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.

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.

Asbestos induces apoptosis of human and rabbit pleural mesothelial cells via reactive oxygen species.

Mesothelial cells, the progenitor cell of the asbestos-induced tumor mesothelioma, are particularly sensitive to the toxic effects of asbestos, although the molecular mechanisms by which asbestos induces injury in mesothelial cells are not known. We asked whether asbestos induced apoptosis in mesothelial cells and whether reactive oxygen species were important. Pleural mesothelial cells (rabbit or human) were exposed to asbestos (crocidolite, amosite, or chrysotile) or control particles at moderate doses (1-10 microg/cm2) over 24 h and evaluated for oligonucleosomal DNA fragmentation, loss of membrane phospholipid asymmetry, and nuclear condensation. Asbestos fibers, not control particles, induced apoptosis in mesothelial cells by all assays and induction of apoptosis was dose dependent for all types of asbestos, with crocidolite (5 microg/cm2) inducing 15.0+/-1.1% (mean+/-SE; n = 12) apoptosis versus control particles < 4%. Apoptosis induced by asbestos, but not by actinomycin D, was inhibited by extracellular catalase, superoxide dismutase in the presence of catalase, hypoxia (8% oxygen), deferoxamine, 3-aminobenzamide [an inhibitor of poly(ADP-ribosyl) polymerase], and cytochalasin B. Only catalase and cytochalasin B decreased fiber uptake. We conclude that asbestos induces apoptosis in mesothelial cells via reactive oxygen species. Escape from this pathway could allow the abnormal survival of mesothelial cells with asbestos-induced mutations.

Immune responses in tuberculosis.

The initial interaction of Mycobacterium tuberculosis with phagocytes through Toll-like receptors can affect induction of the adaptive response by inflammatory cytokine production, as well as begin the process of bacterial containment by induction of antimycobacterial functions. Advances in T cell research in tuberculosis include identification of antigens recognized by CD8(+) T cells in infected hosts, elucidation of antimycobacterial mechanisms of T cells and the discovery of CD8(+) T cells that recognize antigens presented by a variety of non-classical molecules.

Crocidolite asbestos induces apoptosis of pleural mesothelial cells: role of reactive oxygen species and poly(ADP-ribosyl) polymerase.

Mesothelial cells, the progenitor cells of the asbestos-induced tumor mesothelioma, are particularly sensitive to the toxic effects of asbestos, although the molecular mechanisms by which asbestos induces injury in mesothelial cells are not known. We asked whether asbestos induced apoptosis in mesothelial cells and whether reactive oxygen species were important. Rabbit pleural mesothelial cells were exposed to crocidolite asbestos or control particles (1-10 micrograms/cm2) over 24 hr and evaluated for oligonucleosomal DNA fragmentation, loss of membrane phospholipid asymmetry, and nuclear condensation. Asbestos fibers, not control particles, induced apoptosis in mesothelial cells by all assays. Induction of apoptosis was dose dependent; crocidolite (5 micrograms/cm2) induced apoptosis (15.0 +/- 1.1%, mean +/- SE; n = 12) versus control particles (< 4%), as measured by appearance of nuclear condensation. Apoptosis induced by asbestos, but not by actinomycin D, was inhibited by extracellular catalase, superoxide dismutase in the presence of catalase, hypoxia (8% oxygen), deferoxamine, and 3-aminobenzamide (an inhibitor of the nuclear enzyme, poly(adenosine diphosphate-ribosyl) polymerase). We conclude that asbestos induces apoptosis in mesothelial cells via reactive oxygen species. We speculate that escape from this pathway could allow the abnormal survival of mesothelial cells with asbestos-induced mutations.

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.

Initiation and regulation of T-cell responses in tuberculosis.

Tuberculosis (TB) poses a great challenge to immunologists, as it represents a chronic infection characterized by persistence of the pathogen despite development of antigen-specific immune responses. Among the characteristics of adaptive immune responses to Mycobacterium tuberculosis is a delay in the onset of detectable T-cell responses, in both humans and experimental animals. Recent studies have revealed mechanisms that contribute to this delay, including pathogen inhibition of apoptosis, delayed migration of dendritic cells from the lungs to the local lymph node, and influence of regulatory T cells. In addition, novel features of M. tuberculosis antigen-specific T-cell differentiation have been discovered, which reveal pathways that limit and promote immune control of infection. Taken together, these results highlight the need for additional basic research and provide optimism for the development of TB vaccines with greater efficacy.

