SEARCH

SEARCH BY CITATION

Keywords:

  • phagocytosis;
  • oxidative burst;
  • killing;
  • phagocytes;
  • Staphylococcus aureus;
  • Candida albicans

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Objective  Polymorphonuclear leukocytes (PMN) play a central role in the elimination of most extracellular pathogens, and an impairment of their functions predisposes an individual towards local and systemic bacterial and fungal infections. Here we describe a rapid and easy-to-perform cytofluorometric assay for investigation of PMN activity using Candida albicans and Staphylococcus aureus as target organisms.

Methods  Phagocytes were stained with anti-CD13-RPE antibody, and microorganisms were stained with calcein-AM. Oxidative burst production was measured by oxidation of dihydroethidium. The percentage of killed target organisms after ingestion was determined by staining with ethidium-homodimer-1 after lysis of human cells. The dyes and procedures used in this method were chosen after comparison of different stains and cell preparation techniques described in previous assays.

Results  Concerning phagocytosis, the percentages of active phagocytes and of ingested microorganisms were determined. Furthermore, the method allowed measurement of the resulting percentage of PMNs producing respiratory burst, and of the percentage of killed microorganisms. We minimized artifactual changes, which might have been the reason for the difficulties and conflicting results of other cytofluorometric methods.

Conclusions  The described method provides a new whole blood cytofluorometric assay, which combines rapid and simple handling with high reproducibility of results obtained by investigation of PMN activity using Candida albicans and Staphylococcus aureus as target organisms.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Phagocytes play an essential role as a first line of defense in the human host. They strike against foreign matter, in particular bacteria, viruses or fungi which invade the human body. A variety of complex mechanisms helps the phagocyte to achieve the elimination of microorganisms [1,2]. Basically, phagocytes ingest target cells by phagocytosis followed by the development of toxic oxygen substances, such as hydrogen peroxide or hydrohalites; this is known as the oxidative burst, and leads, together with other toxic products, to the destruction of the microorganisms—referred to as killing. Impairment of these processes predisposes an individual towards local and systemic infections [3]. Various flow cytometric test methods have been developed to determine the phagocyte activity [1,2,4,5–7], but they all have restrictions and differ widely in their results. These differences arise mainly from non-uniform sample preparation and the interpretation of flow cytometric histograms. There are variations, such as the types of phagocytes used (monocytes, polymorphonuclear leukocytes or macrophages of blood, bronchial lavage, tissue, etc., of human or animal origin), different anticoagulants, and the amount and sort of stimulating agent (e.g. latex particles, living or dead Gram-positive or Gram-negative bacteria or fungi). The heterogeneity is obvious, and in many cases the investigated cells are not treated in a particularly gentle manner during the required separation procedures. Furthermore, a comparison of phagocytosis, oxidative burst and killing functions by a simultaneous method is still lacking, despite the need for this in the therapeutic surveillance of several diseases. We describe a new whole blood cytofluorometric method, which combines rapid and simple handling with high reproducibility of results. The assay allows measurement of the percentage of phagocytosing PMNs, the percentage of ingested microorganisms (either Candida albicans or Staphylococcus aureus), the percentage of PMNs with burst production after phagocytosis, and the resulting percentage of killed target organisms, enabling investigation of different phagocyte functions. Analysis of all parameters can be performed using a blood sample of 150 µL and can be completed within 2 h.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Materials

CD13-RPE antibody was obtained from Coulter, Krefeld, Germany. Dihydroethidium (DHE), N-ethyl-maleinacidamide (NEM), phosphate-buffered-saline (PBS), Tween-20, Triton X-100, azure A and RPMI-1640 were purchased from Sigma, St Louis, MO, USA. Calcein-AM and ethidium-homodimer-1 (EthD-1) were obtained from Molecular Probes, Eugene, Oregon, USA, and tryptic soy broth was purchased from Difco laboratories, Detroit, Michigan, USA. All other reagents were on the best grade commercially available and were used without further purification.

