Kinetics of Phagocytosis and Intracellular Killing of Staphytococcus aureus and Escherichia coli by Human Monocytes

Tie kinetic patterns of the phagocytosis and intracellular killing of Staphytococcus and Echerichia coli by monocytes were investigated separately to acquire more insight into the total process, i.e. from the ingestion to the death of the micro-organisms. Phagocytosis proved to be dependent on: (1) both the bacteria-to-monocyte ratio and the monocyte concentration; a concentration of at least 5 × 10⁵ monocytes/ml proved necessary for the measurement of ingestion, whereas the rate of ingestion was found to be proportional to the number of extracellular bacteria until a maximum rate is reached, (2) the serum concentration in the incubation medium, which influenced both the rate of phagocytosis and the maximum number of bacteria taken up by one monocyte, and (3) the temperature, the highest rate of phagocytosis being reached at 37–41°C The intracellular killing proved to be dependent on: (1) the number of bacteria ingested; the rate of killing was proportional to the number of ingested bacteria until a maximum rate was reached; (2) the temperature, since a maximum rate of killing is only reached at 37–41°C: at tower and higher temperatures the rate of killing is lower, in the latter case due to inactivation of extracellular stimuli. These separate data on the ingestion and killing processes made it possible to compute the theoretical numbers of extracellular, viable intracellular, and total intracellular bacteria for a model system consisting of 5×10⁶ monocytes, 5×10⁶ bacteria, and 10% serum. These calculated values are in agreement with the experimental data.     

Participation of C3 in Intracellular Killing of Candida albicans

Using new objective methods for measuring, independently, phagcytosis and killing, it was demonstrated that Candida albicans opsonized by C3-deficient serum was ingested by not killed in vitro by human polymorphonuclear leukocytes. Killing could be induced by adding purified C3 to the C3-deficient serum. It is concluded that C3 participates directely in the intracellular process leading to phagocytic killing of C. albicans.     

Trapping and Killing of Candida albicans by Corynebacterium parvum-Activated Livers

Corynebacterium parvum vaccination significantly increased the number of leukocytes adherent to hepatic vessels. Perfused C. parvum-treated livers killed significantly more Candida albicans than did livers not treated with C. parvum, an effect reversed by the macrophage inhibitors silica, phenylbutazone, and iodoacetate.     

Phagocytosis and Killing of Streptococcus pyogenes by Human Alveolar Macrophages

In contrast to human polymorphonuclear leukocytes and monocytes, alveolar macrophages were able to readily phagocytose and kill an M protein-positive Streptococcus pyogenes strain after opsonization in normal human serum.     

Phagocytosis of Candida albicans by concanavalin-A activated peritoneal macrophages.

We evaluated the effect of treatment of mice with concanavalin-A (Con-A) on the phagocytosis of glutaraldehyde-fixed Candida albicans by peritoneal macrophages. The mean number of unopsonized C. albicans blastoconidia phagocytosed in vitro by peritoneal macrophages was doubled (from 1.3+/-0.1 to 2.7+/-0.14) by pre-treatment of the donor mice with Con-A. The percent of peritoneal cells phagocytosing the blastoconidia in vitro was increased about four times (from 22.3+/-8.6 to 80.3+/-3.2) by Con-A. This increase in phagocytosis was about 50% inhibited by addition of mannan (50 microg) plus mannose (50 mM) to the assay medium, suggesting that it was mediated by mannose receptors (MR). Phagocytosis in vitro in the presence of fresh non-immune serum (5%) was also increased, from 84.3+/-5.0 for untreated macrophages to 100% for Con-A activated peritoneal macrophages and the mean number of opsonized C. albicans blastoconidia increased from 2.3+/-0.1 to 4. 6+/-0.1. These results suggest that treatment of mice with Con-A increased both the phagocytosis of C. albicans blastoconidia mediated by mannose receptors and by complement receptors.     

