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.

Cholesterol esterification by host and parasite is essential for optimal proliferation of Toxoplasma gondii.

Upon host cell invasion the apicomplexan parasite Toxoplasma gondii resides in a specialized compartment termed the parasitophorous vacuole that is derived from the host cell membrane but modified by the parasite. Despite the segregation of the parasitophorous vacuole from the host endocytic network, the intravacuolar parasite has been shown to acquire cholesterol from the host cell. In order to characterize further the role of sterol metabolism in T. gondii biology, we focused our studies on the activity of acyl-CoA:cholesterol acyltransferase (ACAT), a key enzyme for maintaining the intracellular homeostasis of cholesterol through the formation of cholesterol esters. In this study, we demonstrate that ACAT and cholesterol esters play a crucial role in the optimal replication of T. gondii. Moreover, we identified ACAT activity in T. gondii that can be modulated by pharmacological ACAT inhibitors with a consequent detrimental effect on parasite replication.

Chemokine receptor 2 serves an early and essential role in resistance to Mycobacterium tuberculosis.

Although the protective cellular immune response to Mycobacterium tuberculosis requires recruitment of macrophages and T lymphocytes to the site of infection, the signals that regulate this trafficking have not been defined. We investigated the role of C-C chemokine receptor 2 (CCR2)-dependent cell recruitment in the protective response to M. tuberculosis. CCR2(-/-) mice died early after infection and had 100-fold more bacteria in their lungs than did CCR2(+/+) mice. CCR2(-/-) mice exhibited an early defect in macrophage recruitment to the lung and a later defect in recruitment of dendritic cells and T cells to the lung. CCR2(-/-) mice also had fewer macrophages and dendritic cells recruited to the mediastinal lymph node (MLN) after infection. T cell migration through the MLN was similar in CCR2(-/-) and CCR2(+/+) mice. However, T cell priming was delayed in the MLNs of the CCR2(-/-) mice, and fewer CD4(+) and CD8(+) T cells primed to produce IFN-gamma accumulated in the lungs of the CCR2(-/-) mice. These data demonstrate that cellular responses mediated by activation of CCR2 are essential in the initial immune response and control of infection with M. tuberculosis.

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.

Phagocytosis of crocidolite asbestos induces oxidative stress, DNA damage, and apoptosis in mesothelial cells.

Phagocytosis of asbestos fibers may be a necessary step for asbestos-induced injury to mesothelial cells, but this has not been established because quantification of fiber uptake is difficult and ways to increase fiber phagocytosis without also increasing total dose were not available. We quantified phagocytosis by counting intracellular fibers after removing adherent fibers with trypsin; we selectively increased fiber phagocytosis by coating crocidolite asbestos fibers with the adhesive serum protein vitronectin (VN), which we have shown increases fiber uptake via integrins. We measured various aspects of asbestos-induced cytotoxicity: intracellular oxidation by the shift of fluorescence of cells loaded with an oxidative probe, DNA strand breakage by the alkaline unwinding ethidium bromide fluorometric assay, apoptosis by annexin V binding and by nuclear morphology, and cell-cycle progression. We found that, compared with control fibers or particles, asbestos increased intracellular oxidation, DNA strand breakage, and apoptosis. Selective increases in fiber uptake by VN-coating of the fibers further increased the oxidation, DNA strand breakage, and apoptosis, and induced a cell-cycle arrest in G2/M. Selective decreases in fiber uptake by cytochalasin or by integrin blockade with RGD peptides inhibited several of these measures of injury. We conclude that phagocytosis is important and perhaps necessary for asbestos-induced injury to mesothelial cells.

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.

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.

Mycobacterium tuberculosis inhibits IFN-gamma transcriptional responses without inhibiting activation of STAT1.

IFN-gamma activates macrophages to kill diverse intracellular pathogens, but does not activate human macrophages to kill virulent Mycobacterium tuberculosis. We tested the hypothesis that this is due to inhibition of IFN-gamma signaling by M. tuberculosis and found that M. tuberculosis infection of human macrophages blocks several responses to IFN-gamma, including killing of Toxoplasma gondii and induction of FcgammaRI. The inhibitory effect of M. tuberculosis is directed at transcription of IFN-gamma-responsive genes, but does not affect proximal steps in the Janus kinase-STAT pathway, as STAT1alpha tyrosine and serine phosphorylation, dimerization, nuclear translocation, and DNA binding are intact in M. tuberculosis-infected cells. In contrast, there is a marked decrease in IFN-gamma-induced association of STAT1 with the transcriptional coactivators CREB binding protein and p300 in M. tuberculosis-infected macrophages, indicating that M. tuberculosis directly or indirectly disrupts this protein-protein interaction that is essential for transcriptional responses to IFN-gamma. Gamma-irradiated M. tuberculosis and isolated cell walls reproduce the effects of live bacteria, indicating that the bacterial component(s) that initiates inhibition of IFN-gamma responses is constitutively expressed. Although lipoarabinomannan has been found to exert effects on macrophages, it does not account for the inhibitory effects of cell walls. These results indicate that one mechanism for M. tuberculosis to evade the human immune response is to inhibit the IFN-gamma signaling pathway, and that the mechanism of inhibition is distinct from that reported for Leishmania donovani or CMV, in that it targets the interaction of STAT1 with the basal transcriptional apparatus.

Preparation and characterization of an endogenously fluorescent annexin for detection of apoptotic cells.

