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J D Ernst

Publications and source records attributed to J D Ernst.

At least 37 records · Page 2Linked to original sources
PubMed1996

Detection of altered membrane phospholipid asymmetry in subpopulations of human red blood cells using fluorescently labeled annexin V.

The phospholipids of the human red cell are distributed asymmetrically in the bilayer of the red cell membrane. In certain pathologic states, such as sickle cell anemia, phospholipid asymmetry is altered. Although several methods can be used to measure phospholipid organization, small organizational changes have been very difficult to assess. Moreover, these methods fail to identify subpopulations of cells that have lost their normal phospholipid asymmetry. Using fluorescently labeled annexin V in flow cytometry and fluorescent microscopy, we were able to identify and quantify red cells that had lost their phospholipid asymmetry in populations as small as 1 million cells. Moreover, loss of phospholipid organization in subpopulations as small as 0.1% of the total population could be identified, and individual cells could be studied by fluorescent microscopy. An excellent correlation was found between fluorescence-activated cell sorter (FACS) analysis results using annexin V to detect red cells with phosphatidylserine (PS) on their surface and a PS-requiring prothrombinase assay using similar red cells. Cells that bound fluorescein isothiocyanate (FITC)-labeled annexin V could be isolated from the population using magnetic beads covered with an anti-FITC antibody. Evaluation of blood samples from patients with sickle cell anemia under oxygenated conditions demonstrated the presence of subpopulations of cells that had lost phospholipid asymmetry. While only a few red cells were labeled in normal control samples (0.21% +/- 0.12%, n = 8), significantly increased (P < .001) annexin V labeling was observed in samples from patients with sickle cell anemia (2.18% +/- 1.21%, n = 13). We conclude that loss of phospholipid asymmetry may occur in small subpopulations of red cells and that fluorescently labeled annexin V can be used to quantify and isolate these cells.

Anemia, Sickle Cell↗
PubMed1996

Phagosome-lysosome fusion is a calcium-independent event in macrophages.

Phagosome-lysosome membrane fusion is a highly regulated event that is essential for intracellular killing of microorganisms. Functionally, it represents a form of polarized regulated secretion, which is classically dependent on increases in intracellular ionized calcium ([Ca2+]i). Indeed, increases in [Ca2+]i are essential for phagosome-granule (lysosome) fusion in neutrophils and for lysosomal fusion events that mediate host cell invasion by Trypanosoma cruzi trypomastigotes. Since several intracellular pathogens survive in macrophage phagosomes that do not fuse with lysosomes, we examined the regulation of phagosome-lysosome fusion in macrophages. Macrophages (M phi) were treated with 12.5 microM bis-(2-amino-S-methylphenoxy) ethane-N,N,N',N',-tetraacetic acid tetraacetoxymethyl ester (MAPT/AM), a cell-permeant calcium chelator which reduced resting cytoplasmic [Ca2+]; from 80 nM to < or = 20 nM and completely blocked increases in [Ca2+]i in response to multiple stimuli, even in the presence of extracellular calcium. Subsequently, M phi phagocytosed serum-opsonized zymosan, staphylococci, or Mycobacterium bovis. Microbes were enumerated by 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) staining, and phagosome-lysosome fusion was scored using both lysosome-associated membrane protein (LAMP-1) as a membrane marker and rhodamine dextran as a content marker for lysosomes. Confirmation of phagosome-lysosome fusion by electron microscopy validated the fluorescence microscopy findings. We found that phagosome-lysosome fusion in M phi occurs noramlly at very low [Ca2+]i (< or = 20 nM). Kinetic analysis showed that in M phi none of the steps leading from particle binding to eventual phagosome-lysosome fusion are regulated by [Ca2+]i in a rate-limiting way. Furthermore, confocal microscopy revealed no difference in the intensity of LAMP-1 immunofluorescence in phagolysosome membranes in calcium-buffered vs. control macrophages. We conclude that neither membrane recognition nor fusion events in the phagosomal pathway in macrophages are dependent on or regulated by calcium.

Animals↗
PubMed1996

The (alpha2-->8)-linked polysialic acid capsule of group B Neisseria meningitidis modifies multiple steps during interaction with human macrophages.