Regulation of Mycobacterium tuberculosis whiB3 in the mouse lung and macrophages.

Mycobacterium tuberculosis is a highly successful human pathogen, with approximately 2x10(9) individuals infected globally. To understand the responses of M. tuberculosis to the in vivo environment, we studied the in vivo regulation of M. tuberculosis genes whose M. marinum homologs are induced in chronically infected frog tissues. The expression of 16S rRNA was shown to remain constant in M. tuberculosis under in vivo and in vitro conditions and therefore could be used for internal normalization in quantitative reverse transcription-PCR assays. We found whiB3, a putative transcriptional regulator implicated in mediating tissue damage, to be maximally induced at 2 weeks postinfection in the lungs of wild-type and immunodeficient (gamma interferon receptor-/-, Rag1-/-, and tumor necrosis factor alpha-/-) mice. At later time points in wild-type mice, whiB3 induction was decreased and gradually declined over the course of infection. In immunodeficient mice, whiB3 induction declined rapidly and was completely abolished in moribund animals. whiB3 was also found to be induced in naïve bone marrow-derived macrophages after 6 h of infection. whiB3 expression in vivo and in vitro was found to be inversely correlated with bacterial density. These results indicate that M. tuberculosis regulates the expression of whiB3 in response to environmental signals present in vivo and are consistent with a model of regulation by quorum sensing.

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.

Bacterial inhibition of phagocytosis.

The concerted study of molecular mechanisms of phagocytosis and the inhibition of phagocytosis by specific products of extracellular bacterial pathogens has borne considerable fruit. The importance of tyrosine phosphorylation and of the Rho family of GTPases has become clear to cell biologists, but pathogenic bacteria recognized the importance of these signalling pathways in phagocytic cells long ago. The discoveries described in this review are only the beginning. The simultaneous pursuit of the mechanisms and molecules involved in the initiation and regulation of phagocytosis and that pathogenic bacteria use to inhibit phagocytosis will surely identify more interesting pathways on each side of the contest. Are there any obvious possibilities? There are several bacterial factors that have the potential to inhibit known mechanisms of phagocytosis. Clostridium species, for example, make a number of exotoxins of interest. Clostridium botulinum and Clostridium tetani neurotoxins inactivate the regulated secretory machinery by proteolytic cleavage of SNARE proteins, and targets of tetanus toxin and botulinum b toxin inhibit the exocytotic delivery of membrane vesicles needed for phagocytosis of large particles (Hackam et al., 1998). Moreover, the C3 exotoxin of C. botulinum catalyses ADP ribosylation and inactivation of rho family GTPases (Wiegers et al., 1991), and toxins A and B of C. difficile UDP-glucosylate and inactivate rho GTPases and thereby disrupt the actin cytoskeleton (Just et al., 1995a,b). However, as Clostridia lack the machinery for type III secretion, these proteins are not rapidly targeted to the phagocyte cytoplasm. More searching may reveal a pathogen that has combined the type III secretory machinery with clostridia toxin-like substrates. A potentially unique strategy for remaining outside phagocytes is exhibited by Helicobacter pylori, which contain a type IV secretion system. Unopsonized virulent strains of H. pylori bind readily to macrophages but are only internalized after a delay of several minutes. Such a delay appears to be sufficient for the bacteria to remain extracellular (Allen et al., 2000). Elucidation of the mechanism used by H. pylori to delay phagocytosis may reveal one or more novel virulence factors as well as one or more novel targets in the phagocyte that will add to the understanding of a fundamental process in host defence. Another field ripe for further mechanistic investigation is complement receptor-mediated phagocytosis. Dedicated study of the molecular events and molecular mediators of phagocytosis downstream of CR3 is likely to reveal interesting differences from FcgammaR phagocytosis and is just as likely to reveal that microbes have discovered unique mechanisms for circumventing them. Study of extracellular pathogens and the mechanisms that they use to remain outside phagocytic cells has revealed a great deal about the initial encounter between pathogen and phagocyte. We can look forward to additional discoveries about the host-pathogen interactions and the mechanisms and factors that each side uses to battle against the other.

Toward the development of antibacterial vaccines: report of a symposium and workshop. Organizing Committee.

