Altered membrane trafficking in activated bone marrow-derived macrophages.

Activation of macrophages with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) leads to increased intracellular resistance to microbes and increased major histocompatibility complex class II-restricted antigen presentation, processes that both use the vacuolar compartment. Despite the requirement of the macrophage vacuolar compartment for microbicidal activities and antigen processing, the rates of endocytosis and membrane trafficking in activated macrophages are not clearly defined. In this study, vacuolar compartment dynamics were analyzed in murine bone marrow-derived macrophages activated with LPS and/or IFN-gamma, conditions that increased macrophage nitric oxide production and resistance to infection by Listeria monocytogenes. Relative to nonactivated cells, activated macrophages showed diminished rates of fluid-phase pinocytosis and phagocytosis and delayed progression of macropinosomes and phagosomes to late endosomes and lysosomes. In contrast to the slowing of membrane trafficking, rates of macropinosome acidification were similar between activated and nonactivated cells. One consequence of this slowed membrane trafficking in activated macrophages was a prolonged exposure of incoming molecules to an acidic nonlysosomal compartment, a condition which may facilitate microbicidal chemistries or antigen processing.

Proteolytic activation of receptor-bound anthrax protective antigen on macrophages promotes its internalization.

Immunofluorescence and other methods have been used to probe the self-assembly and internalization of the binary toxin, anthrax lethal toxin (LeTx), in primary murine macrophages. Proteolytic activation of protective antigen (PA; 83 kDa, the B moiety of the toxin) by furin was the rate-limiting step in internalization of LeTx and promoted clearance of PA from the cell surface. A furin-resistant form of PA remained at the cell surface for at least 90 min. Oligomerization of receptor-bound PA63, the 63 kDa active fragment of PA, was manifested by its conversion to a pronase-resistant state, characteristic of the heptameric prepore form in solution. That oligomerization of PA63 triggers toxin internalization is supported by the observation that PA20, the complementary 20 kDa fragment of PA, inhibited clearance of nicked PA. The PA63 prepore, with or without lethal factor (LF), cleared slowly from the cell surface. These studies show that proteolytic cleavage of PA, in addition to permitting oligomerization and LF binding, also promotes internalization of the protein. The relatively long period of activation and internalization of PA at the cell surface may reflect adaptation of this binary toxin that maximizes self-assembly.

Early Bacillus anthracis-macrophage interactions: intracellular survival survival and escape.

This study describes early intracellular events occurring during the establishment phase of Bacillus anthracis infections. Anthrax infections are initiated by dormant endospores gaining access to the mammalian host and becoming engulfed by regional macrophages (Mphi). During systemic anthrax, late stage events include vegetative growth in the blood to very high titres and the synthesis of the anthrax exotoxin complex, which causes disease symptoms and death. Experiments focus on the early events occurring during the first few hours of the B. anthracis infectious cycle, from endospore germination up to and including release of the vegetative cell from phagocytes. We found that newly vegetative bacilli escape from the phagocytic vesicles of cultured Mphi and replicate within the cytoplasm of these cells. Release from the Mphi occurs 4-6 h after endospore phagocytosis, timing that correlates with anthrax infection of test animals. Genetic analysis from this study indicates that the toxin plasmid pXO1 is required for release from the Mphi, whereas the capsule plasmid pXO2 is not. The transactivator atxA, located on pXO1, is also found to be essential for release, but the toxin genes themselves are not required. This suggests that Mphi release of anthrax bacilli is atxA regulated. The putative 'escape' genes may be located on the chromosome and/or on pXO1.

Cell membrane orientation visualized by polarized total internal reflection fluorescence.

