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.

Janus kinase 2 (V617F) mutation status, signal transducer and activator of transcription-3 phosphorylation and impaired neutrophil apoptosis in myelofibrosis with myeloid metaplasia.

An activating point mutation in Janus kinase 2 (JAK2 V617F) was recently identified in myelofibrosis with myeloid metaplasia (MMM). To further elucidate the pathogenic significance, we examined the JAK2 mutation burden, phosphorylation of JAK2 substrates and neutrophil apoptotic resistance. Immunoblotting revealed phosphorylation of signal transducer and activator of transcription-3 (STAT3) in all four JAK2 with high V617F mutant allele burden and seven of eight with intermediate mutant allele burden, but only one of eight with wild-type JAK2 (P<0.001). In contrast, STAT5 phosphorylation was undetectable in patient MMM neutrophils; and phosphorylation of Akt and extracellular signal-regulated kinases (ERKs) failed to correlate with JAK2 mutation status. Apoptosis was lower in MMM neutrophils (median 41% apoptotic cells, n=50) compared to controls (median 66%, n=9) or other myeloproliferative disorder patients (median 53%, n=11; P=0.002). Apoptotic resistance in MMM correlated with anemia (P=0.01) and the JAK2-V617F (P=0.01). Indeed, apoptotic resistance was greatest in MMM neutrophils with high mutant allele burden (median 22% apoptosis, n=5) than with intermediate burden (median 39%, n=23) or wild-type JAK2 (median 47%, n=22; P=0.008). These results suggest that mutant JAK2 contributes to MMM pathogenesis by constitutively phosphorylating STAT3 and diminishing myeloid cell apoptosis.

Peptide-based analysis of amino acid sequences important to the biological activity of eosinophil granule major basic protein.

Synthetic peptides corresponding to amino acid sequences in eosinophil granule major basic protein (MBP) were evaluated for cytotoxic activity toward K562 cells and for ability to stimulate basophil mediator release. Results obtained using 14 peptides spanning the 117-amino acid sequence of MBP in overlapping fashion indicated that the activities mapped to peptide sequences near the amino and carboxy termini of MBP. The activity of these regions was confirmed using two peptides corresponding to MBP residues 18-45 and 89-117. A 20-h incubation with 5 microM peptide 18-45 or peptide 89-117 caused approximately the same levels (>60%) of cytotoxicity in K562 cells as 5 microM MBP. Similarly, a 30-min incubation with peptides 18-44 and 89-117 stimulated basophil histamine release in a concentration-dependent manner over the range of 5-20 microM. The level of release stimulated by 20 microM peptide 89-117 approached that stimulated by 2 microM MBP. A 20 microM concentration of peptide 89-117 also stimulated leukotriene C4 (LTC4) production by the basophils. Neither peptide 18-45 nor peptide 89-117 was cytotoxic for basophils under the experimental conditions for histamine and LTC4 release, as determined by 51Cr release. These results indicate that two MBP peptide sequences, including one (89-117) that contains a unique carbohydrate-binding region, share the biologic activities of MBP.

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.

Fcgamma-receptor signaling augments the LPS-stimulated increase in serum tumor necrosis factor-alpha levels.

The phagocytosis of IgG-coated erythrocytes (EIgG) has been shown to augment the bacterial lipopolysaccharide (LPS)-stimulated increase in serum tumor necrosis factor-alpha (TNF-alpha) levels. The present study evaluated the role of Fcgamma-receptor (FcgammaR) signaling and complement activation in the effect of EIgG on the TNF-alpha response to LPS. The role of FcgammaR was determined using FcR gamma-chain knockout mice that lack functional FcgammaRI and FcgammaRIII. In wild-type animals, EIgG caused a 16-fold augmentation of the serum TNF-alpha response to LPS, whereas there was no augmentation in the FcgammaR-deficient animals. Heat-damaged erythrocytes also augmented the TNF-alpha response to LPS. This effect was absent in FcgammaR-deficient animals. An IgG antibody against heated erythrocytes was detected in mouse serum. The complement activation caused by EIgG had little effect on the LPS-stimulated increase in serum TNF-alpha levels as indicated by activation of complement with cobra venom factor or IgM-coated erythrocytes as well as studies with C5-deficient mice. These results indicate that FcgammaR signaling primarily mediates the augmented serum TNF-alpha response to LPS caused by EIgG.

