Human Leukocyte Phagocytosis Research Articles

A newly synthesized 17-epi-NeuroProtectin D1/17-epi-Protectin D1: Authentication and functional regulation of Inflammation-Resolution

The production of specialized pro-resolving mediators (SPMs) during the resolution phase in the inflammatory milieu is key to orchestrating the resolution of the acute inflammatory response. 17-epi-neuroprotectin D1/17-epi-protectin D1 (17-epi-NPD1/17-epi-PD1: 10R,17R-dihydroxy-4Z,7Z,11E,13E,15Z,19Z-docosahexaenoic acid) is an SPM of the protectin family, biosynthesized from docosahexaenoic acid (DHA), that exhibits both potent anti-inflammatory and neuroprotective functions. Here, we carried out a new commercial-scale synthesis of 17-epi-NPD1/17-epi-PD1 that enabled the authentication and confirmation of its potent bioactions in vivo and determination of its ability to activate human leukocyte phagocytosis. We provide evidence that this new synthetic 17-epi-NPD1/17-epi-PD1 statistically significantly increases human macrophage uptake of E. coli in vitro and confirm that it limits neutrophilic infiltration in vivo in a murine model of peritonitis. The physical properties of the new synthetic 17-epi-NPD1/17-epi-PD1, namely its ultra-violet absorbance, chromatography, and tandem mass spectrometry fragmentation pattern, matched those of the originally synthesized 17-epi-NPD1/17-epi-PD1. In addition, we verified the structure and complete stereochemical assignment of this new synthetic 17-epi-NPD1/17-epi-PD1 using nuclear magnetic resonance (NMR) spectroscopy. Together, these results authenticate this 17-epi-NPD1/17-epi-PD1 for its structure and potent pro-resolving functions.

Pseudomonas aeruginosa N-3-oxo-dodecanoyl-homoserine Lactone Elicits Changes in Cell Volume, Morphology, and AQP9 Characteristics in Macrophages.

Quorum sensing (QS) communication allows Pseudomonas aeruginosa to collectively control its population density and the production of biofilms and virulence factors. QS signal molecules, like N-3-oxo-dodecanoyl-L-homoserine lactone (3O-C12-HSL), can also affect the behavior of host cells, e.g., by modulating the chemotaxis, migration, and phagocytosis of human leukocytes. Moreover, host water homeostasis and water channels aquaporins (AQP) are critical for cell morphology and functions as AQP interact indirectly with the cell cytoskeleton and signaling cascades. Here, we investigated how P. aeruginosa 3O-C12-HSL affects cell morphology, area, volume and AQP9 expression and distribution in human primary macrophages, using quantitative PCR, immunoblotting, two- and three-dimensional live imaging, confocal and nanoscale imaging. Thus, 3O-C12-HSL enhanced cell volume and area and induced cell shape and protrusion fluctuations in macrophages, processes tentatively driven by fluxes of water across cell membrane through AQP9, the predominant AQP in macrophages. Moreover, 3O-C12-HSL upregulated the expression of AQP9 at both the protein and mRNA levels. This was accompanied with enhanced whole cell AQP9 fluorescent intensity and redistribution of AQP9 to the leading and trailing regions, in parallel with increased cell area in the macrophages. Finally, nanoscopy imaging provided details on AQP9 dynamics and architecture within the lamellipodial area of 3O-C12-HSL-stimulated cells. We suggest that these novel events in the interaction between P. aeruginosa and macrophage may have an impact on the effectiveness of innate immune cells to fight bacteria, and thereby resolve the early stages of infections and inflammations.

The effect of serum opsonins on the phagocytosis of Staphylococcus aureus and zymosan particles, measured by flow cytometry.