Group B Neisseria meningitidis causes systemic disease, including meningitis, after initial colonization and subsequent penetration of nasopharyngeal mucosa, a tissue which is richly populated by macrophages. In an initial effort to characterize the interaction of N. meningitidis and mature human macrophages, the influence of the alpha2-->8) -linked polysialic acid capsule on the interaction of N. meningitidis with human monocyte-derived macrophages was investigated with a capsulate case isolate and an isogenic Tn916-derived noncapsulate transformant. The capsulate strain was fourfold less adherent to the macrophage surface after cold incubation, although adherence of both strains was significantly increased after opsonization with nonimmune C5-depleted serum. When opsonized inocula were adjusted so that they adhered to macrophages in equal numbers, the two strains were internalized at equivalent rates and both entered membrane-bound compartments (phagosomes). Colocalization of bacteria with the late endosomal and lysosomal marker lysosome-associated membrane protein revealed that fusion of lysosomes with phagosomes containing the capsulate organism was significantly reduced 10 and 30 min after entry, but by 1 h, no difference between the strains was observed. Once internalized, meningococci were effectively killed, although more rapid killing of the capsulate strain was observed over the first 3 h. These results indicate that the (alpha2-->8)-linked polysialic acid capsule modifies the interaction of meningococci with human macrophages at multiple steps, including adherence to the macrophage surface and phagosome-lysosome fusion. Moreover, the discordance between the kinetics of phagosome- lysosome fusion and bacterial killing suggests that a nonlysosomal mechanism may be responsible for a significant fraction of macrophage killing of N. meningitidis.

Antigens, CD↗
PubMed1996

Selective receptor blockade during phagocytosis does not alter the survival and growth of Mycobacterium tuberculosis in human macrophages.

Mycobacterium tuberculosis survives and replicates within human macrophages, but the mechanisms whereby tubercle bacilli resist killing are incompletely understood. We tested the general model in which M. tuberculosis evades killing by entering naive macrophages through receptors that are unable to activate cellular microbicidal activities. Complement receptor types 1 (CR1), 3 (CR3), and 4 (CR4) were blocked with monoclonal antibodies, and mannose receptors were blocked with a competitive ligand, mannosylated bovine serum albumin (MBSA). Survival and replication of M. tuberculosis (Erdman) were evaluated after the bacteria were phagocytosed in the presence of blocking agents (directing binding to the unblocked receptors). Although there was significant variation in the growth rate of virulent M. tuberculosis in monocyte-derived macrophages from different donors, the intracellular survival and replication of mycobacteria were equivalent regardless of the receptor(s) used for binding and phagocytosis. We conclude that the mechanisms whereby M. tuberculosis evades killing by human macrophages are independent of the receptor-mediated route of entry, and operate at one or more steps common to all entry pathways. Blocking complement and mannose receptors in combination did not completely abrogate binding of M. tuberculosis to macrophages. However, we found that two polyanionic scavenger-receptor ligands exhibited a concentration-dependent ability to block binding of M. tuberculosis to macrophages. Moreover, blocking class A scavenger receptors abrogated nearly all binding that persisted after blocking complement and mannose receptors. This indicates that class A scavenger receptors are quantitatively important mediators of M. tuberculosis-macrophage interactions. M. tuberculosis has evolved multiple mechanisms to promote its efficient entry into macrophages. This suggests that passage of the organism through macrophages may be an essential early step in the pathogenesis of tuberculosis.

Antibodies, Monoclonal↗
PubMed1995

Calcium signalling initiated by CR1 (CD35) crosslinking is mediated by phagocyte Fc gamma receptors in cis.

We have examined the role of complement receptor type 1 (CR1; CD35) in generating intracellular signals in phagocytic cells, particularly human neutrophils. We find that crosslinking of CR1 alone fails to result in an increase in intracellular ionized calcium [Ca2+]i. However, IgG bound to CR1 is able to interact with Fc gamma receptors to stimulate increases in [Ca2+]i. By examining signals generated in mixed populations of cells, we determined that calcium increases are observed only when CR1 and Fc gamma receptors are engaged on the same cell. Functional interactions between complement receptors and receptors for immunoglobulin G may promote clearance of pathogenic bacteria and fungi that bind limited quantities of complement and IgG.