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.

Differential requirement for classic and novel PKC isoforms in respiratory burst and phagocytosis in RAW 264.7 cells.

The binding of Ab (IgG)-opsonized particles by FcgammaRs on macrophages results in phagocytosis of the particles and generation of a respiratory burst. Both IgG-stimulated phagocytosis and respiratory burst involve activation of protein kinase C (PKC). However, the specific PKC isoforms required for these responses have yet to be identified. We have studied the involvement of PKC isoforms in IgG-mediated phagocytosis and respiratory burst in the mouse macrophage-like cell line, RAW 264.7. Like primary monocyte/macrophages, their IgG-mediated phagocytosis was calcium independent and diacylglycerol sensitive, consistent with novel PKC activation. Respiratory burst in these cells was Ca2+ dependent and inhibited by staurosporine and calphostin C as well as by the classic PKC-selective inhibitors Gö 6976 and CGP 41251, suggesting that classic PKC is required. In contrast, phagocytosis was blocked by general PKC inhibitors but not by the classic PKC-specific drugs. RAW 264.7 cells expressed PKCs alpha, betaI, delta, epsilon, and zeta. Subcellular fractionation demonstrated that PKCs alpha, delta, and epsilon translocate to membranes during phagocytosis. In Ca2+-depleted cells, only novel PKCs delta and epsilon increased in membranes, and the time course of their translocation was consistent with phagosome formation. Confocal microscopy of cells transfected with green fluorescent protein-conjugated PKC alpha or epsilon confirmed that these isoforms translocated to the forming phagosome in Ca-replete cells, but only PKC epsilon colocalized with phagosomes in Ca2+-depleted cells. Taken together, these results suggest that the classic PKC alpha mediates IgG-stimulated respiratory burst in macrophages, whereas the novel PKCs delta and/or epsilon are necessary for phagocytosis.

IgG-coated erythrocytes augment LPS-stimulated TNF-alpha secretion, TNF-alpha mRNA levels, and TNF-alpha mRNA stability in macrophages.

Previous studies have shown that IgG-coated erythrocytes (EIgG) augment the LPS-stimulated increase in serum TNF-alpha levels in animals and the LPS-stimulated secretion of TNF-alpha by isolated macrophages. The present study evaluated the mechanism for the effect of EIgG on LPS-stimulated TNF-alpha secretion in the murine macrophage cell line, RAW 264.7. Incubation of the macrophages with EIgG or IgG-coated glass beads caused a dose-dependent augmentation of LPS-stimulated TNF-alpha secretion. The addition of EIgG increased the rate of LPS-stimulated TNF-alpha protein secretion between 2 and 4 hr after LPS. Accordingly, EIgG increased the levels of TNF-alpha mRNA at 2 and 3 hr after LPS. The increase in the LPS-stimulated TNF-alpha mRNA levels caused by EIgG was associated with an increase in TNF-alpha mRNA stability. Thus, the augmentation of LPS-stimulated TNF-alpha secretion by EIgG was associated with an increase in TNF-alpha mRNA levels which at least partly resulted from an increase in the stability of TNF-alpha mRNA.

A phagocytic challenge with IgG-coated erythrocytes depresses macrophage respiratory burst and phagocytic function by different mechanisms.