Microorganisms

Culture of C. albicans (DSM 1386) as well as of S. aureus (ATCC 25923) was performed in tryptic soy broth. For fluorescence labeling, the broth was centrifuged at 500g for 3 min, and the pellet was resuspended in 1 mL of PBS with 5 µL of calcein-AM and incubated for 50 min at 37 °C in a thermomixer (Eppendorf-Nehler-Hinz GmbH, Germany) at 1100 rev/min. In previous experiments, calcein-AM had proved to be most suitable after comparison with different dyes (for explicit data see Husfel [8]). The stained microorganisms were washed three times in 1 mL of PBS, resuspended in RPMI supplemented with 0.4% glucose, and stored in a refrigerator. The amount of C. albicans per mL in RPMI was determined with Neubauer's counting chamber and the count of staphylococci was obtained by using a spectrophotometer: 7 × 107 bacteria/mL gave a photometric extinction of 0.100 at 350 nm in 1-cm cuvettes.

Determination of PMNs

Blood was drawn from healthy volunteers with an age range of 20–40 years and anticoagulated with 5 IE of heparin per 1 mL of blood. The number of PMNs per mL of blood was determined with Neubauer's counting chamber.

Cell labeling and activation

In the first step, the blood was incubated with DHE (20 µL of PBS containing 0.5 µg of DHE per 1 mL of blood) for 10 min at 37 °C in a shaking water bath, allowing the still colorless dye to diffuse inside the PMNs. In the presence of oxidative substances released later in the burst reaction, the dye is oxidized, becomes red fluorescent and can be detected by the flow cytometer. To achieve previously established optimal ratios of 1 : 1 between PMNs and C. albicans and 1 : 50 between PMNs and S. aureus, respectively, the RPMI solutions containing the microorganisms were diluted with PBS and mixed carefully. According to the chosen kinetics for incubation (0, 2, 4, 6, 8, 10, 15, 20 and 25 min for phagocytosis and burst production, and an additional 40 and 60 min for killing), polypropylene tubes were prepared with 50 µL of RPMI supplemented with 0.4% glucose and stored on ice. Microorganisms and blood were mixed, aliquots of 100 µL were quickly dispensed into the test tubes, and incubation was started at 37 °C for C. albicans. When staphylococci were used as target organisms, the incubation temperature had to be reduced to 30 °C, since at 37 °C the immune reaction proved to be too fast to allow us to obtain optimal kinetics.

After comparison of different fluorescence stains in previous experiments (see Husfeld [8] for explicit data), the chosen DHE for measurement of oxidative burst activity, CD13-RPE antibody for marking the phagocytes and EthD-1 for detection of dead microorganisms appeared to be most suitable and were used further in the described concentrations.

Procedure for phagocytosis and oxidative burst activity

At appropriate times, the reaction was stopped by addition of 200 µL of ice-cold NEM (the tube for 0 min had been immediately mixed with 200 µL of NEM and was left on ice). The tubes were then centrifuged together at 4 °C at 250g for 5 min. To stain the PMNs with orange fluorescence, the pellet of each tube was resuspended with 100 µL of PBS containing 0.80 µL of CD13-RPE antibody and incubated at room temperature for 20 min. Then 1000 µL of lysing buffer for erythrocytes (containing 8.27 mg of ammonium chloride, 1 mg of potassium hydrogencarbonate and 0.04 mg of sodium-EDTA) as described in previous investigations [9,10] was added to each tube and incubated at room temperature for 10 min. Afterwards the cells were stored on ice and measured with a maximum delay of 2 h. Stable flow cytometric results were obtained during this time.