In living cells, variations in membrane orientation occur both in easily imaged large-scale morphological features, and also in less visualizable submicroscopic regions of activity such as endocytosis, exocytosis, and cell surface ruffling. A fluorescence microscopic method is introduced here to visualize such regions. The method is based on fluorescence of an oriented membrane probe excited by a polarized evanescent field created by total internal reflection (TIR) illumination. The fluorescent carbocyanine dye diI-C(18)-(3) (diI) has previously been shown to embed in the lipid bilayer of cell membranes with its transition dipoles oriented nearly in the plane of the membrane. The membrane-embedded diI near the cell-substrate interface can be fluorescently excited by evanescent field light polarized either perpendicular or parallel to the plane of the substrate coverslip. The excitation efficiency from each polarization depends on the membrane orientation, and thus the ratio of the observed fluorescence excited by these two polarizations vividly shows regions of microscopic and submicroscopic curvature of the membrane, and also gives information regarding the fraction of unoriented diI in the membrane. Both a theoretical background and experimental verification of the technique is presented for samples of 1) oriented diI in model lipid bilayer membranes, erythrocytes, and macrophages; and 2) randomly oriented fluorophores in rhodamine-labeled serum albumin adsorbed to glass, in rhodamine dextran solution, and in rhodamine dextran-loaded macrophages. Sequential digital images of the polarized TIR fluorescence ratios show spatially-resolved time-course maps of membrane orientations on diI-labeled macrophages from which low visibility membrane structures can be identified and quantified. To sharpen and contrast-enhance the TIR images, we deconvoluted them with an experimentally measured point spread function. Image deconvolution is especially effective and fast in our application because fluorescence in TIR emanates from a single focal plane.

Childhood farm injuries.

During a recent 6-year period (1991-1997), 143 children and adolescents less than 18 years of age were admitted to a Level I trauma center for agriculture-related trauma. Mechanized pieces of equipment were responsible for half of the injuries. The pattern of injury was clearly seasonal and a daytime occurrence. Half of the patients came from the scene and half from rural hospital emergency rooms, with only 25 per cent being transported via advanced life support. Rural geography led to both long distance (mean, 55 miles) and long transport time to definitive care (mean, 2 hours, 15 minutes). There was a predilection for fractures, amputations, head injuries, and soft-tissue infections. Operative intervention was required immediately in two-thirds, and one-third were admitted to an intensive care unit. Whereas hospital mortality was low at 1.4 per cent, most childhood farm deaths during the study period occurred in the field. Severe permanent disability was present in one-third of children, and 7 per cent incurred a second injury during the study period. Using this review, prevention programs have been developed to minimize death and disability in children sustaining farm injuries.

Ratiometric and fluorescence-lifetime-based biosensors incorporating cytochrome c' and the detection of extra- and intracellular macrophage nitric oxide.

Ratiometric and lifetime-based sensors have been designed for cellular detection of nitric oxide. These sensors incorporate cytochrome c', a hemoprotein known to bind nitric oxide selectively. The cytochrome c' is labeled with a fluorescent reporter dye, and changes in this dye's intensity or fluorescence lifetime are observed as the protein binds nitric oxide. The ratiometric sensors are composed of dye-labeled cytochrome c' attached to the optical fiber via colloidal gold, along with fluorescent microspheres as intensity standards. These ratiometric sensors exhibit linear response, have fast response times (< or = 0.25 s), and are completely reversible. The sensors are selective over numerous common interferents such as nitrite, nitrate, and oxygen species, and the limit of detection is 8 microM nitric oxide. The lifetime-based measurements are made using free, dye-labeled cytochrome c' in solution and have a limit of detection of 30 microM nitric oxide. The use of these two techniques has allowed measurement of intra- and extracellular macrophage nitric oxide. Employing the ratiometric fiber sensors gave a multicell culture average extracellular nitric oxide concentration of 210 +/- 90 microM for activated macrophages, while an average intracellular concentration of 160 +/- 10 microM was determined from the lifetime-based measurements of dye-labeled cytochrome c' in the macrophage cytosol. Microscopic adaptation of the lifetime-based methods described here would allow direct correlation of intracellular nitric oxide levels with specific cellular activities, such as phagocytosis.

A contractile activity that closes phagosomes in macrophages.

