Quantitative evaluation of macrophage phagocytosing capacity by a fluorometric assay

This paper reviews sensitive and simple quantitative evaluation of macrophage phagocytosing capacity by applying fluoresecin-labeled Sacharomyces cerevisiae cells. Yeast cells were conjugated with fluoresceinisothiocyanate (FITC) and used as fluorescent particles. A time course analysis within this method showed that phagocytosis of yeast cells was temperature dependent and that the number of that ones ingested by macrophages increased rapidly during the initial 60 min of incubation at 37 degrees C. Free fluorescent cells can be effectively removed by aspiration from the well. Furthermore, yeast cells required preopsonization with serum to achieve optimal uptake of the cells. The uptake of nonopsonized yeast cells by macrophages was significantly lower than that of opsonized cells (P < 0.05). We propose that about 50% of mouse macrophages can carry functionally active FcR responsible for phagocytosis.     

MEASUREMENT OF RATES OF PHAGOCYTOSIS : The Use of Cellular Monolayers

A method has been developed for measuring the rate of phagocytosis rather than the quantity of particles ingested per cell when the process is virtually complete. The method, which is simpler and more rapid than those described previously, utilizes cellular monolayers, radioactive particles, and short incubation times. Under the conditions described, the rate of uptake of particles by either guinea-pig peritoneal or human blood leukocytes was proportional to both cell concentration and the time of incubation, and was independent of changes in the concentration of particles during the measurement. The particles were retained by the cells for at least 90 min. The most suitable particles so far used have been ³²P-labeled Salmonella typhimurium, and acetyl-¹⁴C- or methyl-¹⁴C-labeled starch particles. The oxidation of ¹⁴C-labeled glucose has been studied under the same conditions that were used for the assays of phagocytosis: the greatest increase in formation of ¹⁴CO₂ from glucose-1-¹⁴C occurred a few minutes after the most rapid period of phagocytosis.     

The protective action of disodium cromoglycate on alveolar macrophages in phagocytosis of fibrogenic silica.

Treatment of fibrogenic silica (DQ-12) with Disodium Cromoglycate (DSCG) prior to its in-vitro and in-vivo phagocytosis by alveolar macrophages prevents the destruction of the cells and the fibrosis of lung tissue which are a consequence of phagocytosis. However, the treatment of alveolar macrophages with DSCG before phagocytosis of the silica had no, or a negligible, protective effect on the cells. Acid phosphatase activity which was significantly enhanced above the control in cells phagocytosing the silica was returned to the range found in phagocytosis of inert dust when silica treated with DSCG was phagocytized. The inhibitor of DNA- dependent RNA synthesis actinomycin D caused an increase of acid phosphatase activity. DSCG did not depress the phagocytic ability of alveolar macrophages. It appears that the catabolic enzyme process predominant in cells phagocytosing DQ-12 was under control in cells phagocytosing DQ-12 treated with DSCG and that DSCG probably acted as a regulator of the factors permitting catabolism. From the results it is suggested that the equilibrium of the enzyme reactions which accompany phagocytosis was such that the integrity of the phagocytes was preserved.     

Transient increase in cyclic AMP localized to macrophage phagosomes

Cyclic AMP (cAMP) regulates many biological processes and cellular functions. The importance of spatially localized intracellular gradients of cAMP is increasingly appreciated. Previous work in macrophages has shown that cAMP is produced during phagocytosis and that elevated cAMP levels suppress host defense functions, including generation of proinflammatory mediators, phagocytosis and killing. However, the spatial and kinetic characteristics of cAMP generation in phagocytosing macrophages have yet to be examined. Using a Förster resonance energy transfer (FRET)-based cAMP biosensor, we measured the generation of cAMP in live macrophages. We detected no difference in bulk intracellular cAMP levels between resting cells and cells actively phagocytosing IgG-opsonized particles. However, analysis with the biosensor revealed a rapid decrease in FRET signal corresponding to a transient burst of cAMP production localized to the forming phagosome. cAMP levels returned to baseline after the particle was internalized. These studies indicate that localized increases in cAMP accompany phagosome formation and provide a framework for a more complete understanding of how cAMP regulates macrophage host defense functions.     