Procedure for killing

After incubation, the test tubes were placed on ice to stop the reaction, mixed immediately with 300 µL of lysing buffer for human cells (consisting of 1.2 µL of Triton X-100 and 1.2 µL of Tween-20 in distilled water) and incubated at 37 °C for 25 min in the thermomixer at 1100 rev/min. The tubes were then centrifuged at 4 °C at 800 g for 5 min. The pellets were each mixed thoroughly with 400 µL of EthD-1-solution (2 µM), incubated for 10 min at room temperature and, after addition of 700 µL of PBS, stored on ice. Stable flow cytometric results were obtained within a period of 2 h. During the set-up of our method, the use of Triton X-100 and Tween-20 had been compared to other lysing procedures, such as distilled water, gelatine, sodium deoxycholate, lysolecithin, digitonin and ultrasonics, and had proved to be the most suitable. Furthermore, we had conducted an investigation to determine possible binding of EthD-1 to fragments of incompletely lysed human cells. Assays in which yeasts and bacteria were used, respectively, were compared with assays using the usual mixture of blood and microorganisms. After addition of the appropriate amount of lysing solution and incubation for 30 min at 37 °C and 1100 rev/min in the thermomixer, the described staining with EthD-1 had been performed and no significant difference in the number of detectable dead cells was found. Therefore, perturbation of the measurement of dead cells by DNA-containing fragments seemed unlikely. However, incubation with EthD-1 at higher temperatures (e.g. 37 °C instead of 20 °C) or for longer than 60 min resulted in an increased number of living cells stained with EthD-1, so these conditions had to be avoided. To exclude a possible influence of the detergent treatment on the microorganisms, the test procedure for killing had also been performed with both microorganisms in plasma without phagocytes. After comparison of the percentage of dead microorganisms with and without detergent treatment, no significant difference had been found after staining with EthD-1. These findings seemed to exclude a significant influence of the detergents. Apparently, the lysing solution was affecting only human cells [8]. To confirm the cytofluorometric determination of the percentage of killed microorganisms, phagocytes together with microorganisms were incubated in parallel with samples for the cytofluorometric assay; the phagocytes were then lysed and the microorganisms were investigated by fluorescence microscopy after staining with acridine orange. Similar results were obtained with both methods [8].

Flow cytometry

A Profile II with powerpack (Coulter Electronics, Miami, Florida, USA) with a 5-W argon ion laser operating at 350 mW and tuned to a 488-nm exciting wavelength was used. A minimum of 5000 phagocytes was measured per sample. Fluorescence and side scatter parameters were collected by logarithmic amplification after setting the threshold on linear forward scatter to avoid debris. Green fluorescence from calcein was collected through a band-pass filter of 525 nm, CD13-RPE was analyzed by a band-pass filter of 575 nm, and ethidium (E) and Eth-D1 were detected with a band-pass filter of 635 nm. Data were displayed as single-parameter histograms or two-parameter plot analysis and acquired by using an instrument status with a linear data mode for the forward scatter (FS) and a logarithmic data mode for the sideward scatter (LSS), the green fluorescence 1 (LFL1), the orange fluorescence 2 (LFL2) and the red fluorescence 3 (LFL3). Measurement of phagocytosis and burst production was performed with the same reaction tube, and determination of killing with a separate one.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Phagocytosis

Figure 1 shows an example of the flow cytometric analysis. In the upper right quadrant of dot plot A (LFL2 versus LSS), the PMNs are selected in a bitmap, while the target organisms appear in the lower left. As soon as the phagocytosis starts, an increasing number of PMNs take up one or more microorganisms, generating additional signals in FL1. In histogram B (count versus LFL1), the percentage of phagocytosing PMNs is detected at each time point. The green fluorescing target organisms are selected in dot plot C (LFL1 versus LSS), while in histogram D (count versus LFL2) the percentage of these cells with an additional orange fluorescence obtained after ingestion is detected. Thus, for each time point, the percentage of PMNs which have phagocytosed (histogram B) and the percentage of ingested microorganisms (histogram D) were automatically determined in selected gates represented by the markers 1–4. After 25 min of incubation, the percentage of phagocytosing PMNs from 25 healthy individuals reached a mean of 46.4% ± 4.3% (SD) when investigating with yeasts and 85.3% ± 5.6% when using bacteria. The investigation of ingested microorganisms resulted in percentages of 79.9% ± 4.9% for Candida and 91.4% ± 3.2% for Staphylococcus.

image

Figure 1. Analysis of phagocytosis; the four top diagrams represent the results at the beginning and the bottom panels show the results after incubation. In dot plot A the PMNs are selected in a bitmap; in histogram B the percentage of the previously selected cells with an additional green fluorescence (phagocytosing PMNs) along the kinetics is detected. The target organisms are selected in a bitmap in dot plot C; in histogram D the percentage of these cells with additional orange fluorescence (ingested microorganisms) is detected.