Human leukocyte phagocytosis of S. aureus and zymosan particles was measured by flow cytometry (FCM). Killing of bacteria was measured by a standard microbiological method. When pooled human serum was heated to 56 degrees C for 30 min, the percentage of phagocytosing polymorphonuclear neutrophilic leukocytes (PMNLs), the rate of phagocytosis and killing of S. aureus by the whole leukocyte population were reduced to about 50% of the control values. The results indicate that the impaired phagocytosis and killing were due to the lack of attachment of bacteria opsonized with heat-stabile serum opsonins, mainly IgG, to 50% of the PMNLs. A prey-predator model was used to compare phagocytosis of S. aureus in heated and control serum. The rate of phagocytosis by those PMNLs that were active in heated serum was the same as that of all the leukocytes in control serum, suggesting that heat-labile serum opsonins did not affect the rate of phagocytosis by these PMNLs. PMNLs and monocytes phagocytosed zymosan particles in control serum, but in heated serum only a fraction of the leukocytes, corresponding to the fraction of monocytes phagocytosed. Thus, all PMNLs seem to be capable of phagocytosis by heat-labile serum opsonins. The combined use of S.aureus and zymosan particles may be of advantage in rapid screening of serum opsonin activities and phagocyte function in infectious and haematological disorders.

Group B Streptococcus suppression of phagocyte functions by protein-mediated engagement of human Siglec-5

Group B Streptococcus (GBS) is a leading cause of invasive bacterial infections in human newborns. A key GBS virulence factor is its capsular polysaccharide (CPS), displaying terminal sialic acid (Sia) residues which block deposition and activation of complement on the bacterial surface. We recently demonstrated that GBS Sia can bind human CD33-related Sia-recognizing immunoglobulin (Ig) superfamily lectins (hCD33rSiglecs), a family of inhibitory receptors expressed on the surface of leukocytes. We report the unexpected discovery that certain GBS strains may bind one such receptor, hSiglec-5, in a Sia-independent manner, via the cell wall–anchored β protein, resulting in recruitment of SHP protein tyrosine phosphatases. Using a panel of WT and mutant GBS strains together with Siglec-expressing cells and soluble Siglec-Fc chimeras, we show that GBS β protein binding to Siglec-5 functions to impair human leukocyte phagocytosis, oxidative burst, and extracellular trap production, promoting bacterial survival. We conclude that protein-mediated functional engagement of an inhibitory host lectin receptor promotes bacterial innate immune evasion.

Dose-dependent influence of barbiturates but not of propofol on human leukocyte phagocytosis of viable Staphylococcus aureus

Deep sedation with barbiturates or propofol is a standard therapy for patients with critically elevated intracranial pressure. Such patients are prone to infectious complications, especially to pneumonias, which are most commonly caused by Staphylococcus aureus. Although various immunomodulatory effects of barbiturates have been described in vitro, their influence on the phagocytosis of viable S. aureus has yet to be investigated. Therefore, we examined the effects of thiopentone, methohexitone, and propofol on the phagocytosis of viable S. aureus. Laboratory study. University laboratory. Ten healthy volunteers aged 32.5 +/- 7 yrs. Blood sampling. Whole blood samples were preincubated with different concentrations of thiopentone, methohexitone, and propofol, which is an isopropylphenol derivate. After viable S. aureus was added, phagocytosis was stopped at different time points. Leukocytes were then stained with monoclonal antibodies for flow cytometric analysis of granulocyte recruitment (ratio of ingesting granulocytes) and phagocytosis activity (fluorescence intensity of ingested bacteria). Both barbiturates inhibited granulocyte recruitment and phagocytosis activity in a dose-dependent manner, whereas propofol did not affect any of the investigated variables. At concentrations higher than 7.6 x 10(-3) M (for thiopentone, p < .008) and 1.1 x 10(-3) M (for methohexitone, p < .04), granulocyte recruitment and phagocytosis activity were significantly inhibited. The calculated inhibitory concentrations (IC50) of thiopentone for granulocyte recruitment and for phagocytosis activity were 1.3 x 10(-2) M and 1.1 x 10(-2) M, respectively. The corresponding values for methohexitone were 3.6 x 10(-3) M and 1.1 x 10(-3) M. Our in vitro model points at substantially different effects of barbiturates and propofol on phagocytosis of S. aureus, which is one of the most important pathogens in patients who need neuroprotective therapy. The inhibitory effects of both barbiturates demonstrate a strong dose-dependency, with more pronounced effects for methohexitone. Impairment of phagocytosis activity was more pronounced than granulocyte recruitment.