Inactivation during phagocytosis of leucine aminopeptidase, an ecto-enzyme of polymorphonuclear neutrophils

Changes in enzyme activities of the plasma membrane makers were examined during phagocytosis using guinea-pig polymorphonuclear neutrophils. Incubation of neutrophils with fresh serum-opsonized zymosan particles showed a significant reduction in leucine aminopeptidase activity, whereas 5′-nucleotidase and alkaline phosphodieterase activities remained unchanged. Inactivation of leucine aminopeptidase activity was also observed by exposure of neutrophils to complement-opsonized zymosan particles, but not to non-opsonized zymosan, IgG-coated zymosan or polysterene latex particles. Pretreatment of neutrophils with cytochalasin B, which prevents phagocytosis but not surface binding of particles, provoked inactivation to the same degree as when the cells were allowed to phagocytose the particles. However, the inactivation during phagocytosis was protected by serine protease inhibitors. These findings suggest that loss of leucine aminopeptidase activity from phagocytosing cells may be mediated by certain serine protease inhibitor-sensitive factor(s) which are probably activated by the attachment of an opsonized zymosan particle to a specific membrane receptor, probably the C3b receptor.     

Glucose-6-phosphate dehydrogenase and mouse Kupffer cell activation: an ultrastructural dual staining enzyme-cytochemical study

Glucose-6-phosphate dehydrogenase (G6PD) plays an important role in Kupffer cell function, especially in phagocytosis activity. Although it was suggested that Kupffer G6PD may be upregulated in Kupffer phagocytosis/activation, direct morphological evidence has been lacking. Acid phosphatase (ACP), a representative lysosomal enzyme, can be used as a cytochemical marker for phagocyte activation. Using an ultrastructural enzyme-cytochemical dual staining method, I simultaneously localized G6PD and ACP activity in mouse Kupffer cells on a cell-by-cell basis, and examined whether or not cytochemically detectable G6PD activity increases in phagocytosing/activated mouse Kupffer cells. Glucose-6-phosphate dehydrogenase labelings were observed in the cytoplasm and on the cytosolic side of the endoplasmic reticulum, and ACP labelings were seen in the lysosomes. In phagocytosing Kupffer cells, in which ACP deposits were observed not only in the lysosomes but also on the phagosomal membranes and phagosomal contents, G6PD labelings were denser than dormant Kupffer cells. Enzyme-cytochemically detectable G6PD activity increases in phagocytosing/activated mouse Kupffer cells. Kupffer cell G6PD, activated in phagocytosing Kupffer cells, may play an important role not only in liver defense but also in liver disease pathogenesis/pathophysiology.     

Visualization of reactive oxygen species formation by phagocytizing macrophages

Activated peritoneal macrophages exhibiting phagocytosing capacity produced an electron-dense precipitate of formazan in contact sites of macrophage plasmalemma and phagocytosed yeast cells. No production of formazan occurred, when non-opsonized latex particles were ingested by macrophages. Formazan precipitation could be prevented by anaerobiosis but not by addition of cyanide.     

Role of the cellular attachment domain of fibronectin in the phagocytosis of beads by human gingival fibroblasts in vitro

Summary To study the role of phagocytosis in periodontal tissues, internalization of fibronectin-coated latex beads by Gin-1 fibroblast populations was investigated. Demonstration of phagocytosis by internalization of beads was confirmed by immunofluorescence microscopy, electron microscopy, and flow-cytometry. The percent of cells phagocytosing beads measured by flow-cytometry was negligible at 4° and 23°C, but increased to approximately 17% at 37°C. As measured by automated image analysis, the percentage of phagocytosing cells increased linearly from 8 to 22 with increasing fibronectin concentration of the incubation solution from 30 ng to 300 g/ml. Similar linear increases in the percentage of phagocytosing cells were observed when beads were incubated with cells for periods ranging from 2 h to 2 days. To examine the role of the Arg-Gly-Asp receptor in mediating phagocytosis, fibronectin-coated beads were first coated with either Gly-Arg-Gly-Asp-Ser-Pro or Gly-Arg-Gly-Glu-Ser-Pro peptides at concentrations of 0.125, 0.5, and 1 mg/ml, or with control vehicle, and then incubated with cells. Phagocytosis was completely blocked at 1 mg/ml of the Gly-Arg-Gly-Asp-Ser-Pro peptide, but the Gly-Arg-Gly-Glu-Ser-Pro peptide showed no significant inhibition compared to control values. Blocking antibodies to the cell attachment domain of the fibronectin molecule also reduced the percentage of phagocytosing cells significantly. The data show that these phagocytic assays are sensitive enough to detect the influence of incubation temperature and time, cellular heterogeneity, ligand type, and ligand concentration on the percentage of phagocytosing cells. Further, the mechanisms which determine internalization of fibronectin-coated beads rely in part on the initial binding of ligand to the Arg-Gly-Asp receptor present on fibroblasts.     