Download figure to PowerPoint

Oxidative burst

An example of the analysis is shown in Figure 2. In dot plot A (LFL1 versus FS), microorganisms are selected. After phagocytosis, the compounds released during the oxidative bursts subsequently convert the colorless precursor DHE in the phagocytes into the fluorescent ethidium (E), which can be registered by the FL3 detector. In histogram B (count versus LFL3), the percentage of the cells in the bitmap with an additional red fluorescence (oxidized dye) is detected. The selection of the target cells in dot plot A ensures that only PMNs which have phagocytosed microorganisms are tested for their oxidative burst activity. The measured events with significant fluorescence 3 signal represent the activated PMNs. Therefore, for each incubation time point, the percentage of PMNs displaying oxidative burst activity after phagocytosis of target organisms is automatically determined in selected gates represented by the markers 1–4.

image

Figure 2. Analysis of the oxidative burst; the top panels represent the results at the beginning, and the bottom panels show the results after incubation. In dot plot A, the microorganisms are selected in a bitmap. Oxidative compounds create the fluorescent ethidium bromide, which is scored in histogram B. The measured events represent the PMNs displaying an oxidative burst after phagocytosis.

Download figure to PowerPoint

By investigation of the same 25 healthy donors at the end of the incubation period (25 min), a mean of active PMNs of 79.3% ± 4.0% (SD) after phagocytosis of yeasts and 57.7% ± 4.3% when using bacteria was obtained.

Killing

The analysis of killing is performed using the dye EthD-1, a high-affinity red DNA stain detectable in FL3, which cannot pass intact membranes and thus is used to mark the dead microorganisms after lysis of the surrounding phagocytes, while the calcein-AM slowly diffuses out. In a first dot plot (LFL3 versus LFL1) at the beginning only living microorganisms positive in FL1 are present in the samples. Upon incubation, the cells wander from FL1 via FL1/FL3 to FL3. This dot plot can be used to control the optimal adjustments of the flow cytometer. In a second dot plot (LSS versus FS), the population of microorganisms is detected and selected in a bitmap. Furthermore, the completeness of the lysis of human cells can be controlled. In a histogram (count versus LFL3), the percentage of dead microorganisms stained with EthD-1 in the population selected in the bitmap in the second dot plot is automatically determined.

From investigation of 25 healthy individuals, the percentage of dead microorganisms reached a mean of 61.4% ± 10.6% (SD) for C. albicans and a mean of 79.3% ± 7.2% for S. aureus after 60 min of incubation.

Examples of the results from the investigation of PMN phagocytosis, oxidative burst and killing for one donor are shown in Figure 3A using C. albicans and in Figure 3B using S. aureus. The results obtained by investigation of bacteria and yeasts showed a quicker increase and a nearly two-fold higher final percentage of phagocytosing PMNs when using staphylococci, despite the lower incubation temperature. Also, the percentage of ingested microorganisms showed a much quicker increase for bacteria with similar final results. The investigation of the percentage of dead microorganisms showed a quicker increase and slightly higher final results for staphylococci. The measurement of the percentage of PMNs displaying an oxidative burst after phagocytosis, however, showed a quicker increase and much higher final results when using yeasts.

image

Figure 3. The kinetics of phagocytosis, burst production and killing using (A) Candida albicans and (B) Staphylococcus aureus; percentages of phagocytosing PMNs, ingested microorganisms, oxidative burst-producing PMNs and dead microorganisms. Data of one of 25 experiments with similar results are shown.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

In recent years increasing interest has been shown in the evaluation of PMN functions, and many investigators have developed flow cytometric approaches to assay their activation. However, there are still surprisingly conflicting results regarding neutrophil phagocytosis and oxidative burst activity. We describe a simple and rapid assay, which reduced handling difficulties, allowed measurement at multiple time intervals of incubation and minimized artefactual changes. The assay was designed to investigate the phagocytosis of C. albicans and S. aureus, the resulting percentage of PMNs producing respiratory burst and the percentage of killed microorganisms.

Many of the procedures used for isolation of PMNs for analysis [5–7, 11] themselves cause artefactual changes such as altered expression of surface antigens [12], morphologic changes [13], aggregation and activation of PMNs [10] and reduction of the oxidative burst, chemotaxis and increased release of lysosomal enzymes [14]. The use of whole blood for investigation reduced these effects.

Anticoagulation was performed using 5 IE of sodium-heparin/mL blood. EDTA, citrate as used by Model et al [2], and oxalate form complexes with calcium ions and therefore impair PMN functions [15]. Fluoride inhibits enzymes involved in oxidative metabolism [15], and higher concentrations of heparin reduce phagocytosis and the respiratory burst [16].