Non-Coplanar PCB-Mediated Modulation of Human Leukocyte Phagocytosis: A New Mechanism for Immunotoxicity

Organochlorine (OC) contaminants, notably polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are ubiquitous in all ecosystems and found in the tissues of humans and wildlife. Although the immunotoxicity of coplanar, dioxinlike PCBs is well documented, the adverse effects exerted by non-coplanar, non-dioxinlike PCBs have received little attention. Direct causal relationship between PCB and dioxin exposure and the observed detrimental effects on the immune system has yet to be fully established in humans. The immunomodulatory potential of toxic coplanar PCB 169 and TCDD and abundant non-coplanar PCBs 138, 153, and 180 on human leukocyte phagocytosis, an important innate immune function that initiates the clearance of pathogens, was tested upon in vitro exposure. Mixture and concentration-response experiments demonstrated a suppression of phagocytosis by non-coplanar PCBs suggesting a previously unrecognized aryl hydrocarbon receptor (AhR)-independent pathway. Regression analysis revealed that reduction of phagocytosis was mostly explained by the non-coplanar congeners. The effects on phagocytosis could not be accurately predicted by either the currently used toxic equivalence (TEQ) approach or the mouse model, thus undermining the use of the traditional models in the risk assessment for OC mixtures containing non-coplanar congeners. Our results are cause for concern as they suggest an AhR-independent pathway through which non-coplanar PCBs modulate phagocytosis, the immune system's first line of defense, possibly increasing the risk to developing infectious disease.

A novel exopolysaccharide from a clinical isolate of Prevotella nigrescens: purification, chemical characterization and possible role in modifying human leukocyte phagocytosis

Prevotella nigrescens, a gram-negative black-pigmented anaerobic rod, has frequently been isolated from periodontitis and periapical periodontitis lesions. We have isolated an exopolysaccharide-producing P. nigrescens, strain 22, from a chronic periodontitis lesion. The purpose of this study was to determine the chemical composition and function of the exopolysaccharide associated with this clinical isolate. The chemical composition and structure of the purified exopolysaccharide from strain 22 were determined by high performance liquid chromatography and methylation analysis. To define the biological function of this exopolysaccharide, a chemically induced exopolysaccharide nonproducing mutant, strain 328, which was derived from strain 22, was established. The biological effects of exopolysaccharide were determined by comparing the ability of strain 22, strain 328 or heat-killed strain 22 to form abscesses in mice and to interfere with the phagocytic activity of peripheral blood polymorphonuclear leukocytes. Chemical analysis showed that isolated exopolysaccharide consisted of mannose (521.6 microg/mg), glucose (25.6 microg/mg), fructose (65.8 microg/mg), galactose (12.5 microg/mg), arabinose (6.2 microg/mg), xylose (3.2 microg/mg), rhamnose (6.1 microg/mg), and ribose (0.6 microg/mg). Methylation analysis of exopolysaccharide indicated that the linkages of mannose were primarily (1-->2, 1-->6) (1-->2) (1-->6), and (1-->3). Strain 22 and, to a lesser extent, its heat-killed counterpart induced greater abscess formation in mice than strain 328, even though the enzymatic profile of strain 22 was similar to that of strain 328. The ability of strain 328 to induce abscess formation was restored by adding the purified exopolysaccharide isolated from strain 22 to the cell suspension of strain 328. Exopolysaccharide alone failed to induce abscess formation in mice. Further, strain 328 but not the untreated or heat-killed strain 22, was phagocytosed by polymorphonuclear leukocytes both in the presence and in the absence of opsonic factors. The results suggest that these polysaccharides isolated from strain 22, which primarily consisted of mannose, may play a key role in the development of the chronic inflammatory lesion from which this strain was isolated.

Exocytosis of zymosan particles by human phagocytes.