On 26 and 27 October 1998, the Department of Medicine at the University of California, San Francisco (UCSF), hosted a symposium and workshop on bacterial vaccines. The symposium featured invited speakers who are internationally recognized authorities in their fields and who discussed selected topics related to specific pathogens or specific principles of bacterial vaccine development. The workshop, held on the day following the symposium, brought together the invited speakers and members of the organizing committee, who came from UCSF and the University of California, Berkeley, to discuss 4 specific topics and to define priorities for future vaccine development. Considerable knowledge has been gained from successful and unsuccessful vaccine development efforts, and large gains in knowledge relevant to vaccine development have resulted from studies of basic immunology and microbial pathogenesis. This report summarizes the presentations at the symposium and the discussions of the workshop sessions.

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.

Calcium-dependent neutrophil secretion: characterization and regulation by annexins.

To gain direct access to the secretory machinery and study the regulation, mechanisms, and effectors of Ca2+-dependent neutrophil secretion, we developed an efficient and reproducible method of plasma membrane permeabilization using streptolysin O. We confirmed previous studies that permeabilized neutrophils secrete in response to calcium alone, but we also found that the Ca2+ dose-response is biphasic. Secretion is detectable at <1.0 microM Ca2+ and reaches a plateau between 1.0 and 60 to 80 microM. When stimulated with >80 microM Ca2+, secretion is two- to threefold greater than at lower [Ca2+], suggesting that two distinct mechanisms of Ca2+-dependent secretion that differ in their affinity for Ca2+ exist in neutrophils. Although permeabilization allows 100% leak of lactate dehydrogenase, maximum secretion from permeabilized cells is 80% that of f-met-leu-phe-stimulated intact cells, indicating that the essential components of the Ca2+-dependent secretory apparatus are predominantly, if not entirely, membrane bound. Permeabilization causes leakage of 100% of annexins V and VI, but 41% of annexin I and 12% of annexin III are retained. Immunofluorescence microscopy revealed that retained annexins I and III are associated with granule membranes. Addition of soluble annexins I and III to permeabilized cells increased Ca2+-induced secretion up to 15% and 90%, respectively, implying that both annexins participate in this secretory pathway. While annexin V is not required for secretion, it inhibits the low Ca2+-affinity mechanism of secretion.

Antibiotic combinations in experimental infections in animals.

The study of experimental infections has afforded the opportunity to examine the response to antimicrobial therapy in the complex milieu of living animals. This review summarizes the information gained from studies of combination antibiotic therapy for experimental endocarditis, meningitis, and other infections in which host defenses are unable to participate in effecting cure. The studies cited suggest that antibiotic combinations that yield rapid microbial killing are more effective in the treatment of these selected infections than are those combinations that do not give enhanced killing. In addition, the relevance of various methods of demonstrating antimicrobial synergy is examined, and in light of the results of treatment of experimental infections, suggestions for the applications of these methods to the evaluation of the efficacy of antibiotic combinations on infections in humans are given.

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.

GTP-dependent permeabilized neutrophil secretion requires a freely diffusible cytosolic protein.

Guanosine triphosphate (GTP) has been implicated in the regulation of Ca(2+)-mediated secretion from neutrophils. We further examined the role of GTP in neutrophil secretion using streptolysin O permeabilized cells. We found that, in the presence of GTP, 1.0 microM free Ca(2+) causes maximum secretion-equivalent to that achieved with 100 microM free Ca(2+)-whereas GTPgammaS inhibits Ca(2+)-stimulated secretion. Interestingly, GTP by itself stimulates secretion. These results indicate the existence of a GTP-regulated mechanism of secretion in neutrophils that requires GTP hydrolysis to stimulate secretion in the presence and absence of Ca(2+). The stimulatory effect of GTP is only observed when GTP is present during permeabilization. Addition of GTP after permeabilization, when the cytosolic contents have leaked out from cells, gives no stimulatory response, implying that the GTP-dependent secretory apparatus requires at least one cytosolic protein. GTP-dependent secretion can be reconstituted with crude HL-60 and bovine liver cytosol. The reconstituting activity binds to GTP-agarose, suggesting that the cytosolic factor is a GTP-binding protein or forms a complex with a GTP-binding protein. However, it is not a member of the rho or rac families of GTPases. By gel filtration chromatography, the secretion-reconstituting activity eluted at 870 and 200 kDa, but in the presence of GTP, eluted at 120 kDa, indicating that it is part of a high-molecular-weight complex that dissociates in the presence of GTP. Retention of adenosine diphosphate-ribosylation factor (ARF) in permeabilized cells and insensitivity of the cytosolic reconstituting activity to brefeldin A led to our speculation that ARF6 may be the GTPase involved in GTP-dependent secretion, and that activity from a BFA-insensitive ARF6 guanine nucleotide exchange factor reconstitutes secretion.

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.

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.