Annexin proteins specifically bind anionic phospholipids such as phosphatidylserine, which are normally confined to the cytoplasmic leaflet of cellular membranes. During programmed cell death, or apoptosis, this phospholipid asymmetry is lost, and anionic phospholipids are exposed on the extracellular leaflet of the plasma membrane where they are accessible to exogenously added, labeled annexins. Chemically [e.g., fluoroscein isothiocyanate (FITC)]-modified annexin V has been widely used to detect and enumerate apoptotic cells by flow cytometry. We prepared chimeric proteins containing green fluorescent protein (GFP) fused to annexin V. A chimera containing GFP fused to the C-terminus of annexin V was soluble and fluorescent, but was unable to bind phospholipids. In contrast, a chimera containing GFP fused to the N-terminus of annexin V specifically bound apoptotic cells. GFP-annexin V represents a sensitive and facile alternative to FITC-annexin V for studies of apoptosis.

Roles of calcium and annexins in phagocytosis and elimination of an attenuated strain of Mycobacterium tuberculosis in human neutrophils.

The phagocytic function of neutrophils is a crucial element in the host defence against invading microorganisms. We investigated phagocytosis and intracellular killing of an attenuated strain of Mycobacterium tuberculosis(H37Ra) by human neutrophils focusing on the role of the cytosolic free calcium concentration [Ca2+]i and certain cytosolic calcium-dependent membrane-binding proteins annexins. Phagocytic uptake did not trigger a calcium rise and occurred independently of different calcium conditions, and in a serum-dependent manner. Changes in the viability of H37Ra were determined by agar plate colony count and a radiometric assay. Neutrophils showed a capacity to kill ingested mycobacteria and this occurred without a rise in [Ca2+]i. The ability to kill H37Ra decreased in the absence of extracellular calcium and when intra-extracellular calcium was reduced. Immunofluorescence staining revealed that during phagocytosis of H37Ra, annexins III, IV and VI translocated from cytoplasm to the proximity of the H37Ra-containing phagosomes, whereas the localization of annexin I and V remained unchanged. The translocation of annexin IV occurred even when Ca2+-depleted neutrophils ingested H37Ra in the absence of extracellular calcium. We concluded that neutrophil-mediated killing of mycobacteria is a Ca2+-dependent process. The fact that the association of certain annexins to the membrane vesicle containing H37Ra differ from other phagosomes suggests a selective regulatory mechanism during phagocytosis of mycobacteria by neutrophils.

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.

Transfer of phagocytosed particles to the parasitophorous vacuole of Leishmania mexicana is a transient phenomenon preceding the acquisition of annexin I by the phagosome.

The eukaryotic intracellular pathogen Leishmania mexicana resides inside macrophages contained within a membrane bound parasitophorous vacuole which, as it matures, acquires the characteristics of a late endosomal compartment. This study reports the selectivity of fusion of this compartment with other particle containing vacuoles. Phagosomes containing zymosan or live Listeria monocytogenes rapidly fused with L. mexicana parasitophorous vacuoles, while those containing latex beads or heat killed L. monocytogenes failed to do so. Fusigenicity of phagosomes was not primarily dependent on the receptor utilized for ingestion, as opsonization with defined ligands could not overcome the exclusion of either latex beads or heat killed organisms. However modulation of intracellular pH by pharmacological agents such as chloroquine and ammonium chloride increased delivery of live Listeria and also induced transfer of previously excluded particles. The absence of fusion correlated with the acquisition of annexin I, a putative lysosomal targeting, molecule, on the phagosome membrane. We propose that the acquisition of cellular membrane constituents such as annexin I during phagosome maturation can ultimately direct the fusion pathway of the vesicles formed and have described a model system to further document changes in vesicle fusigenicity within cells.

Role of annexins in endocytosis of antigens in immature human dendritic cells.

We have evaluated the uptake of a soluble protein antigen, denitrophenylated human serum albumin (DNP-HSA), and two different intracellular bacteria; Chlamydia trachomatis serovar L2 and Mycobacterium tuberculosis strain H37Ra, by immature human dendritic cells. These were generated by culturing progenitor cells from blood in the presence of cytokines (granulocyte-macrophage colony-stimulating factor and interleukin-4). Dendritic cells play a crucial part in antigen presentation for the induction of T-cell-dependent immune responses in various tissues. Recently, macropinocytic and phagocytic activity has been shown for immature dendritic cells of mouse, rat and human origin. In the present study, macropinocytosis characterized the uptake of the soluble protein-antigen DNP-HSA, whereas the C. trachomatis were ingested via receptor-mediated endocytosis in coated pits, and opsonized M. tuberculosis via phagocytosis. To follow the intracellular routes of the antigens, their positions were compared with the localization of annexins, a family of Ca(2+)-and phospholipid-binding proteins, involved in membrane fusion, aggregation and transport of different vesicles. To elucidate further the intracellular pathway of the antigens, two other proteins, lysosome-associated membrane protein-1 (LAMP-1) and cathepsin D, were labelled. They are known to colocalize with major histocompatibility complex class II compartments in the immature dendritic cells. We observed a distinct translocation of annexin V to DNP-HSA containing endosomes, and annexin III to vesicles with C. trachomatis. Furthermore, annexin III, IV and V redistributed to phagosomes with M. tuberculosis. Both LAMP-1 and cathepsin D colocalized with DNP-HSA endosomes, and with phagosomes with M. tuberculosis. Thus, immature human dendritic cells have the capacity to phagocytose. Moreover, the handling of these antigens by dendritic cells may represent three distinct intracellular pathways, albeit some properties and compartments are shared.

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.

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.

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.