Studies of Fc-mediated phagocytosis by mouse macrophages identified a contractile activity at the distal margins of forming phagosomes. Time-lapse video microscopic analysis of macrophages containing rhodamine-labeled actin and fluorescein dextran showed that actin was concentrated at the distal margins of closing phagosomes. Phagocytosis-related contractile activities were observed when one IgG-opsonized erythrocyte was engaged by two macrophages. Both cells extended pseudopodia until they met midway around the erythrocyte. It was then constricted and pulled into two phagosomes, which remained interconnected by a string of erythrocyte membrane. Butanedione monoxime, an uncompetitive inhibitor of class II and perhaps other myosins, and wortmannin and LY294002, inhibitors of phosphoinositide 3-kinase, prevented the constrictions without inhibiting the initial pseudopod extension. Immunofluorescence microscopy showed the presence of myosins IC, II, V and IXb in phagosomes. Of these, only myosin IC was concentrated around the strings connecting shared erythrocytes, suggesting that myosin IC mediates the purse-string-like contraction that closes phagosomes. The sequential processes of pseudopod extension and contraction can explain how macropinosomes and spacious phagosomes form without guidance from a particle surface.

Multiple genetic controls on Rhizobium meliloti syrA, a regulator of exopolysaccharide abundance.

Exopolysaccharides (EPS) are produced by a wide assortment of bacteria including plant pathogens and rhizobial symbionts. Rhizobium meliloti mutants defective in EPS production fail to invade alfalfa nodules. Production of EPS in R. meliloti is likely controlled at several levels. We have characterized a new gene of this regulatory circuit. syrA was identified by its ability to confer mucoid colony morphology and by its ability to suppress the colonial phenotype of an exoD mutant. Here we show that syrA encodes a 9-kD hydrophobic protein that has sequence similarity to two other EPS regulatory proteins: ExoX of Rhizobium NGR234 and R. meliloti, and Psi of R. leguminosarum bv. phaseoli. The syrA transcription start site lies 522 nucleotides upstream of a non-canonical TTG start codon. The syrA promoter region is similar to the promoter region of the nodulation regulatory protein, nodD3. We found that in free-living bacteria, syrA expression is activated by the regulatory locus, syrM, but not by nodD3. In planta, syrM is not required for expression of syrA. Instead, expression of the nitrogen fixation (nifHDKE) genes upstream of syrA plays a role. Specific and distinct sets of genetic controls may operate at different times during nodule invasion.

pH-dependent perforation of macrophage phagosomes by listeriolysin O from Listeria monocytogenes.

The pore-forming toxin listeriolysin O (LLO) is a major virulence factor implicated in escape of Listeria monocytogenes from phagocytic vacuoles. Here we describe the pH-dependence of vacuolar perforation by LLO, using the membrane-impermeant fluorophore 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) to monitor the pH and integrity of vacuoles in mouse bone marrow-derived macrophages. Perforation was observed when acidic vacuoles containing wild-type L. monocytogenes displayed sudden increases in pH and release of HPTS into the cytosol. These changes were not seen with LLO-deficient mutants. Perforation occurred at acidic vacuolar pH (4.9-6.7) and was reduced in frequency or prevented completely when macrophages were treated with the lysosomotropic agents ammonium chloride or bafilomycin A1. We conclude that acidic pH facilitates LLO activity in vivo.

Use of a raw meat-based diet or a dry kibble diet for sand cats (Felis margarita).

Limited information is available on the utilization of different types of diets by captive exotic felid species. Utilization of diets by small exotic felids may differ depending on the diet fed. Eight sand cats (Felis margarita), which are small, 2- to 4-kg cats, were used to examine the digestibility of two types of diets: a raw meat-based diet and a dry kibble diet. Dry matter, crude protein and energy intakes and digestibilities were evaluated. Digestibilities for dry matter, energy, and crude protein were 83.5 +/- 4.8, 89.6 +/- 5.2, 92.4 +/- 5.3% for the raw meat-based diet and 72.7 +/- 12.3, 76.8 +/- 14.5, and 77.9 +/- 13.5% for the kibble diet. Physiological variables also were examined and included plasma taurine, vitamin A, retinyl palmitate, beta-carotene, calcium, and phosphorus. Plasma taurine means were 91.4 +/- 8.4 mumol/L in cats consuming the raw meat-based diet and 248.0 +/- 23.2 mumol/L in cats consuming the kibble diet. Plasma phosphorus was 5.2 +/- .1 and 4.5 +/- .1 mg/dL, respectively, in cats consuming raw meat-based and kibble diets. beta-Carotene was 25.2 +/- 2.9 and 2.9 +/- .3 micrograms/dL, respectively, for cats consuming the raw meat-based and kibble diets. These results indicate that diets formulated for small captive exotic felid species should be evaluated with respect to diet type and nutrient utilization.