Phagocytosis and Killing of Francisella tularensis by Human Polymorphonuclear Leukocytes

Bacteria of a wild strain of Francisella tularensis were less efficiently killed by human polymorphonuclear leukocytes than were bacteria of an attenuated strain. This finding was explained to some extent by a less efficient phagocytosis, but bacteria of the wild strain also seemed to be more resistant to killing after ingestion.     

Phagocytosis measured as inhibition of uridine uptake by Candida albicans.

Inhibition of 3H-uridine incorporation into Candida albicans can be used as a sensitive index of phagocytic function because: 1) there is a linear correlation between uridine incorporation and yeast number; 2) phagocytic cells do not incorporate significant amounts of uridine in short term cultures; and 3) C. albicans replicating inside phagocytic cells does not take up uridine from culture medium. Appropriate conditions for measuring phagocytic capacity of human polymorphonuclear cells (PMN's) were 5 x 10(5) C. albicans and 5 x 10(4) PMNs in 0.5 ml of medium containing 2.5% AB serum. This mixture was incubated for 30 min at 37 degrees C. Aliquots were then transferred into microtiter wells and incubated for a further 60 min in the presence of 3H-uridine. Under these conditions PMN leucocytes from 25 healthy individuals caused suppression of uridine incorporation ranging from 33 to 75% (50 +/- 12).     

Pertussis Toxin and Lipopolysaccharide Influence Phagocytosis of Bordetella pertussis by Human Monocytes

The potential of human monocytes to mediate the clearance of Bordetella pertussis infection was examined. Bacteria expressing green fluorescent protein were incubated with adherent peripheral blood monocytes, and phagocytosis was quantified by using fluorescence microscopy. Monocytes internalized only a small percentage of the adherent bacteria. Surface-associated Bvg-regulated virulence factors, including adenylate cyclase toxin and filamentous hemagglutinin, did not affect attachment or phagocytosis. However, 1-h pretreatment with purified pertussis toxin inhibited the ability of monocytes to internalize wild-type bacteria. Mutations affecting the terminal trisaccharide of lipopolysaccharide resulted in reduced internalization without affecting adherence of bacteria to monocytes. Opsonization with human serum played only a modest role in promoting phagocytosis. The viability of internalized bacteria was determined by colony counts following treatment with polymyxin B and gentamicin. Less than 1% of internalized bacteria remained viable. These results suggest that pertussis toxin plays a role in the evasion of monocyte phagocytosis and that these cells represent a potential mediator of the clearance of B. pertussis infection.     

Electron Microscopy of Group A Streptococci After Phagocytosis by Human Monocytes

Group A streptococci were added to cultures of isolated human blood monocytes. The bacteria were readily sequestered within phagocytic vacuoles after being coated with flocculent material, apparently derived from the plasma-containing medium. Progressive lysis of intravacuolar streptococci was observed, characterized by plasmolysis, internal disruption, and eventual plasma membrane dissolution. However, the cell walls remained essentially identical to those of nonphagocytized streptococci, showing no signs of dissolution within the limited in vitro survival time of the monocytes. These results indicate that streptococcal cell walls may persist in migrating human phagocytes in vivo and may be deposited in body tissues. This cell wall material, known to be toxic to animal tissues, may be an important determinant in the pathogenesis of poststreptococcal sequelae in man.     