Selective down-regulation of alveolar macrophage oxidative response to opsonin-independent phagocytosis

We have compared the oxidative response of alveolar macrophages (AM) during opsonin-dependent and independent phagocytosis by using multiparameter flow cytometry. The respiratory burst of AM during phagocytosis was quantitated by the intracellular oxidation of the nonfluorescent precursors dichlorofluorescin diacetate (DCFH) or hydroethidine (HE, a reduced precursor of ethidium) to their fluorescent (oxidized) counterparts. After loading freshly isolated normal hamster AM with DCFH or HE, red or green fluorescent beads, respectively, were added to the shaking cell suspensions. Ingestion of opsonized particles by AM caused a marked increase in oxidation of both DCFH and HE proportional to the number of beads ingested. In contrast, uptake of one to three unopsonized particles per cell led to inhibition of oxidative activity compared to control cells incubated without particles. AM ingesting four or more unopsonized particles showed some increase in oxidative metabolism, but far less than that with identical numbers of particles in opsonin-dependent ingestion. Similar results were obtained using fluorescent labeled staphylococcal bacteria. Using three-color flow cytometry to study cells ingesting both types of particles, cells first ingesting unopsonized beads were also found to have an inhibited oxidative response to subsequently ingested opsonized particles. The mitochondrial poison antimycin inhibited most of the intracellular oxidative response to either type of phagocytosis. The remaining antimycin-insensitive, membrane derived respiratory burst of AM was also substantially diminished after phagocytosis of unopsonized particles vs similar numbers of opsonized particles. The greatly increased mitochondrial respiration in AM during phagocytosis of opsonized particles may be related to bactericidal mechanisms. Killing of ingested Staphylococcus by AM was markedly impaired in the presence of antimycin. The results suggest that AM may ingest the numerous, unopsonized inert particles that are inhaled without generation of potentially toxic oxygen metabolites, while retaining the capacity to undergo a respiratory burst after ingesting opsonized particles and bacteria. The mechanism(s) for this distinct response may include generation of an inhibitor of intracellular oxidative metabolism.     

The unsuitability of the uridine incorporation assay for the measurement of phagocytosis of Escherichia coli

The uridine incorporation technique for assaying phagocytosis is based on the fact that polymorphonuclear leucocytes are impermeable to labelled uridine, and therefore ingested bacteria inside phagocytic vacuoles will be unable to take it up. Extracellular bacteria, including those adherent to the phagocytic cell surface, can do so however. Differences in uptake between bacteria alone and in the presence of phagocytic cells can be used to measure ingestion. The present paper describes the application of this technique to Escherichia coli O-86 as the test organism. It appears that with this test species, the method is unsuccessful, because exposure of the non-ingested bacteria to some soluble product of the triggered polymorphonuclear leucocytes causes a large increase in their uridine uptake rates, over that of the control bacteria. The nature of the product responsible is unknown. It is unconnected with change in the pH of the medium, is heat stable, and is only produced by polymorphonuclear leucocytes which are actively phagocytosing. It may be that a release of phagolysosome contents is responsible.     

Altered circadian rhythms of corticosterone, melatonin, and phagocytic activity in response to stress in rats