The efficiency of antigen delivery from macrophage phagosomes into cytoplasm for MHC class I-restricted antigen presentation.

Macrophages can present exogenous antigen in association with MHC class I molecules. Indirect evidence indicates that antigens internalized by phagocytosis can enter cytoplasm before following the conventional MHC class I presentation pathway. However, little is known about how common such entry is, or to what extent it depends on the kind of particle ingested. This study reports quantitative and morphological characterization of antigen delivery from phagosomes into cytoplasm for MHC class I-restricted antigen presentation. Ovalbumin (OVA) was associated with polystyrene particles (PS), biodegradable poly-e-caprolactone particles (PCL), and sheep red blood cells (SRBC), and its delivery into macrophage cytoplasm, via phagocytosis was assessed with a T hybridoma assay for MHC class I-restricted presentation of OVA-derived peptides. Although direct introduction of antigen into cytoplasm by scrape-loading produced the most efficient presentation, comparable signals could be obtained after phagocytosis of PCL or PS. Phagocytosis of OVA-loaded SRBC, and OVA internalized by pinocytosis, did not deliver efficiently. MHC class I-restricted presentation of phagosome-derived OVA required cytoplasmic processing, as it was inhibited by proteasome inhibitors and brefeldin A. Morphological studies showed that biotinylated OVA originating in PCL phagosomes could be delivered into the cytoplasm of 90% of the macrophages. These results indicate that phagocytosis per se is not sufficient to deliver antigen into cytoplasm, but that phagocytosis of solid, synthetic polymeric particles delivers antigen efficiently into cytoplasm for MHC class I processing.

The impact of caffeine use on tobacco cessation and withdrawal.

Continuous caffeine consumption with smoking cessation has been associated with more than doubled caffeine plasma levels. Such concentrations may be sufficient to produce caffeine toxicity symptoms in smoking abstinence conditions. To test whether caffeine abstinence influences smoking cessation, 162 caffeine-using smokers were enlisted from American Lung Association smoking cessation programs. Volunteers were randomly assigned by clinic to caffeine-use and caffeine-abstinence conditions and measured for 3 weeks post-smoking cessation, at 6 months and one year. Results showed a significant linear increase in caffeine sputum levels across 3 weeks post cessation for those who quit smoking and continued using caffeine. Three weeks after cessation, concentrations reached 203% of baseline for the caffeine user. Typical nicotine withdrawal symptoms occurred during the first 16 days of cessation. The caffeine abstainers, but not continued users of caffeine, reported increased fatigue during the first 3 days of cessation. Among complete caffeine abstainers, compared with caffeine users, there was a significant increase in fatigue, a decrease in stimulation, and a marginal increase in caffeine craving immediately following tobacco cessation. There were no differences between the groups on other withdrawal symptoms or in cessation success at 16 days, 6 months, or 12 months.

A role for phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages.

