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.

Crystal structure of the eosinophil major basic protein at 1.8 A. An atypical lectin with a paradigm shift in specificity.

The eosinophil major basic protein (EMBP) is the predominant constituent of the crystalline core of the eosinophil primary granule. EMBP is directly implicated in epithelial cell damage, exfoliation, and bronchospasm in allergic diseases such as asthma. Here we report the crystal structure of EMBP at 1.8 A resolution, and show that it is similar to that of members of the C-type lectin superfamily with which it shares minimal amino acid sequence identity (approximately 15--28%). However, this protein lacks a Ca(2+)/carbohydrate-binding site. Our analysis suggests that EMBP specifically binds heparin. Based on our results, we propose a possible new function for this protein, which is likely to have implications for EMBP function.

Comparative structure, proximal promoter elements, and chromosome location of the human eosinophil major basic protein genes.

Human eosinophil major basic protein (MBP) is strongly implicated as a mediator of disease, especially bronchial asthma. We recently isolated a highly divergent human homologue of MBP (MBPH). Given human MBP's importance in disease and the restricted expression of it and human MBPH, we isolated the 4.6-kb human MBPH gene (HGMW-approved symbol PRG3). Comparisons among the human MBP (PRG2), human MBPH, and murine MBP-1 (mMBP-1; Prg2) genes suggest that the human MBP and mMBP-1 genes are more closely related than either is to the human MBPH gene. Proximal promoters of these three genes show conservation of potential binding sites for IK2 and STAT and of a known GATA site. However, a known C/EBP site is altered in the human MBPH gene's proximal promoter. The human MBP and MBPH genes localized to chromosome 11 in the centromere to 11q12 region. Thus, the human MBP and MBPH genes have diverged considerably, probably following a gene duplication event. Furthermore, the identified conserved and distinct proximal promoter elements likely contribute to the eosinophil-restricted and relatively reduced transcription of the human MBPH gene.

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.

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.

A novel and highly divergent homolog of human eosinophil granule major basic protein.

Eosinophils are important effector cells in defense against helminth infection and in allergic diseases. To identify novel eosinophil proteins, large scale sequencing of a cDNA library prepared from interleukin-5-stimulated umbilical cord precursor cells was performed, and the major genes expressed by maturing eosinophils were determined. This resulted in the identification of a cDNA with 64% identity to human prepro-major basic protein (hprepro-MBP). This cDNA was designated hprepro-MBP homolog (hprepro-MBPH). Interestingly, the calculated pI values for hMBPH and hMBP differed by >100-fold, with pI values of 8.7 and 11.4, respectively. Given this pronounced basicity difference, the homolog transcript's abundance (1.1%), and MBP's critical role in eosinophil biological activity, we further characterized the homolog. Reverse transcription-polymerase chain reaction detected transcription of hprepro-MBPH in bone marrow only, and this result was confirmed by analysis of a large cDNA data base (electronic Northern). hMBPH was isolated from human eosinophil granule lysates, and its identity was verified by amino acid sequencing and by mass spectrometry. Analyses of the biological activities showed that hMBPH had effects similar to hMBP in cell killing and neutrophil (superoxide anion production and interleukin-8 release) and basophil (histamine and leukotriene C4 release) stimulation assays, but usually with reduced potency. Overall, this novel homolog's unique physical properties indicated that the high net positive charge of hMBP is important but not essential for biological activity.

Cytotoxic properties of eosinophil granule major basic protein for tumor cells.

BACKGROUND: Eosinophil granule major basic protein (MBP) mediates many eosinophil-associated immune functions and it adheres eosinophils to parasite targets. METHODS: We compared the toxicities of MBP and melittin to K562 and HL-60 cells using five cytotoxicity methods. RESULTS: Trypan blue staining, propidium iodide/ CellTrackertrade markGreen staining and incorporation of 14C-leucine assays indicated that MBP damages most cells by 1 h. In contrast, 51Cr and lactic dehydrogenase (LDH) release assays indicated that MBP damages most cells only at 20 h. All five methods indicated that melittin damages nearly all cells by 1 h. To resolve these discrepancies, the procedures were modified. Without cell transfer, dye staining methods showed that MBP produces very little cytotoxicity at 4 h. 51Cr and LDH assays, modified to mimic cell transfer, showed increased cytotoxicities at 4 h. The 14C-leucine assay modified by solubilization of cells with SDS and by trichloroacetic acid precipitation showed increased recovery of labeled protein and, thus, lower cytotoxicity, about 50%, at 4 h. CONCLUSION: Overall, MBP's ability to cause molecular and cellular adhesion confounds cytotoxicity measurements. A modified 14C-leucine assay overcame MBP's adhesiveness and provided an accurate measure of cytotoxicity.