Corticosterone is thought to be the main glucocorticoid secreted in response to stressful exercise, while melatonin buffers the adverse immunological effects of stress. The present work was aimed to evaluate whether swimming-exercise-induced stress leads to changes in the chronobiology parameters of the circadian rhythms of melatonin and corticosterone, and in the number and phagocytosis of peritoneal macrophages in 3-month-old male Wistar rats. The animals were subjected to a physical activity trial consisting of 2 h of free swimming. Radioimmunoassay was used to determine the plasma levels of melatonin and corticosterone. Phagocytosis was measured by the latex-bead phagocytosis index (PI), i.e., the number of latex beads ingested by 100 macrophages, the phagocytosis percentage (PP), i.e., the percentage of cells that had phagocytosed at least one latex bead, and the phagocytosis efficiency (PE), i.e., the ratio PI: PP which indicates how effectively the phagocytes ingested the particles. Stress significantly decreased the MESOR and amplitude of the melatonin rhythm, and significantly increased the MESOR of the corticosterone rhythm. The control animals' peritoneal macrophage number and PI showed a circadian rhythm with maxima at 02:00 and 03:00, respectively. The stressed group displayed higher values of PI than the controls at most hours of the night, but the number of cells in the peritoneal cavity was practically the same at all hours studied. These data confirm that melatonin and corticosterone act as modulators of the innate immune response, and that the circadian rhythm of the two hormones are altered in situations of stress.     

HEMA-particles phagocytosis of rat neutrophils

A micromethod for testing of phagocytosis employing synthetic hydrophilic HEMA particles seems to be suitable for an examination of rat neutrophils. The normal phagocytic activity was 53.7 +/- 1.4%, phagocytic index 8.2 +/- 0.7, and the number of phagocytosing neutrophils was found to be 74 +/- 5.10(6)/l. Using HEMA particles, an experimentally induced depression of rat neutrophil phagocytosis after cyclophosphamide or benflurone was well detected and quantified.     

THE ADHESIVENESS OF LEUCOCYTES TO SOLID SURFACES

1. Methods are described for measuring the stickiness of cells to solid surfaces. 2. The effect of various factors such as temperature, serum concentration, sodium chloride concentration, etc., on the adhesiveness of leucocytes to solid surfaces and the phagocytosis of solid particles are compared, and certain marked differences pointed out. 3. On account of these differences it is concluded that forces of surface tension, though undoubtedly operative in determining the behavior of cells in contact with solid bodies, are not the only factors involved. 4. Ways in which changes in the structure or rigidity of the protoplasm might affect phagocytosis and adhesiveness in opposite directions are suggested.     

Suppressive effect of delta 9-tetrahydrocannabinol in vitro on phagocytosis by murine macrophages

Incubation of normal mouse peritoneal cells consisting of over 90% phagocytizing macrophages with delta 9-tetrahydrocannabinol (THC) resulted in a inhibition of phagocytic function. The THC in a dose-related manner suppressed the percentage of macrophages per culture which ingested yeast and the average number of yeast particles ingested by the phagocytizing macrophages. The vehicle used to suspend the THC in vitro, i.e., DMSO, had no detectable effect on macrophage function. Suppression of phagocytosis with no effects on viability or cell number occurred with doses of 10 micrograms or less THC per milliliter culture medium. Measurable suppression also occurred after 24- to 48-hr treatment of the macrophages with the THC. This compound had little if any detectable effect on phagocytosis when added directly to the cultures shortly before testing for phagocytosis. Further studies concerning the effects of THC on macrophage function appear warranted.     

Simultaneous measurement by flow cytometry of phagocytosis and metabolic burst induced in phagocytic cells in whole blood by Borrelia burgdorferi

Abstract This paper describes the interactions between a strain of Borrelia burgdorferi and phagocytic cells, measured in whole blood, by a two-color flow cytometric method, which allowed the simultaneous quantification of both the phagocytosis rate and the oxidative burst activation. The data obtained indicated that: a) phagocytosis and metabolic activation increased as a function of spirochete concentration; b) the number of ingesting cells peaked within 10 min but activation followed later, and did not involve all the phagocytosing cells; c) opsonization of borreliae with a patient's serum enhanced the two cellular activities, mostly phagocytosis. The intensity of such functions was lower than those found for Staphylococcus aureus . The flow cytometric assay of phagocytosis interactions with Borrelia burgdorferi assessed in whole blood represents an experimental approach which simulates the physiological conditions in nature.     

Stereological analysis of phagocytosing cells

Various complexes of phagocytes with adsorbed (I) and entrapped (J) particles are formed during phagocytosis. Method of stereological reconstruction is proposed that allows to demonstrate the actual distribution of these complexes on the basis of morphometric analysis of their ultrathin sections. The principle of the method lies on the probability simulation of section distribution for a given distribution of complexes and on the solution of the reverse problem by stepwise determination of the relative quantity of each complex type (from the most complicated to the most simple one, when I = 0 and J = 0). The stereological analysis of phagocytosing murine peritoneal macrophages revealed an absolutely different and more adequate kinetical picture of phagocytosis, as compared to the morphometric data.     