For the provision of a sufficient nutrient supply to the cells during incubation, a percentage of 0.4% glucose in the RPMI solution proved necessary. The yeasts seemed to be particularly sensitive to stressful conditions and tended to develop pseudohyphae [17], which is why some investigators added 0.5 µg of amphotericin B per mL to the samples. This addition led, in our investigations, to an approximately 10% higher percentage of dead yeasts (data not shown), corresponding to the findings of Schaumann and Shah [18]. Contrary findings, however, are reported by Martin and Bhakdi [11] and Nugent and Couchot [19], who excluded an effect of low concentrations of amphotericin B. In any case, our results indicate that, microscopically, there is no development of pseudohyphae after addition of 0.4% glucose.

For the staining of living bacteria and yeasts we used calcein-AM, which appeared to be most suitable after comparison of different dyes for microorganisms. Previously used fluorescence stains such as FITC [1–4,6,20] and BCECF-AM [5–11] showed a high sensitivity for pH changes, which occur in the phagolysosomes and cause a change in the emitted wavelength of light. Others, such as Texas Red [21], showed toxic effects on the microorganisms (data not shown). Additionally, calcein-AM stains only the cytoplasm of living cells, causes no damage to the cell membrane and has a high fluorescence and diffusion rate.

Our investigations confirmed the results of Perticarari et al [1] that no preopsonization of microorganisms was necessary, since the incubation time within whole blood during the test ensures sufficient opsonization of target cells for phagocytosis in a physiologic environment.

The staining of phagocytes was performed using a CD13 antibody linked to R-phycoerythrine (RPE), which is selective for granulocytes and monocytes. In contrast to other studies, we marked the phagocytes after the incubation with the microorganisms, because the staining of PMNs involves washing and centrifugation procedures, which affect the phagocytes [12–14]. Furthermore, the linking of antibodies to PMNs might itself cause an alteration of their functions. Fluorescence microscopic investigations with the chosen concentration of antibodies showed no antibody-capping, which is probably due to the low concentration of antibodies used and the presence of human plasma in the samples. Therefore, there was no necessity to remove surplus antibodies or to add bovine serum albumin.

The determination of the percentage of ingested microorganisms gives important additional information about the efficiency of PMNs. This applies even more for bacteria than for yeasts, because each phagocyte can ingest widely differing numbers of staphylococci during the incubation. To facilitate this measurement, it was necessary to omit the quenching procedure usually performed in cytofluorometric experiments.

Fluorescence microscopic investigations showed that, after incubation, an average of 6% of the phagocytes had microorganisms attached to their surface. We centrifuged samples after incubation with yeasts, as well as with bacteria at different accelerations (60–300g). The pellets were resuspended in PBS and again investigated microscopically. After centrifugation at at least 200g and storage of the samples on ice (which suppresses the mobility of cells) for 45 min, no attached microorganisms were detectable [8]. Centrifugation at 250g is performed in any case in the course of the PMN antibody staining. Accordingly, no quenching procedure seemed necessary. We could nevertheless demonstrate that the fluorescence of calcein-AM could be suppressed by using 2 mg of azure A in PBS per sample for quenching after the incubation.

For the determination of the oxidative burst activity a similar principle is used in nearly all previously described assays: during the oxidative burst, a colorless dye is oxidized and is then detectable by the flow cytometer [1–4,9]. Dihydrorhodamine 123 (DHR), used by Rothe and Valet [7], Vowells et al [3] and Hirt et al [4], is a very sensitive intracellular dye, which, however, emits a green fluorescence and therefore interferes with the detection of the microorganisms in our assay. As it was an important aim to measure all parameters in one assay, a different dye had to be found. Model et al [2] used 4-carboxydihydrotetramethylrosamine succinimidyl ester (RS-SE) in a method for measuring only the oxidative burst. RS-SE showed the lowest non-specific oxidation and very sensitive detection of the PMN oxidative burst compared with DHR, DHE and dihydrotetramethylrosamine (RS) [8]. However, due to its labeling of the plasma membrane and a consequent possible influence on PMN function, it could not be used. DHE was used by Perticarari et al [1] and its fluorescence is detectable in both the orange and red channels. Perticarari et al detected its fluorescence in FL2, which would interfere with the emission of the RPE dye of the phagocytes. In our method, therefore, the fluorescence was detected in FL3. We used a concentration 150 times lower than that of Rothe and Valet [7], which may account for the fact that, in contrast to their findings, no non-specific oxidation of DHE was observed [8].