Sulfonylureas inhibit cytokine-induced eosinophil survival and activation.

Eosinophils play a key role in the pathogenesis of asthma and other allergic inflammatory diseases. We have previously shown that treatment of eosinophils with lidocaine preferentially inhibits IL-5-induced survival. This inhibition cannot be overcome by increasing concentrations of IL-5 and is not due to the blocking of Na+ channels by lidocaine. Here we report that one class of K+ channel blockers, the sulfonylureas, inhibits eosinophil survival in a manner similar to lidocaine. The sulfonylurea glyburide inhibits eosinophil survival even at high concentrations of IL-5. In contrast, increasing concentrations of IL-3 or granulocyte-macrophage CSF overcome glyburide inhibition. Glyburide also blocks cytokine-induced eosinophil superoxide production. Similar results were seen with the sulfonylureas tolbutamide and glipizide. Interestingly, the effects of glyburide are not antagonized by the ATP-sensitive K+ channel openers cromakalim, pinacidil, or diazoxide. Although Scatchard analysis of [3H]glyburide binding to eosinophil membranes indicated that the high affinity sulfonylurea receptor (SUR1) is not present on eosinophils, human eosinophils do express mRNA homologous to the sulfonylurea receptor family, in keeping with the presence of a sulfonylurea receptor. Finally, coculture of eosinophils with combinations of glyburide, lidocaine, and dexamethasone resulted in synergistic inhibition of cytokine-mediated eosinophil survival and superoxide production. These results have intriguing clinical implications for the treatment of eosinophil-associated diseases.

Expression, purification, and characterization of the recombinant proform of eosinophil granule major basic protein.

The cDNA for the highly toxic eosinophil granule major basic protein (MBP) encodes a 25-kDa acidic precursor (proMBP) that is processed to form the 14-kDa mature MBP. To characterize the biochemical and biological properties of proMBP, and compare these to the known properties of MBP, we expressed recombinant proMBP in Chinese hamster ovary cells and purified the secreted form from supernatants. We developed a mAb specific for proMBP, J163-15E10, and by using a proMBP-specific RIA we found that recombinant proMBP was expressed quite efficiently at levels between 10 and 100 mg/l. By SDS-PAGE and immunoblotting analyses of bulk Chinese hamster ovary supernatants, recombinant proMBP was electrophoretically heterogeneous with an apparent molecular mass ranging from 3 x 10(4) to 1 x 10(5) daltons. Despite difficulties encountered because of the extreme molecular heterogeneity of the proform, two methods for purification of a predominant 33-kDa form of recombinant proMBP are presented. Glycosylation analysis of purified 33-kDa proMBP indicated that approximately 5 kDa is likely accounted for by the addition of one glycosaminoglycan group, three O-linked, and one N-linked complex type carbohydrate groups. Functional studies of purified recombinant proMBP were also conducted. Using amounts of proMBP determined to be optimal for MBP activity, it was shown that proMBP not only lacked the ability to inhibit protein synthesis in K562 cells, but it also lacked the ability to stimulate basophil histamine release or generate neutrophil superoxide anion release. Furthermore, proMBP inhibited in a dose-responsive manner the basophil histamine release and superoxide anion generation stimulated by MBP. The development of a mAb and RIA specific for proMBP will now make it possible to analyze biologic fluids for the presence of this protein, especially in pregnancy, when proMBP is increased.

Expression of eosinophil-granule major basic protein messenger ribonucleic acid in placental X cells.