Animals↗
PubMed1995

Mobilization of annexin V during the uptake of DNP-albumin by human dendritic cells.

Dendritic cells play a crucial role in antigen presentation in various tissues. The endocytic capacity of these cells has been regarded as minimal, but recent work on dendritic cells from mouse spleen has disclosed that the fluid-phase traffic through late endosomes is as active in dendritic cells as in other antigen-presenting cell types. We show that cultured human dendritic cells express the annexins I, III, IV, V and VI, as detected by immunofluorescence staining. The annexins are cytosolic Ca(2+)-dependent proteins with the ability to promote vesicle aggregation and membrane fusion through their capacity to bind to membrane phospholipids. Annexin I and VI appeared to outline the cytoskeleton and the plasma membrane in cultured human dendritic cells. Studies using confocal laser scanning microscopy showed that during the endocytosis of fluorescent dinitrophenyl-conjugated albumin by dendritic cells, there was a redistribution of annexin V which was found to colocalize with vesicles containing dinitrophenyl-FITC-conjugated albumin.

Albumins↗
PubMed1994

Annexins possess functionally distinguishable Ca2+ and phospholipid binding domains.

All annexins bind Ca2+ and phospholipids, although individual annexins differ markedly in their affinities for these ligands. Annexin I binds phosphatidylserine (PS) at lower [Ca2+] than annexin V, while annexin V exhibits a higher affinity for PS than does annexin I. To identify the structural determinants of these properties, we characterized a series of chimeric annexins. A chimera containing repeat 1 of annexin V used to repeats 2, 3, and 4 of annexin I exhibited a Ca2+ requirement for PS binding close to that of annexin I, while chimeras containing repeat 1 of annexin I fused to repeats 2, 3, and 4 of annexin V required higher [Ca2+], similar to that of annexin V. In contrast, the overall affinity for PS vesicles was determined by the source of repeat 1. The chimera that contained repeat 1 of annexin V exhibited a high affinity for PS, while a chimera that contained repeat 1 of annexin I had a low affinity for PS similar to that of annexin I. We conclude that the [Ca2+] requirement for phospholipid binding and the overall phospholipid affinity of annexins are determined by distinct domains.

Annexins↗
PubMed1994

Selective translocation of annexins during intracellular redistribution of Chlamydia trachomatis in HeLa and McCoy cells.

When Chlamydia trachomatis elementary bodies enter epithelial cells, they occupy membrane-bound vesicles that aggregate with each other in a calcium-dependent manner but that do not fuse with lysosomes. As members of the annexin family of calcium- and membrane-binding proteins have been implicated in mediating calcium-regulated membrane traffic during endo- and exocytosis, we examined the intracellular localization of certain annexins following invasion of HeLa and McCoy cells by C. trachomatis serovar L2. Immunofluorescence staining with a panel of polyclonal antibodies against five human annexins revealed that annexins III, IV, and V translocate within the cytoplasm to the proximity of intracellular chlamydiae whereas the distribution of annexins I and VI was unaffected. The distinct distribution of annexins I and III was further analyzed by confocal microscopy, which revealed an intimate association between chlamydial aggregates or inclusions and annexin III. Confocal microscopy also confirmed the nonassociation of annexin I with chlamydial aggregates. Depletion of intracellular Ca2+ did not prevent association of annexin III with individual elementary body-containing endosomes but did prevent formation of chlamydial aggregates and translocation of annexin III. Furthermore, chloramphenicol-treated cells also showed association between chlamydial aggregates and annexin III, indicating that the annexins are of host cell origin. These data suggest that certain cytosolic annexins may be involved in the Ca(2+)-dependent aggregation and fusion of chlamydia-containing vesicles. The fact that these Ca(2+)-binding proteins differ in their ability to associate with chlamydia-containing vesicles and inclusions implies that the factors that regulate the interaction of annexin I and annexin III with membrane are different and suggests a selective regulatory mechanism for endosome aggregation and avoiding lysosome fusion during chlamydia infection.