For the determination of the percentage of dead microorganisms, it was necessary to lyse the phagocytes. In the recent literature different techniques are described, but most have proved to have major or minor disadvantages [8]. Use of distilled water [22,23] led to complete lysis of granulocytes, while the monocytes were not affected. Gelatine [24], sodium deoxycholate (DOC) [25], lysolecithin [26] and digitonin [27] resulted in satisfactory lysis of phagocytes, but the concentrations required caused severe damage to the microorganisms. Especially for DOC, the findings stand in contrast to the results of Martin and Bhakdi [5], who did not, however, use counterstaining with a dye, especially for detection of dead microorganisms. The use of ultrasonics [28] gave satisfactory results but was too time-consuming for routine measurement. With the chosen combination of Triton X-100 and Tween-20, we obtained results corresponding in quality to those of ultrasonic treatment, with a minimum of handling difficulties and time consumption.

Martin and Bhakdi [11] determined the percentage of dead target cells by measuring the decrease in calcein-AM-fluorescence compared to living microorganisms. Our own experiments, however, showed that the kinetics of the killing were faster than the diffusion of the dye out of the damaged microorganisms. It was not possible to use propidium iodide [29], because this dye stained even living cells after the necessary duration of the killing assay. Finally, with adapted cytometric parameters, satisfactory results were obtained by using EthD-1, in contrast to the findings of Haugland [30], who stated that only mammalian cells could be stained with that dye.

The described assay, in contrast to previous investigations [1–7,31], focuses only on the presence of the different fluorescent signals in the specific tests rather than on the quantity of the detected fluorescence. Therefore, no statement could be made about a possible correlation between the degree of particle uptake and the degree of DHE oxidation. The reason for this is the use of living microorganisms, which, in contrast to, for example, latex particles, makes a calibration impossible. This is due, on the one hand, to the fact that the staining capacity of living microorganisms is a priori variable, and on the other hand, to the fact that the signal may vary as a consequence of diffusion of the dye or morphologic changes of the cells during the assay.

A typical feature of cytofluorometric investigations is the high statistical significance of the results obtained. For every single evaluated parameter we measured about 5000 PMNs. The intra-assay variation of the results is around 1–2%, obtained by investigation of six samples of the same donor, which were independently processed in parallel. The inter-assay variability of a single donor amounts to 5–10% between 10 blood samples investigated over a period of 4 weeks and is therefore normally significantly larger than the intra-assay variability. Concerning the phagocytosis, burst activity and killing, the inter-assay variations of 10 different donors were about 7–14% [8]. It seems that individual variability influences the kinetics of the reaction as well as the end results.

For investigations with bacteria, an optimal target cell/PMN ratio of 50 : 1 had to be used. Furthermore, it proved necessary to perform incubation with the bacteria at 30 °C instead of 37 °C, because at 37 °C the immune reaction with staphylococci is too fast to obtain optimal kinetics. Despite the lower incubation temperature the assay with S. aureus still showed a quicker increase and a nearly two-fold higher final percentage of phagocytically active PMNs. The percentage of ingested microorganisms also showed a quicker increase with bacteria with similar final results for both microorganisms. These results might be due to the higher target-to-effector cell ratio and the resulting higher frequency of collisions between bacteria and PMNs. Furthermore, phagocytes can also ingest more than one yeast, and therefore the maximum achievable proportion of phagocytosing PMNs is already, on a statistical basis, lower when using a PMN/yeast ratio of 1 : 1. In addition, the smaller cell size of bacteria may accelerate the phagocytosis, and with that the percentage of phagocytosing PMNs as well as that of ingested staphylococci.