BACKGROUND: The human eosinophil-granule major basic protein (MBP) is a 13.8-kilodalton cationic polypeptide constituting the core of the eosinophil granule. MBP is cytotoxic to parasites and numerous mammalian cells and is a potent secretagogue for platelets, basophils, mast cells, and neutrophils. Concentrations of a molecule immunochemically similar to eosinophil granule MBP are present in maternal plasma, and MBP has been localized by immunofluorescence to placental X cells. EXPERIMENTAL DESIGN: To determine whether X cells produce MBP, the expression of MBP messenger RNA (mRNA) was investigated in placentas by Northern blot analyses and by in situ hybridization with 35S-labeled RNA probes. RESULTS: Northern blot analyses of RNA from placental septa and villi showed the existence of a 1.0-kb RNA band that hybridized with the MBP anti-sense probe; no MBP mRNA was detected in whole blood of normal or pregnant women or in cord blood. Analyses of placentas by in situ hybridization showed MBP mRNA in X cells of placental septa and anchoring villi, but not in other cellular elements such as syncytiotrophoblasts, cytotrophoblasts, villous stromal cells, and fetal endothelial cells. RNase pretreatment abolished X-cell hybridization signals; treatment of sections with an excess of nonradiolabeled anti-sense RNA also blocked binding of the 35S-labeled anti-sense RNA probe. Additional evidence supporting the production of MBP by X cells was obtained using a combination of in situ hybridization and immunofluorescence, which showed colocalization of MBP and its mRNA. CONCLUSIONS: The presence of MBP mRNA and MBP protein in placental X cells indicates that X cells synthesize this biologically active molecule.

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.

Eosinophil-derived neurotoxin and human liver ribonuclease. Identity of structure and linkage of neurotoxicity to nuclease activity.

Eosinophil-derived neurotoxin (EDN) and human liver RNase were found to be indistinguishable from each other but distinct from the pancreatic ribonucleases in their nucleolytic activity on polynucleotides or small defined substrates. Antibodies to EDN and liver RNase showed identical cross-reactivities in assays of nuclease inhibition and in a radioimmunoassay. In each instance, EDN and liver RNase were easily distinguished from bovine or human pancreatic RNase. When injected intrathecally into rabbits, 5-10 micrograms of EDN or liver RNase each was neurotoxic as judged by induction of the Gordon phenomenon. Human pancreatic RNase was less neurotoxic, and up to 20-fold higher levels of bovine pancreatic RNase showed no effect. Treatment of EDN, liver RNase, and eosinophil cationic protein with iodoacetic acid at pH 5.5 resulted in inactivation of their RNase activity and also destroyed their neurotoxicity. EDN conformation was not greatly affected by iodoacetate treatment since interaction of the modified protein with antibodies was only slightly altered. We conclude that RNase activity is necessary but not sufficient to induce neurotoxic action.

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.

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.

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

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

An investigation of the cause of the eosinophilia-myalgia syndrome associated with tryptophan use.

BACKGROUND: The eosinophilia-myalgia syndrome is a newly recognized illness that has been associated with the consumption of tryptophan products. It is not known whether the cause is related to the tryptophan itself or to chemical constituents introduced by the manufacturing process. METHODS: To describe the epidemiology of the eosinophilia-myalgia syndrome further and elucidate a possible association with the manufacturing process, we conducted surveillance for the syndrome in Minnesota, a community survey of tryptophan use in Minneapolis-St. Paul, and a case-control study to assess potential risk factors, including the use of tryptophan from different manufacturers. We performed high-performance liquid chromatography on tryptophan samples to identify other chemical constituents. RESULTS: The prevalence of tryptophan use increased from 1980 to 1989 and was highest among women. Among the subjects for whom the source of the tryptophan was known, 29 of 30 case patients (97 percent) and 21 of 35 controls (60 percent) had consumed tryptophan manufactured by a single company (odds ratio, 19.3; 95 percent confidence interval, 2.5 to 844.9; P less than 0.001). This company used a fermentation process involving Bacillus amyloliquefaciens to manufacture tryptophan. Analysis of the manufacturing conditions according to the retail lot demonstrated an association between lots used by case patients and the use of reduced quantities of powdered carbon in a purification step (odds ratio, 9.0; 95 percent confidence interval, 1.1 to 84.6; P = 0.014), as well as the use of a new strain of B. amyloliquefaciens (Strain V) (odds ratio, 6.0; 95 percent confidence interval, 0.8 to 51.8; P = 0.04). There was a significant correlation (r = 0.78, P less than 0.001) between the reduced amount of powdered carbon used during manufacturing and the use of the new bacterial strain. High-performance liquid chromatography of this company's tryptophan demonstrated one absorbance peak (peak E) that was present in 9 of the 12 retail lots (75 percent) used by patients and 3 of 11 lots (27 percent) used by controls (odds ratio, 8.0; 95 percent confidence interval, 0.9 to 76.6; P = 0.022). CONCLUSIONS: The outbreak of the eosinophilia-myalgia syndrome in 1989 resulted from the ingestion of a chemical constituent that was associated with specific tryptophan-manufacturing conditions at one company. The chemical constituent represented by peak E may contribute to the pathogenesis of the eosinophilia-myalgia syndrome, or it may be a surrogate for another chemical that induces the syndrome.