Phagocytosis of Candida albicans by alveolar macrophages from patients with cystic fibrosis

Cystic fibrosis (CF) children suffer from chronic and progressive lung disease characterized in most cases by persistent Pseudomonas aeruginosa colonization. In the present work, a kinetic assay using CF alveolar macrophages (CF-AM) and polymorphonuclear leukocytes (PMN) was designed in order to investigate the effect of CF serum on the phagocytic function of these cells. Bronchopulmonary lavages were performed on eight CF children, eight non-CF children with infectious lung disease, and five normal children who were diagnosed as having a foreign body in the air passages, without infection. Serum samples were taken from eight CF and eight normal children; all the controls were age and sex matched with CF patients. The CF serum had no effect on the kinetics of phagocytosis of Candida albicans by CF and control AM and PMN. A suggestive finding was the decrease in the Michaelis-Menten constant (K_m) for the interaction of CF-AM with Candida, measured as the abscissa to the origin of a Lineweaver-Burk plot. The Michaelis-Menten constant obtained from the challenge of AM with yeasts in a medium containing normal fresh serum was 9.28 for the noninfected controls, 2.86 for the infected controls, and 21.38 for the CF patients; the last was significantly different from both controls (P < 0.01).     

Phagocytosis by Human Monocytes of Particles Activating the Alternative Pathway of Complement

The phagocytosis of particles activating the alternative pathway of complement by human monocytes cultured under serum-free conditions was studied. In contrast to native zymosan particles, which were easily ingested, rabbit erythrocytes and agarose beads had to be coated with C3b or C3bi to be engulfed by the monocytes. The binding and ingestion by monocytes of particles coated with C3bi were greater than for the same particles coated with the equivalent amount of C3b. The binding and uptake of rabbit erythrocytes and agarose beads were proportional to the amount of C3b or C3bi on the particles. In contrast to the complement activator particles, C3b- and C3bi-coated sheep erythrocytes, which are non-activators, were not ingested by the monocytes, although attachment to the monocytes took place. The presence of methylamine or cobra venom factor, which are complement inhibitors, strongly reduced the ingestion of native zymosan by the monocytes, whereas the uptake of C3b- or C3bi-coated zymosan particles were only weakly affected. This suggests that the binding of native zymosan to monocytes is sensitive to interference from a cell-derived alternative pathway C3 convertase (C3bBb). Binding and uptake of activators by human monocytes via complement receptor(s) are discussed.     

Diazepam Inhibits Phagocytosis and Killing Exerted by Polymorphonuclear Cells and Monocytes from Healthy Donors. In Vitro Studies

The effect of a benzodiazepine (BDZ), diazepam on human polymorphonuclear cell (PMN) and monocyte pha gocytosis and killing from healthy volunteers has been evaluated. Diazepam is able to inhibit in vitro both functions exerted by PMN and monocytes at 10^-5 and 10^-6 M concentrations/ 4 × 10⁶ phagocytes. 10^-7 M con centration was not effective in all the instances.

These results are discussed for their possible clinical implications, since previous studies have shown that in patients with phobic disorder there is evidence for reduced phagocytosis and killing capacities.     

Toll-Like Receptor Stimulation Enhances Phagocytosis and Intracellular Killing of Nonencapsulated and Encapsulated Streptococcus pneumoniae by Murine Microglia

