBP killed 75% or more of the larvae within the first hour of culture. ECP was an effective toxin for these larvae after 3 hr of culture, and by 12 hr, dose-related toxicity was evident. After 24 hr, 100% of the larvae were killed at 5 X 10(-5) M ECP. EDN was much less toxic; after 12 hr, 90% of the larvae survived at concentrations of 1 X 10(4) M, while 5 X 10(-4) M EDN killed all the larvae. At the optimal toxic concentrations of 5 X 10(-5) M ECP and 5 X 10(-4) M EDN, kinetics of killing by these 2 proteins were essentially the same. Thus, on a molecular basis, both MBP and ECP appear to be potent helminthotoxins whereas EDN is much less so.

Phosphatidylserine exposure during apoptosis reflects bidirectional trafficking between plasma membrane and cytoplasm.

Phosphatidylserine (PS) exposure on the external leaflet of the plasma membrane is widely observed during apoptosis and forms the basis for the annexin V binding assay to detect apoptotic cell death. Current efforts to explain PS exposure focus on two potential mechanisms, activation of a phospholipid scramblase or calcium-mediated trafficking of lysosomes to the cell surface. Here, we provide evidence that apoptotic PS exposure instead reflects bidirectional trafficking of membrane between the cell surface and cytoplasm. Using a series of cell lines, some of which expose large amounts of PS during apoptosis and some of which do not, we demonstrate that accumulation of plasma membrane-derived cytoplasmic vesicles in a dynamin-, clathrin- and Cdc42-independent manner is a previously undescribed but widely occurring feature of apoptosis. The apoptotic exposure of PS occurs when these vesicles traffic back to cell surface in a calcium-dependent process that is deficient in a substantial fraction of human cancer cell lines. These observations provide a new model for PS externalization during apoptosis and simultaneously identify an altered step that accounts for the paucity of apoptotic PS exposure in many cell lines.