Phosphoinositide 3-kinase (PI 3-kinase) has been implicated in growth factor signal transduction and vesicular membrane traffic. It is thought to mediate the earliest steps leading from ligation of cell surface receptors to increased cell surface ruffling. We show here that inhibitors of PI 3-kinase inhibit endocytosis in macrophages, not by interfering with the initiation of the process but rather by preventing its completion. Consistent with earlier studies, the inhibitors wortmannin and LY294002 inhibited fluid-phase pinocytosis and Fc receptor-mediated phagocytosis, but they had little effect on the receptor-mediated endocytosis of diI-labeled, acetylated, low density lipoprotein. Large solute probes of endocytosis reported greater inhibition by wortmannin than smaller probes did, indicating that macropinocytosis was affected more than micropinocytosis. Since macropinocytosis and phagocytosis are actin-mediated processes, we expected that their inhibition by wortmannin resulted from deficient signaling from macrophage colony-stimulating factor (M-CSF) receptors or Fc receptors to the actin cytoskeleton. However, video microscopy showed cell surface ruffling in wortmannin-treated cells, and increased ruffling after addition of M-CSF or phorbol myristate acetate. Quantitative measurements of video data reported slightly diminished ruffling in wortmannin-treated cells. Remarkably, the ruffles that formed in wortmannin-treated macrophages all receded into the cytoplasm without closing into macropinosomes. Similarly, wortmannin and LY294002 did not inhibit the extension of actin-rich pseudopodia along IgG-opsonized sheep erythrocytes, but instead prevented them from closing into phagosomes. These findings indicate that PI 3-kinase is not necessary for receptor-mediated stimulation of pseudopod extension, but rather functions in the closure of macropinosomes and phagosomes into intracellular organelles.

Rapid and complete fusion of macrophage lysosomes with phagosomes containing Salmonella typhimurium.

The virulence of Salmonella typhimurium for mice results, in part, from its ability to survive after phagocytosis by macrophages. Although it is generally agreed that intracellular bacteria persist in membrane-bound phagosomes, there remains some question as to whether these phagosomes fuse with macrophage lysosomes. This report describes the maturation of phagosomes containing S. typhimurium inside mouse bone marrow-derived macrophages. Macrophages were infected briefly and incubated for various intervals; then they were examined by fluorescence microscopy for colocalization of bacteria with lysosomal markers. These markers included LAMP-1, cathepsin L, and fluorescent proteins or dextrans preloaded into lysosomes by endocytosis. By all measures, phagosomes containing S. typhimurium merged completely with the lysosomal compartment within 20 min of phagocytosis. The rate of phagosome-lysosome fusion was similar to the rate for phagocytosed latex beads. Phagolysosomes remained accessible to fluid-phase probes and contained lysosomal markers for many hours. Moreover, a large percentage of the wild-type bacteria that were viable 20 min after infection survived longer incubations inside macrophages, indicating that the survivors were not a minor subpopulation that avoided phagosome-lysosome fusion. Therefore, we conclude that S. typhimurium survives within the lysosomal compartments of macrophages.

Delivery of macromolecules into cytosol using liposomes containing hemolysin from Listeria monocytogenes.

The cytosolic space of cells is an important but relatively inaccessible target for the delivery of therapeutic macromolecules. Here we describe the efficient delivery of macromolecules into the cytosolic space of macrophages from liposomes that contain listeriolysin O (LLO), the hemolytic protein of Listeria monocytogenes that normally mediates bacterial passage from phagosomes into cytosol. LLO was purified and encapsulated inside pH-sensitive liposomes, along with other molecules to be delivered. When internalized by bone marrow-derived macrophages, these liposomes rapidly released encapsulated fluorescent dye, first into endosomes and then into the cytosol, without measurably harming the cells. Furthermore, these liposomes efficiently delivered encapsulated ovalbumin to the cytosolic pathway of antigen processing and presentation, as measured by the major histocompatibility complex (MHC) class I-restricted presentation of peptides derived from ovalbumin. Delivery was significantly better than that obtained by other currently available liposome formulations. LLO-containing liposomes should therefore provide an efficient vehicle for delivery of antigens or therapeutic molecules in vivo.

Different fates of phagocytosed particles after delivery into macrophage lysosomes.