In contrast to previous assays [5–11], which determined the decrease of the non-phagocytosed target cells, we performed a direct measurement of the ingested microorganisms. The advantage is that perturbation of the measurement by debris is excluded. Investigation of the percentage of PMNs displaying oxidative burst activity after phagocytosis revealed a quicker increase and much higher final results using yeasts. A possible explanation might be the fact that C. albicans is exclusively killed by the oxidative burst [32], and other toxic substances tend to play no role [33]. However, the lower incubation temperature used with bacteria might possibly affect the burst production, but, on the other hand, the fast kinetics of phagocytosis at the same temperature indicate that this may not be the only reason. Using Candida, the kinetics for the oxidative burst-producing PMN correspond to those of ingested yeasts ( Figure 3A). This implies that, after phagocytosis of yeasts, nearly all PMNs start the production of oxygen-dependent toxic substances, while after ingestion of bacteria, even if the oxidative burst takes place, the more important part of the destruction of target cells is mediated by non-oxygen-dependent mechanisms. This view is supported by the finding that the percentage of dead microorganisms showed a quicker increase and slightly higher final results for staphylococci. An additional explanation for these results lies in the observation of other authors [34,35] that, in contrast to bacteria, yeasts are not damaged in plasma, but only within phagocytes.

Our findings demonstrate the necessity of the simultaneous determination of all the investigated parameters and the importance of different target organisms to obtain objective results.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References
  • 1
    Perticarari S, Presani G, Banfi E. A new flow cytometric assay for the evaluation of phagocytosis and the oxidative burst in whole blood. J Immunol Methods 1994; 170: 117 24.
  • 2
    Model M, KuKuruga M, Todd III, R. A sensitive flow cytometric method for measuring the oxidative burst. J Immunol Methods 1997; 202: 105 11.
  • 3
    Vowells S, Sekhsaria S, Malech H, Shalit M, Fleisher T. Flow cytometric analysis of the granulocyte respiratory burst: a comparison study of fluorescent probes. J Immunol Methods 1995; 178: 89 97.
  • 4
    Hirt W, Nebe T, Birr C. Phagotest und Bursttest (Phagoburst), Testkits zur Untersuchung von Phagozytenfunktionen. Wien Klin Wochenschr 1994; 106/8: 250 2.
  • 5
    Martin E & Bhakdi S. Quantitative analysis of opsono-phagocytosis and of killing of Candida albicans by human peripheral blood leukocytes by using flow cytometry. J Clin Microbiol 1991; 29: 2013 23.
  • 6
    Plasman N & Vray B. Quantification of bacterial phagocytosis by flow cytometry and spectrofluorimetry. J Immunol Methods 1994; 174: 195 202.
  • 7
    Rothe G & Valet G. Flow cytometric assays of oxidative burst activity in phagocytes. Methods Enzymol 1994; 233: 539 48.
  • 8
    Husfeld L. Entwicklung einer durchflusszytometrischen Methode zur Messung der Phagozytose-, Burst- und Killingfaehigkeit humaner Phagozyten. Thesis, Ludwig-Maximilians-University of Munich, 1996.
  • 9
    Böhmer R, Trinkle L, Staneck J. Dose effects of LPS on neutrophils in whole blood. Flow cytometric assay of phagocytosis and oxidative burst. Cytometry 1992; 13: 525 31.
  • 10
    Metcalf J, Gallin J, Nauseef W, Root R. Laboratory manual of neutrophil function. New York: Raven Press, 1986.
  • 11
    Martin E & Bhakdi S. Flow cytometric assay for quantifying opsonophagocytosis and killing of staph. aur. by peripheral blood leukocytes. J Clin Microbiol 1992; 30: 2246 55.
  • 12
    Freischlag J, Colburn M, Quinnones-Baldrich W, Moore W. Alteration of neutrophil function by heparine, dexamethasone and enalapril. J Surg Res 1992; 52: 523 9.
  • 13
    White-Owen C, Alexander JW, Sramkoski RM, Babcocck GF. Rapid whole-blood microassay using flow cytometry for measuring neutrophil phagocytosis. J Clin Microbiol 1992; 30: 2071 6.