Toll-like receptors (TLRs) are crucial pattern recognition receptors in innate immunity that are expressed in microglia, the resident macrophages of the brain. TLR2, -4, and -9 are important in the responses against Streptococcus pneumoniae, the most common agent causing bacterial meningitis beyond the neonatal period. Murine microglial cultures were stimulated with agonists for TLR1/2 (Pam₃CSK₄), TLR4 (lipopolysaccharide), and TLR9 (CpG oligodeoxynucleotide) for 24 h and then exposed to either the encapsulated D39 (serotype 2) or the nonencapsulated R6 strain of S. pneumoniae. After stimulation, the levels of interleukin-6 and CCL5 (RANTES [regulated upon activation normal T-cell expressed and secreted]) were increased, confirming microglial activation. The TLR1/2, -4, and -9 agonist-stimulated microglia ingested significantly more bacteria than unstimulated cells (P < 0.05). The presence of cytochalasin D, an inhibitor of actin polymerizaton, blocked >90% of phagocytosis. Along with an increased phagocytic activity, the intracellular bacterial killing was also increased in TLR-stimulated cells compared to unstimulated cells. Together, our data suggest that microglial stimulation by these TLRs may increase the resistance of the brain against pneumococcal infections.Immunocompromised patients have a higher risk of developing bacterial infections in the central nervous system (CNS) (34, 37, 42). The list of the pathogens includes many organisms with low pathogenicity in the immunocompetent host (34, 37). Moreover, the distribution of the pathogens also differs from the immunocompetent host and depends on the nature of the immune defect. Patients with a decrease in B-lymphocyte function or with a loss of splenic function have an increased risk of meningitis caused by encapsulated bacteria, while patients with an impaired T-lymphocyte-macrophage system are more susceptible to CNS infections caused by intracellular pathogens (7, 42). One additional cause of this increased susceptibility to CNS infections probably is a decreased local immune defense (33).CNS infections not only are more frequent but also are associated with higher mortality rates and more severe long-term sequelae in immunocompromised than in immunocompetent individuals (9, 17, 34, 44). Polymicrobial infections, multiple organ system presentation, and the absence of typical clinical manifestations subsequent to the host''s diminished inflammatory response are challenging aspects in the management of these infections (34, 37, 42).The brain tissue shows a well-organized innate immune reaction in response to bacteria in the cerebrospinal fluid (CSF) (3, 21). Microglial cells, the resident phagocytes of the CNS, express Toll-like receptors (TLRs) that identify pathogen-associated molecular patterns (PAMPs) (41). The receptor-ligand interactions activate microglia to undergo morphological transformation as well as functional changes, such as the production of proinflammatory cytokines, chemokines, and reactive oxygen species, enhanced phagocytic activity, and antigen presentation (15, 39). This immune reaction cannot eliminate high amounts of pneumococci from the CSF but does prevent or minimize the invasion of these pathogens into the brain tissue, thereby limiting tissue destruction and neuronal injury.TLR2, -4, and -9 contribute to the recognition and response to Streptococcus pneumoniae in the CNS (31). A deficiency of TLR2, -4, or -9 or of the coreceptor CD14, which is necessary for TLR4 signaling increases the susceptibility of mice to S. pneumoniae (1, 11, 12, 40).Here, we hypothesized that activation of the innate immune response in microglia could increase the resistance of the brain tissue against CNS pneumococcal infections (14). This may be of particular interest in immunocompromised patients, whose outcome after S. pneumoniae meningitis is worse than that of immunocompetent individuals (9, 44). The aim of the present study was to investigate whether the stimulation of microglia by respective PAMPs can increase their ability to phagocytose and kill intracellular nonencapsulated and encapsulated S. pneumoniae strains, thereby protecting the brain during meningitis. Moreover, by using an encapsulated and a nonencapsulated pneumococcal strain, we assessed the protective effect of the capsule against phagocytosis by microglial cells.     

Influence of Intermittent Hyperglycemic Glucose Levels on the Phagocytosis of Microorganisms by Human Granulocytes in Vitro

Repeated brief exposures to glucose in concentrations of 0.8g/100 ml significantly depresses in vitro phagocytic ingestion of opsonized Gram-positive and Gram-negative bacteria by human peripheral polymorphonuclear granulocytes (PMN's), while the same concentration of L-aminoacid mixtures (Freamine) under the same conditions has no influence on it.     

Influence of Intermittent Hyperglycemic Glucose Levels on the Phagocytosis of Microorganisms by Human Granulocytes in Vitro

Repeated brief exposures to glucose in concentrations of 0.8g/100 ml significantly depresses in vitro phagocytic ingestion of opsonized Gram-positive and Gram-negative bacteria by human peripheral polymorphonuclear granulocytes (PMN's), while the same concentration of L-aminoacid mixtures (Freamine) under the same conditions has no influence on it.     