Annexins↗
PubMed1993

Epitope mapping of annexin I: antibodies that compete with phospholipids and calcium recognize amino acids 42-99.

To understand further the structural basis of phospholipid binding by annexin I, three monoclonal antibodies that compete with Ca2+ and phospholipids for binding of annexin I were used to screen an expression library containing fragments of bovine annexin I cDNA. In all, 15 clones were isolated, and all contain overlapping fragments of the cDNA. The smallest unit common to all of the clones encodes amino acids 42-99 of annexin I, representing a portion of the first repeat domain. This demonstrates that recognition of a single domain of annexin I is sufficient to completely block phospholipid binding, and implies that the first repeat may contribute to phospholipid binding by annexin I.

Amino Acid Sequence↗
PubMed1991

Annexin III translocates to the periphagosomal region when neutrophils ingest opsonized yeast.

After phagocytosis, killing and digestion of ingested microorganisms depend on fusion of phagocytic vesicle membranes with membranes of intracellular vesicles (azurophil and specific granules). There is considerable evidence that phagosome-granule membrane fusion is regulated by transient increases in intracellular ionized Ca2+. In previous studies, we found that a cytosolic Ca2(+)-dependent membrane-binding protein, annexin III, represents over 1% of the total protein of human neutrophils and promotes tight contact between membranes of isolated specific granules in vitro. To determine whether annexin III localizes to the region of phagosome-granule membrane fusion in vivo, we used a monospecific polyclonal antibody to stain fixed, permeabilized neutrophils that had ingested opsonized yeast. We found that annexin III concentrates in the region surrounding the phagosome. Annexin III was concentrated ninefold in the periphagosomal region compared with the cell body, as demonstrated by laser scanning confocal microscopy. Periphagosomal translocation of annexin III occurred whether yeast were opsonized with IgG, complement, or both, and persisted for at least 1 h after phagocytosis. This is not a general phenomenon, inasmuch as calmodulin was as abundant in the cell body as in the periphagosomal region. These findings imply that annexin III plays a specialized role in the metabolic and structural events that accompany phagocytosis.

Annexins↗
PubMed1991

Identification of a domain that mediates vesicle aggregation reveals functional diversity of annexin repeats.

Annexins are structurally related proteins that bind phospholipids in a Ca2(+)-dependent manner and possess at least four conserved 70-amino acid repeat domains. The ability of certain annexins to promote contact between vesicle membranes in vitro has prompted the suggestion that these proteins regulate membrane traffic in exocytosis. We have previously found that annexins I and II promote contact between vesicles whereas annexin V does not. In order to understand the mechanism of annexin I-mediated vesicle-vesicle contact, we prepared a monoclonal antibody that specifically inhibits annexin I-mediated vesicle aggregation. We identified the domain of annexin I recognized by this monoclonal antibody by using it to screen an expression library containing random fragments of annexin I cDNA. The antibody identified a fragment encoding amino acids 41-118 (the first repeat plus 8 residues of the amino-terminal tail). We constructed a chimeric protein containing these amino acids of annexin I fused to the second, third, and fourth repeats of annexin V. Transfer of this domain conferred the ability to promote vesicle aggregation, confirming that this domain participates directly in mediating contact between vesicle membranes.

Antibodies, Monoclonal↗
PubMed1990

Characterization of Ca2(+)-dependent phospholipid binding, vesicle aggregation and membrane fusion by annexins.

The annexins are a family of structurally similar, Ca2(+)-dependent, phospholipid-binding proteins. We compared six members of this family (calpactin I heavy chain, lipocortins I and III, endonexin II, p68 and protein II) to determine their phospholipid-binding specificities, as well as their ability to promote aggregation and fusion of phospholipid vesicles. The Ca2+ requirement for all of the proteins was lowest for binding to vesicles composed of phosphatidic acid, followed by phosphatidylserine and then phosphatidylinositol. Only protein II, p68, lipocortin III and endonexin II bound to vesicles composed of phosphatidylethanolamine, and none bound to phosphatidylcholine. Both calpactin I heavy chain and lipocortin I promoted aggregation of phosphatidylserine- or phosphatidylinositol-containing vesicles in the presence of less than 10 microM-Ca2+. Lipocortin I promoted fusion of liposome membranes by lowering threshold Ca2+ concentrations. Although calpactin I heavy chain did not affect threshold Ca2+ concentrations, it did increase the rate and extent of spontaneous fusion. In contrast, p68 inhibited fusion at threshold Ca2+ concentrations. Whereas previous reports have emphasized properties that the annexins have in common, these findings reveal quantitative and qualitative differences among the annexins which may relate to distinct intracellular functions.