The kinetics of human leucocyte phagocytosis and exocytosis of fluorescein-isothiocyanate (FITC)-labelled zymosan particles were studied by flow cytometry (FCM). The leucocytes rapidly associated with zymosan particles, and internalization was confirmed by a fluorescence quenching technique. For incubation periods longer than about 60 min, exocytosis of ingested particles was observed. All human phagocytes ejected zymosan particles. The rate of exocytosis was about 1 particle per phagocyte per h, and was independent of the number of internalized particles. Exocytosis was dependent on temperature and glucose, but did not require Ca++ and Mg++ ions. Phagocytosis and exocytosis occurred concurrently, and phagocytosis accelerated ejection of previously internalized particles. The exocytosed particles were partially degraded by the phagocytes, and rephagocytosis of ejected zymosan particles was slower than the uptake of control particles. Phagocytes undergoing exocytosis remained intact during the 210 min examined. The findings indicate a heretofore neglected vector in the interaction of prey and phagocytosing cells.

A modified flow microcalorimeter adapted for the study of human leucocyte phagocytosis.

A modified flow microcalorimeter is described, in which the sample fluid that passes through the instrument is in contact only with Teflon®. The heat production (H.P.) from samples with leucocytes and thrombocytes in heparin-dextran-plasma was determined and found to agree with earlier findings using a similar system with gold tubings. A substantial increase of H.P. was found when the leucocytes were phagocytizing certain bacteria or polystyrene latex particles. The increase was related to the number of particles added. When leucocytes were prepared from defibrinated serum, they had no or only very little H.P., although they retained their capacity for phagocytosis, and when stimulated, showed similar H.P. as leucocytes prepared in heparinplasma. It is suggested that the low H.P. corresponds to a resting condition of the leucocytes, and that it was observed because the measurements were made with minimal mechanical disturbance of the cells.

Oxygen consumption during the initial stage of human leucocyte phagocytosis of polystyrene latex particles.

A polarographic method of studying oxygen consumption during phago cytosis by human leucocytes has been evaluated. Oxygen consumption during phagocytosis is markedly increased by raising the temperature from 20°C to 37°C, and inhibited by EDTA. Further, 2-deoxy-D-glucose inhibited the increase, but inhibition was stronger in combination with oligomycin. This indicates that the energy for phagocytosis is mainly derived from glycolysis, oxidative phosphorylation playing a minor role. The increase of oxygen consumption by washed human leucocytes during phagocytosis was greater in heparinized plasma than in serum. The difference was due to heparin. Heat-inactivated serum had a higher stimulatory effect than untreated serum. The combination of both calcium and magnesium, added to dialyzed plasma, showed a greater stimulatory effect than did magnesium alone. Calcium alone had no stimulatory effect.

Oxygen consumption during the initial stage of human leucocyte phagocytosis as a test for chronic granulomatous disease.

It has been demonstrated that an oxygraph technique can be used as a test for chronic granulomatous disease.

The effect of chloramphenicol on human leukocyte phagocytosis and respiration.

SummaryThe respiratory burst associated with phagocytosis can be significantly inhibited by high concentrations of chloramphenicol without impairment of phagocytosis perse. The impaired respiratory burst reflects decreased activation of the HMP shunt probably through the mechanism of enzyme inhibition.

The effect of temperature, mabolic inhibitors, andEDTA on pgocytosis of polystyrene latex particles by human leucocytes.

A modified method of Roberts & Quastel has been used to study the influence of temperature, metabolic inhibitors and EDTA on human leucocyte phagocytosis. The phagocytosis was increased by raising the temperature from 4° to 37° C. 2-deoxy-D-glucose inhibited phagocytosis. Antimycin or oligomycin had no influence. The greatest inhibition of phagocytosis was obtained by a combination of 2-deoxy-D-glucose and antimycin or oligomycin, demonstrating that the energy may derive from both glycolysis and respiration, the main source being glycolytic energy. Increasing concentrations of EDTA from 2 to 4 mM as final concentrations decreased phagocytosis.