Suppression of Candida albicans by Human Oral Streptococci in Gnotobiotic Mice

Mixed human salivary bacteria and strains of Streptococcus salivarius and S. miteor suppressed colonization of Candida albicans in gnotobiotic mice. C. albicans attached in lower numbers to epithelial cells from conventional rats than from germ-free rats, and attachment inhibition by indigenous flora may explain in part the suppression of Candida colonization.     

A Low Concentration of Ethanol Reduces the Chemiluminescence of Human Granulocytes and Monocytes but Not the Tumor Necrosis Factor Alpha Production by Monocytes after Endotoxin Stimulation

The ability of polymorphonuclear neutrophils (PMNs) and monocytes (M) to produce reactive oxygen species (ROS) has been related closely to their potential in the killing of microorganisms. Ethanol has been shown to impair the generation of ROS in these phagocytes after stimulation with some immunogens and to increase the susceptibility of alcohol abusers to infectious diseases. As endotoxemia is common in alcohol abusers, we investigated the effect of ethanol (21.7 mmol/liter) on the luminol-amplified chemiluminescence of PMNs and M after endotoxin stimulation and the release of tumor necrosis factor alpha (TNF-α) from M. Further, the efficiency of ethanol to inactivate chemically generated ROS was tested. Significant stimulation of ROS release occurred at endotoxin concentrations of 1 ng/ml or higher in both PMNs and M. Ethanol significantly suppressed the formation of ROS in both cell types, the decrease being more pronounced in M (−73.8%) than in PMNs (−45.7%). The correlations between endotoxin concentration and the amount of released ROS showed a dose-dependent, sigmoidal course. Concentrations of endotoxin necessary for half-maximum stimulation were nearly identical (6 to 8 ng/ml) in both PMNs and M, independent of the presence of ethanol. In contrast to ROS formation, ethanol had no effect on the amount of TNF-α produced by endotoxin-stimulated M. Ethanol was shown to be unable to decrease the levels of chemically generated ROS under physiological conditions. Therefore, ethanol cannot be assumed to be an “antioxidative” compound but rather seems to modify processes of endotoxin recognition, intracellular signal transduction, or metabolism.     

Altered Phospholipid Metabolism in Escherichia coli Accompanying Killing by Disrupted Granulocytes

The effect of bactericidal concentrations of disrupted rabbit granulocytes and of partially purified granulocyte fractions on phospholipid metabolism by Escherichia coli has been investigated. Previous studies in this laboratory have shown that, during and after killing of E. coli by granulocytes, bacterial macromolecular synthesis continues. Similarly, despite almost complete loss of viability within 15 min, incorporation of [1-¹⁴C]palmitate, [2-¹⁴C]glycerol, and [1-¹⁴C]acetate into E. coli phospholipids, in the presence of granulocyte preparations, remains the same as in control E. coli populations for at least 1 h. Incorporation of [1-¹⁴C]oleate into E. coli phospholipids is actually stimulated during the first 60 min of incubation in the presence of granulocyte preparations (more than twofold at 30 min and 40% at 60 min). With all labeled lipid precursors, bactericidal granulocyte preparations cause a relative increase in the labeling of E. coli cardiolipin, with a corresponding drop in labeled phosphatidyl-glycerol. Labeled lyso-compounds accumulate in the presence of granulocyte preparations when [1-¹⁴C]palmitate, but not when [1-¹⁴C]oleate is the labeled precursor. Since oleate occurs mainly in the 2-acyl position of E. coli phospholipids, whereas at least 50% of palmitate occurs in the 1 position, it appears that a phospholipase A₂ acts on the E. coli phospholipids. These various effects are also seen when E. coli are exposed to highly purified granulocyte preparations that possess potent bactericidal and phospholipase A₂ activities. We speculate that this phospholipase A₂ in the granulocyte preparations stimulates oleate but not palmitate incorporation by initiating increased turnover of the fatty acid in the 2-acyl position of E. coli phospholipids, causing formation of 1-acyl lyso-compounds likely to be preferentially reacylated with unsaturated fatty acids.