Phagocytosis by human macrophages is accompanied by changes in ionic channel currents

The present study has shown that changes in ionic channel currents accompany the phagocytosis of particles by mononuclear phagocytes. The patch-clamp technique in the cell-attached configuration was applied to human monocyte-derived macrophages to measure the activity of single transmembrane ionic channels in intact cells. During such measurements, IgG-opsonized and non-opsonized latex particles were offered for phagocytosis under continuous video-microscopical observation. Single particles were presented to the phagocytes at a membrane location some distance from that of the patch electrode. After a lag period following particle attachment, enhanced inward and outward time-variant single channel currents coinciding with particle engulfment were observed. On the basis of current-voltage characteristics and membrane potential measurements, the outward-directed channels were identified as K+ channels. Phagocytosis was also accompanied by slow transient changes in background membrane currents, probably due to changes in the membrane potential of the phagocytosing cell. Phagocytosis of IgG-coated latex particles differed from phagocytosis of uncoated or albumin-coated particles by a shorter lag time between particle attachment and the onset of enhanced ionic channel activity.     

Simultaneous flow cytometric method to measure phagocytosis and oxidative products by neutrophils.

We developed a rapid and sensitive two-color flow cytometric method which allows the simultaneous quantification of both the phagocytosis rate and the oxidative burst activation of polymorphonuclear leukocytes (PMNLs). The oxidation of hydroethidine (HE) to ethidium bromide (EB) was performed by the oxidative neutrophil products within the cells during the respiratory burst, which was stimulated by phagocytized fluorescein-labeled Staphylococcus aureus. By means of flow cytometry we measured red EB fluorescence emission together with green fluorescence, which was emitted by the ingested fluoresceinated bacteria. The fluorescence intensity was proportional to the number of bacteria ingested. Adherent bacteria were distinguished from the ingested ones. This two-color cellular staining permits measurement of two different functions of neutrophils in one step. This method could be of interest for the determination of the interactions between neutrophils and bacteria and for the investigations on infectious diseases in chronic granulomatous disease patients.     

Phagocytosis of bacteria by human leukocytes measured by flow cytometry

A new method has been developed for the evaluation of the phagocytic activity of human leukocytes using fluorescently labeled bacteria and flow cytometry. By simultaneous measurement of cellular light scatter and fluorescence, extracellular bacteria, phagocytes, and nonphagocytes could be discriminated and quantified. All leukocytes assumed to be capable of phagocytosis were phagocytosing, and about 90% of these cells were polymorphonuclear neutrophilic granulocytes. Within 15 min 85% of the bacteria were phagocytosed and each phagocyte contained an average of 15-20 bacteria. The phagocytic capacity of the leukocytes from healthy individuals showed minor interindividual and day-to-day variations. This method facilitates a rapid and accurate in vitro evaluation of the phagocytic activity of human leukocytes.     

Isolation and quantitative estimation of diesel exhaust and carbon black particles ingested by lung epithelial cells and alveolar macrophages in vitro

A new procedure for isolating and estimating ingested carbonaceous diesel exhaust particles (DEP) or carbon black (CB) particles by lung epithelial cells and macrophages is described. Cells were incubated with DEP or CB to examine cell-particle interaction and ingestion. After various incubation periods, the cells were separated from free extracellular DEP or CB particles by Ficoll density gradient centrifugation and dissolved in hot sodium dodecyl sulfate detergent. Insoluble DEP or CB residues were isolated by high-speed centrifugation, and the elemental carbon (EC) concentrations in the pellets were estimated by a thermal-optical-transmittance method (i.e., carbon analysis). From the EC concentration, the amount of ingested DEP or CB could be calculated. The described technique allowed the determination of the kinetics and dose dependence of DEP uptake by LA4 lung epithelial cells and MHS alveolar macrophages. Both cell types ingested DEP to a similar degree; however, the MHS macrophages took up significantly more CB than the epithelial cells. Cytochalasin D, an agent that blocks actin polymerization in the cells, inhibited approximately 80% of DEP uptake by both cell types, indicating that the process was actin-dependent in a manner similar to phagocytosis. This technique can be applied to examine the interactions between cells and particles containing EC and to study the modulation of particle uptake in diseased tissue.