Annexin A4↗
PubMed1990

Purification and characterization of an abundant cytosolic protein from human neutrophils that promotes Ca2(+)-dependent aggregation of isolated specific granules.

Intracellular ionized calcium has been strongly implicated in mediating several responses of human neutrophils to stimulation. However, proteins that serve as effectors of these responses have not been well characterized. To identify proteins that might serve as mediators of the effects of Ca2+ in human neutrophils, we isolated proteins that bind to membrane phospholipids in a Ca2(+)-dependent manner. The most abundant of these, a protein of 33 kD, was readily purified to homogeneity, and was found to bind to phosphatidylserine vesicles in the presence of 2 microM ionized Ca2+. In addition, this purified protein promoted Ca2(+)-dependent aggregation of isolated specific granules from human neutrophils, indicating that it might mediate membrane-membrane contact during processes such as phagosome-lysosome fusion or degranulation. This protein was localized to the cytoplasm of unstimulated neutrophils and found to account for approximately 1% of the cytosol protein. Amino acid sequence of several peptides derived from the purified protein revealed that it is identical to lipocortin III, a recently described member of the annexin family that is scarce in other cells and tissues. The abundance of this protein, together with its Ca2(+)-dependent membrane effects, suggest that it mediates membrane-localized events in stimulated neutrophils, such as phagosome-lysosome fusion or degranulation.

Amino Acid Sequence↗
PubMed1989

Use of a novel strategy for the preparation and characterization of an antipeptide antibody capable of recognizing members of the annexin family.

Annexins are structurally-related proteins which bind phospholipids in a Ca2+-dependent manner. We have used a novel coupling strategy to prepare an antiserum directed against a 17-amino acid synthetic peptide that resembles the sequence of a highly-conserved portion of these proteins. This antipeptide serum specifically recognizes 5 of 6 human annexins on Western blots, despite differences between the protein and peptide sequences of 3 or 4 amino acids. The antiserum does not recognize endonexin II, whose sequence differs from that of the peptide by 6 amino acids. The availability of multiple proteins with known amino acid sequence has allowed analysis of structural requirements for recognition by this antibody. In some situations, use of such an antibody may allow the identification of a protein as a member of a family.

Amino Acid Sequence↗
PubMed1987

Synexin-like proteins from human polymorphonuclear leukocytes. Identification and characterization of granule-aggregating and membrane-fusing activities.

We have used Ca2+-dependent binding to a phospholipid vesicle affinity column to isolate a mixture of three synexin-like proteins from the cytosol of human polymorphonuclear leukocytes (PMN), with relative molecular weights of approximately 67,000, 47,000, and 28,000. Rabbit antibodies raised against bovine liver synexin recognized the 47,000 molecular weight PMN protein. These PMN proteins, like bovine liver synexin, promoted aggregation of isolated PMN specific granules in the presence of Ca2+ and increased the overall rate of Ca2+-induced fusion of liposomes composed of phosphatidate (PA)/phosphatidylethanolamine (PE) (1:3) and phosphatidylserine/PE (1:3), but decreased the rate of spermine-induced fusion of PA/PE (1:3) liposomes. Using fluorescent lipid probes, rapid fusion of PA/PE liposomes with PMN specific granules (50% maximum signal within a few minutes) was observed when 1 mM Ca2+ was added in the presence of both synexin and free arachidonic acid. Dilution of the aqueous contents of liposomes was also observed under the same conditions. The rate of fusion increased monotonically with Ca2+ and arachidonic acid concentrations, but synexin exhibited an optimum concentration. Lack of any one of the components precluded rapid fusion. These results suggest that PMN contain a protein similar to, or identical with, synexin that may be involved in calcium-dependent fusion of intracellular membranes.

Animals↗