  • 14
    Nitzan D, Pruzanski W, Saito S, Ranadive N. Modulation of locomotor activity of polymorphonuclear cells by cationic substances and cationic lysosomal fractions from human neutrophils. Inflammation 1985; 94: 375 87.
  • 15
    Stossel T. Quantitative studies of phagocytosis. Kinetic effects of cations and heat-labile opsonine. J Cell Biol 1973; 58: 346 52.
  • 16
    Salih H, Husfeld L, Adam D. Inhibitory effect of heparin on neutrophil phagocytosis and burst production using a new whole blood cytofluorometric method for determination. Eur J Med Res 1997; 2: 507 13.
  • 17
    Buffo J, Hermann M, Soll D. A characterization of pH regulated dimorphism in Candida albicans. Mycopathologica 1984; 85: 21 6.
  • 18
    Schaumann R & Shah P. Effect of amphotericin B alone and or in combination with rifampicin on phagocytosis of Candida species by human polymorphonuclear leukocytes. Methods Find Exp Clin Pharmacol 1992; 14: 753 8.
  • 19
    Nugent K & Couchot K. Effects of sublethal concentrations of amphotericin B on Candida albicans. J Infect Dis 1986; 154: 665 9.
  • 20
    Cantinieaux B, Hariga C, Courtoy P, Hupin J, Fondu P. Staphylococcus aureus phagocytosis. J Immunol Methods 1989; 121: 203 8.
  • 21
    Bandres J, Trial T, Musher D, Rossen R. Increased phagocytosis and generation of reactive oxygen products by neutrophils and monocytes of men with stage 1 human immunodeficiency virus infection. J Infect Dis 1993; 168: 75 83.
  • 22
    Sawyer R. Experimental pulmonary candidiasis. Mycopathologica 1990; 109: 99 109.
  • 23
    Smail E, Cronstein B, Meshulam V, Esposito A, Ruggeri R, Diamond R. In vitro, Candida albicans releases the immune modulator adenosine and a second, high molecular weight agent that blocks neutrophil killing. J Immunol 1992; 148: 3588 95.
  • 24
    Papp-Szabo E, Sutherland C, Josephy P. Superoxide dismutase and the resistance of Escherichia coli to phagocytic killing by human neutrophils. Infect Immun 1993; 49: 1442 6.
  • 25
    Raith L, Csato M, Doboz A. Decreased Candida albicans killing activity of granulocytes from patients with diabetes mellitus. Mycopathologica 1983; 26: 557 63.
  • 26
    Dent G, Leglise M, Pryzwandsky K, Ross D. Simultaneous paired analysis by flow cytometry of surface markers, cytoplasmatic antigens, or oncogene expression with DNA content. Cytometry 1989; 4: 192 8.
  • 27
    Fabrizi S & Robins-Browne R. Elimination of extracellular bacteria by antibiotics in quantitative assays of bacterial ingestion and killing by phagocytes. J Immunol Methods 1993; 37: 201 6.
  • 28
    Phillips W, Shelton M, Hosking C. A simple micro-assay for neutrophil bacterial activity. J Immunol Methods 1992; 26: 187 91.
  • 29
    Bjerknes R. Flow cytometric assay for combined measurement of phagocytosis and intracellular killing of Candida albicans. J Immunol Methods 1984; 72: 229 41.
  • 30
    Haugland R. Handbook of fluorescent probes and research chemicals, 5th edn. Eugene: Molecular Probes, 1992.
  • 31
    Andoh A, Fujiyama Y, Kitoh K, Hodohara K, Bamba T, Hosoda S. Flow cytometric assay for phagocytosis of human monocytes mediated via Fcy-receptors and complement receptor CR1 (CD 35). Cytometry 1991; 12: 677 86.
  • 32
    Leijh PCJ, Van Den Barselaar MT, Van Furth R. Kinetics of phagocytosis and intracellular killing of Candida albicans by human granulocytes and monocytes. Infect Immun 1977; 3: 3 8.
  • 33
    Ganz T, Selset M, Szklarek D et al. Defensins: natural peptide antibiotics of human neutrophils. J Clin Invest 1985; 76: 1427 35.
  • 34
    Sasada M & Johnston R. Macrophage microbicidal activity: correlation between phagocytosis associated oxidative metabolism and the killing of Candida by macrophages. J Exp Pathol 1980; 152: 85 9.
  • 35
    Shimokawa O & Nakayama H. Increased sensitivity of Candida albicans cells accumulating 14-α-methylated sterols to active oxygen: possible relevance to in vitro efficacies of azole antifungal agents . Antimicrob Agents Chemother 1992; 31: 1626 9.