A phagocytic challenge with IgG-coated erythrocytes (EIgG) previously has been shown to cause impaired macrophage respiratory burst capacity and FcgammaR-mediated phagocytic function. Because both the respiratory burst and FcgammaR-mediated phagocytosis are dependent on the release of arachidonate (AA), we evaluated the effects of impaired AA release on the depression of macrophage function caused by a phagocytic challenge. Challenge with EIgG caused a depression of A23187-stimulated AA release that was associated with impaired phorbol myristate acetate (PMA)-stimulated H2O2 production and FcgammaR-mediated phagocytic function. In contrast, challenge with IgG-coated glass beads (BIgG) had no effect on either AA release or H2O2 production but did depress phagocytic function. Exogenous AA prevented the depression of H2O2 production but had no effect on phagocytic function. Phospholipase A2 (PLA2) activity was depressed under conditions where AA release was impaired. The depression of phagocytic function was correlated with a depression of both EIgG binding and FcgammaR expression. Thus, a phagocytic challenge with EIgG results in macrophage dysfunction by depressing PLA2 activity and depleting FcgammaR.

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.

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.

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.

IgG-coated erythrocytes augment the lipopolysaccharidestimulated increase in serum tumor necrosis factor-alpha.

Previous studies have shown that the injection of IgG-coated erythrocytes (EIgG) caused an increase in the mortality rate due to bacterial lipopolysaccharide (LPS). This observation led to the present evaluation of the effect of EIgG on the LPS-stimulated increase in serum tumor necrosis factor-alpha (TNF-alpha) levels and TNF-alpha secretion by macrophages. The prior injection of EIgG augmented the increase in LPS-stimulated serum TNF-alpha levels ninefold at 1 h after LPS. Serum TNF-alpha levels were augmented when LPS was injected 2 or 6 h after EIgG but not at 0.5 or 12 h after EIgG. Complement activation caused by EIgG may contribute to the priming for TNF-alpha, because activation of complement with cobra venom factor caused a threefold augmentation of the LPS-stimulated serum TNF-alpha levels. Isolated macrophages that had ingested EIgG or were adherent to immobilized IgG showed augmented TNF-alpha secretion in response to LPS. Thus clearance of immune complexes from the blood can augment the LPS-stimulated increase in serum TNF-alpha levels that is due, in part, to complement activation and signaling via FcgammaR.

Role of an oxidative stress in the macrophage dysfunction caused by erythrophagocytosis.

A phagocytic challenge with immunoglobulin G (IgG)-coated erythrocytes (EIgGs) has been shown to cause a subsequent depression of macrophage respiratory burst capacity and phagocytic function. The present study evaluated the hypothesis that this macrophage dysfunction is caused by an oxidative stress. An oxidative stress induced by ferric ammonium citrate (FAC) plus cumene hydroperoxide (CHP) caused a depression of macrophage function that was attenuated by antioxidants and iron chelators. In contrast, the same antioxidants and iron chelators did not alter changes caused by a challenge with EIgGs. EIgG challenge caused an increase in lipid peroxidation but failed to deplete glutathione (GSH) or decrease the activity of glyceraldehyde-3-phosphate dehydrogenase (GA-3-PD), suggesting that there was only a slight oxidative stress. Inhibition of the Fc gamma receptor (Fc gammaR) stimulated respiratory burst by removing calcium during the challenge did not attenuate the changes caused by an EIgG challenge. A phagocytic challenge with nonerythrocyte particles, IgG-coated beads (BIgGs), did not depress the respiratory burst capacity but did depress phagocytic function. Fc gammaR expression was depressed following a phagocytic challenge but not an oxidative stress. Thus, an oxidative stress can depress macrophage function, but the dysfunction caused by a phagocytic challenge with EIgGs involves Fc gammaR depletion and the erythrocyte contents rather than an oxidative stress.

Regulation and processing of a precursor form of eosinophil granule major basic protein (ProMBP) in differentiating eosinophils.