In vitro killing of microfilariae of Brugia pahangi and Brugia malayi by eosinophil granule proteins.

Eosinophil infiltration and degranulation around the tissue-invasive stages of several species of helminths have been observed. Release of eosinophil granule contents upon the worms is supported by localization of two of the major granule proteins, major basic protein (MBP) and eosinophil peroxidase (EPO), on and around species of trematodes, nematodes, and cestodes. In the case of filarial worms, MBP is deposited on degenerating microfilariae (mf) of Onchocerca volvulus. Here, we performed in vitro assays of the toxicity of four purified eosinophil granule proteins, namely, MBP, EPO, eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN), for the mf of Brugia pahangi and Brugia malayi. MBP, ECP, and EDN killed these worms in a dose-related manner although relatively high concentrations of EDN were necessary. EPO, in the presence of a H2O2-generating system and a halide, was the most potent toxin on a molar basis; here, the most potent halide was I- followed by Br- and Cl-. Surprisingly, EPO in the absence of H2O2 killed mf at concentrations comparable to those required for MBP and ECP. The toxicity of EPO + H2O2 + halide was inhibited by heparin, catalase, or 1% BSA, whereas the toxicity of EPO alone was inhibited only by heparin. Heparin also inhibited killing by both MBP and ECP. Despite the homology of ECP with certain RNases, placental RNasin, an RNase inhibitor, was unable to inhibit ECP-mediated toxicity. These results indicate that all of the eosinophil granule proteins are toxic to mf and they support the hypothesis that eosinophil degranulation causes death of mf in vivo.

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.

Blood eosinophils and eosinophil-derived proteins in allergic asthma.

The concentrations of the eosinophil (EOS)-derived proteins, major basic protein (MBP), EOS-derived neurotoxin (EDN), EOS peroxidase (EPO), and EOS cationic protein (ECP), and EOS counts were measured in the peripheral blood of 12 atopic subjects with asthma and 23 normal control subjects. The same measurements were performed in seven subjects with asthma with previously documented dual (early plus late) asthmatic responses after inhalation challenges with methacholine and allergen. EOSs (p less than 0.001), MBP (p less than 0.01), EDN (p less than 0.01), and ECP (p greater than 0.03) were elevated in the subjects with asthma compared with control subjects, whereas EPO (p less than 0.01) concentrations were reduced. There were no significant differences between baseline measurements of FEV1, EOSs, MBP, EDN, EPO, or ECP on the methacholine- and allergen-challenge days. When the changes in these variables after allergen challenge were compared with the corresponding changes after methacholine challenge, there were no significant differences at 0 to 60 minutes or at 3 hours, whereas EDN (p less than 0.025), EPO (p less than 0.05), and ECP (p less than 0.025) were relatively increased at 6 to 12 hours and accompanied the late falls in FEV1 (p less than 0.001). EOSs (p less than 0.025) were elevated at 24 hours when there was a small relative increase in MBP (p less than 0.05). EOSs appear to be "activated" in subjects with allergic asthma, and further activation may occur during late asthmatic responses.