Phagocytosis in macrophages is often studied using inert polymer microspheres. An implicit assumption in these studies is that such particles contain little or no specific information in their structure that affects their intracellular fate. We tested that assumption by examining macrophage phagosomes containing different kinds of particles and found that although all particles progressed directly to lysosomes, their subsequent fates varied. Within 15 min of phagocytosis, >90% of phagosomes containing opsonized sheep erythrocytes, poly-e-caprolactone microspheres, polystyrene microspheres (PS), or polyethylene glycol-conjugated PS merged with the lysosomal compartment. After that point, however, the characteristics of phagolysosomes changed in several ways that indicated differing degrees of continued interaction with the lysosomal compartment. Sheep erythrocyte phagolysosomes merged together and degraded their contents quickly, poly-e-caprolactone phagolysosomes showed intermediate levels of interaction, and PS phagolysosomes became isolated within the cytoplasm. PS were relatively inaccessible to an endocytic tracer, Texas red dextran, added after phagocytosis. Moreover, immunofluorescent staining for the lysosomal protease cathepsin L decreased in PS phagolysosomes to 23% by 4 h after phagocytosis, indicating degradation of the enzyme without replacement. Finally, PS surface labeled with fluorescein-labeled albumin showed a markedly reduced rate of protein degradation in phagolysosomes, when compared to rates measured for proteins in or on other particles. Thus, particle chemistry affected both the degree of postlysosomal interactions with other organelles and, consequently, the intracellular half-life of particle-associated proteins. Such properties may affect the ability of particles to deliver macromolecules into the major histocompatibility complex class I and II antigen presentation pathways.

Concise review for primary-care physicians.

Acute otitis media (AOM) in young children consumes a substantial amount of medical care services provided by primary-care physicians. A recent increase in the number of young children with AOM prompted a review of the associated risk factors. Eustachian tube dysfunction, bacterial colonization, and host inflammatory response form the basis for the development of AOM. Signs and symptoms of AOM in young children are often nonspecific and subtle, particularly in infants. Physical examination and pneumatic otoscopy verify the diagnosis. New modalities including tympanometry and acoustic reflectometry may be helpful. Amoxicillin remains the drug of choice for AOM, despite recent trends in microbial resistance. Second- and third-line antimicrobial agents might be considered in selected clinical settings. Young children with recurrent episodes of otitis media must be monitored closely. Preventive measures and medical or surgical intervention should be considered in order to minimize the long-term medical and developmental effects of AOM.

Microtubules can modulate pseudopod activity from a distance inside macrophages.

Microtubules are thought to influence cell shape as structural components of an integrated cytoskeletal matrix. Here we show that microtubules can affect the dynamics of macrophage pseudopodia without being integrated into their structure. Macrophages landing on glass surfaces spread within 15 min into flattened circular cells with radial symmetry, and the radial distribution of microtubules reflected this symmetry. Depolymerization of microtubules using nocodazole, colchicine, or vinblastine did not inhibit macrophage spreading or the early establishment of radial symmetry. Shortly after spreading, however, macrophages without microtubules gradually became asymmetric, assuming irregular, lobed profiles. The asymmetry resulted from exaggerated protrusion and retraction of pseudopodia, with net retraction overall. This loss of radial symmetry could be inhibited by treatment of initially spread cells with cytochalasin D, indicating that the change in cell shape was mediated by the actin cytoskeleton. Intact microtubules suppressed the exaggerated pseudopod movements, even when they were separated by a distance from the cell margin. In cells treated with taxol, microtubules remained clustered near the cell center after spreading, yet the dynamics of pseudopodia at the cell margin were reduced and cells maintained a circular profile. Similarly, in cells treated with low concentrations of nocodazole, a much reduced microtubule cytoskeleton nonetheless suppressed pseudopod dynamics. We propose that microtubules act to stabilize cell shape at a distance from the cell edge via a biochemical intermediate that affects the structure or function of the microfilament system.

Macropinocytosis.

Macropinocytosis is a form of endocytosis that accompanies cell surface ruffling. It is distinct in many ways from the better characterized micropinocytosis, which includes clathrin-coated vesicle endocytosis and small uncoated vesicles. Because macropinosomes are relatively large, they provide an efficient route for non-selective endocytosis of solute macromolecules. This route may facilitate MHC-class-II-restricted antigen presentation by dendritic cells. Because the ruffling that leads to macropinocytosis is regulated, it has been exploited by some pathogenic bacteria as a novel route for entry into cells.