The cDNA for eosinophil granule major basic protein (MBP) encodes a prepromolecule with a total length of 222 amino acids (preproMBP). PreproMBP includes a secretory leader of 15 amino acids, an acidic propiece of 90 amino acids, and a basic MBP portion of 117 amino acids. The function of the propiece, which has a predicted pI of 3.9, is unknown, but it gives proMBP an overall acidic charge. Because proMBP is not found in mature eosinophils, we analyzed eosinophil differentiation in interleukin-5 (IL-5)-stimulated umbilical cord stem cells cultured for 24 days. By immunofluorescence, proMBP appeared by day 6 and peaked on day 18, whereas MBP was prominent at days 12 to 24. By day 6, Western blots detected heterogeneous glycosylated 33-kD proMBP; its peak expression occurred on day 12. Western blots showed sequential processing of 33-kD proMBP to an 18-kD intermediate form and finally to 14-kD MBP. By dual label immunoelectron microscopy, proMBP was localized primarily to large uncondensed eosinophil granules, whereas MBP was localized to granules containing a condensed central area. Thus, proMBP is likely expressed and processed as the granule condenses in a multistep process to 14-kD MBP in differentiating eosinophils.

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.

Blood flow and glucose uptake in denervated, insulin-resistant muscles.

To investigate whether changes in blood flow contribute to the insulin resistance in denervated muscles, basal and insulin-stimulated 2-deoxy-D-glucose (2-DG) uptake in vivo and blood flow were measured in soleus (slow twitch), plantaris (fast twitch), and gastrocnemius (fast twitch) muscles at 1 and 3 days after a right hindlimb denervation in the rat. Muscles of the contralateral sham hindlimb served as an internal control. Sham plantaris and gastrocnemius muscles showed 32 and 60% lower basal 2-DG uptake, 46 and 66% lower insulin-stimulated 2-DG uptake, and 79 and 81% lower blood flow, respectively, compared with sham soleus muscle. At 1 day after denervation, soleus, plantaris, and gastrocnemius muscles exhibited an 80, 64, and 42% decrease in insulin-stimulated 2-DG uptake, respectively, in the presence of 63, 323, and 304% higher blood flow, respectively. At 3 days after denervation, soleus muscle showed a 60% decrease in basal 2-DG uptake, complete unresponsiveness to insulin, and an 86% decrease in blood flow. In contrast, the denervated plantaris and gastrocnemius muscles exhibited a 262 and 105% increase in basal 2-DG uptake, respectively, no change in insulin-stimulated 2-DG uptake, and no change in blood flow compared with corresponding contralateral sham muscles. The results demonstrate that muscle blood flow is influenced by muscle fiber population and time after denervation and that changes in blood flow do not contribute to the insulin resistance in the denervated muscles.

Lysosomotropic agents ameliorate macrophage dysfunction following the phagocytosis of IgG-coated erythrocytes: a role for lipid peroxidation.

Phagocytosis of IgG-coated erythrocytes (EIgG) can depress several macrophage functions. Our previous studies have suggested that this macrophage dysfunction may be due to an oxidative stress caused by the interaction of hemoglobin-derived iron with superoxide and/or hydrogen peroxide. Since lysosomotropic agents are capable of altering iron handling by macrophages, the present study evaluated the ability of these agents to prevent the macrophage dysfunction and lipid peroxidation caused by a phagocytic challenge with EIgG. Elicited rat peritoneal macrophages showed a depression of PMA-stimulated hydrogen peroxide production, calcium ionophore-stimulated arachidonate release and Fc receptor-mediated phagocytosis. The lysosomotropic agents; chloroquine, quinacrine, ammonium chloride and methylamine all prevented the depression of hydrogen peroxide production and arachidonate release but did not alter the depression of phagocytic function. These agents also prevented the increase in lipid peroxidation products caused by a phagocytic challenge with EIgG. These results suggest that the ability of lysosomotropic agents to prevent some aspects of macrophage dysfunction after a phagocytic challenge may be due to their ability to block the oxidative stress caused by the challenge.