Vol. 30, No. 9

JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1992, p. 2246-2255 0095-1137/92/092246-10$02.00/0 Copyright X 1992, American Society for Microbiology

Flow Cytometric Assay for Quantifying Opsonophagocytosis and Killing of Staphylococcus aureus by Peripheral Blood Leukocytes EDITH MARTIN* AND SUCHARIT BHAKDI Institute of Medical Microbiology, Johannes Gutenberg University, Hochhaus Augustusplatz, W-6500 Mainz, Germany Received 9 January 1992/Accepted 21 May 1992

We describe a novel flow cytometric method for quantifying opsonophagocytosis and killing of Staphylococcus aureus in cell-rich plasma obtained after dextran sedimentation of erythrocytes. To analyze opsonophagocytosis, phagocytes were labeled with a phycoerythrin-conjugated monoclonal antibody and were incubated with viable staphylococci containing carboxyfluorescein as a vital fluorescent dye. Phagocytosing cells assumed a dual, orange-green fluorescence. The relative numbers of bacteria associating with phagocytes could be determined by quantifying the decrease of free green fluorescent particles. A parallel incubation of fluorescent bacteria with unlabeled cell-rich plasma was performed to assess phagocytic killing. Blood cells were lysed with 3-[(3-cholamidopropyl)-dimethyl-ammoniol-1-propanesulfonate. This detergent spared viable bacteria, and residual green fluorescent particles were counted. The decrease in the number of these particles relative to the controls yielded the degree of killing. At bacteria-to-phagocyte ratios of 1:1 and 10:1, approximately 36 and 75% of the phagocytes participated in opsonophagocytosis, respectively. Over 90%o of the staphylococci were phagocyte associated after 30 to 60 min. Killing rates were on the order of 66% + 12% and 80%o ± 7% after 1 and 2 h of incubation, respectively. These numbers, which were confirmed by colony countings, were significantly lower than those reported in the majority of past reports.

Phagocytes play a central role in the elimination of most extracellular pathogenic microorganisms, and any impairment of their function therefore predisposes an individual toward local and systemic bacterial and fungal infections. It is all the more remarkable that despite impressive advances in diagnostic technologies, there is no generally accepted method for the routine assessment of phagocyte function. This has not been due to a lack of effort. Many investigators have developed clever approaches to assaying opsonophagocytosis and killing of classical target organisms such as Staphylococcus aureus, Escherichia coli, and Candida albicans. The "gold standard" for assaying overall killing involving colony countings has been supplemented by improved procedures that allow one to quantify the association of target particles with phagocytes (3, 4, 14, 16, 21, 23, 25, 29, 34, 42, 43, 50, 52), differentiate between attached and ingested particles (29, 40), and distinguish killed from viable intracellular organisms (4, 25, 26, 31, 42-44). Many of these methods involve the use of radioisotopes (3, 14, 16, 21, 40, 50, 52) or dyes (25, 26, 42) and fluorescent dyes (4, 30, 33, 34) to label or stain the microorganisms. However, their handling usually requires considerable expertise, because cumbersome elements such as the need to isolate phagocytes from whole blood, the use of radioisotopes, the requirement for differential centrifugation steps, and final quantitation by microscopic countings or plating out and determination of CFU are inherent in one form or the other. Several groups have recently introduced flow cytometric approaches to the study of phagocytosis. Methods have been described for analyzing the uptake of different target particles (e.g., bacteria [2, 9, 46], yeasts [6, 11, 13, 51], latex beads [1, 12, 35, 36, 41, 45]), for detecting oxidative products (17, 37, 47) and *

Corresponding author.

the degradation of proteins (18), and for quantifying the killing of yeast cells (5, 8). A simple and rapid assay was developed in our laboratory. The assay minimized handling difficulties and measured both opsonophagocytosis and killing of C. albicans (32). We wished to complement the technique described above with an analogous assay that would permit bacteria to be used as target cells. Here we present the results of a flow cytometric assay that involved the use of viable S. aureus. The method represents an advance over earlier techniques since both opsonophagocytosis and killing can be quantified. Principle of the assay. The assay described here is conceptually related to the Candida killing assay (32) and uses S. aureus cells that are labeled with a vital fluorescent dye. The dye precursor is a membrane-permeable, esterified derivative of carboxyfluorescein that diffuses rapidly into the cells. Intracellular esterases cleave the molecule to its nonpermeable, fluorescent derivative (7) that remains entrapped within the bacteria without impairing their viability. Granulocytes and monocytes present in cell-rich plasma (CRP) are labeled orange with a phycoerythrin-tagged monoclonal antibody. Opsonophagocytosis is quantified by recording the appearance of dually labeled phagocytes. We describe how the flow cytometer readings can be adjusted so that the decrease in free, green fluorescent particles can be determined simultaneously. Killing of phagocytosed S. aureus is assayed in parallel samples with unlabeled phagocytes by solubilizing blood cells with a detergent which does not harm viable bacteria. The viable bacteria are liberated and counted as free, green particles, whereas dead staphylococci disappear from the respective region. The decrease in the numbers of green fluorescent particles directly reflects the degree of phagocytic killing. 2246

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MATERIALS AND METHODS S. aureus Wood 46 was cultured for 6 h in tryptic soy broth. One and a half milliliters of bacterial culture was centrifuged, and pelleted cells were resuspended in 1 ml of NaCl and were incubated for 30 min at 37°C in an Eppendorf thermomixer 5436 (Eppendorf Inc., Hamburg, Germany) with 30 ,ul of a 200 ,uM stock solution of bis-carboxyethyl-

carboxyfluorescein pentaacetoxy-methylester (BCECF-AM; Becton and Dickinson, Heidelberg, Germany). After three washes in NaCl, the cells were suspended in saline and were kept on ice. The cell densities were optically determined by using McFarland standards. Cell-free plasma and CRP were obtained from healthy donors as described previously (32). The numbers of phagocytes were determined by microscopic counting. Assay for opsonophagocytosis. CRP was incubated with phycoerythrin-conjugated monoclonal antibody My7 (Coulter Electronics, Krefeld, Germany) at a final antibody concentration of 5 ,ug/ml for 20 min on ice. Cells were then centrifuged at 500 x g for 5 min at 4°C, washed once with phosphate-buffered saline (PBS), and resuspended in an identical volume of cell-free plasma. Labeled staphylococci were adjusted to 10-fold of their desired final concentration. The ratios of bacteria to phagocytes were generally 1:1 or 10:1. In a few experiments, ratios of 100:1 were also used. Ten volumes of CRP were admixed with 1 volume of bacteria in Eppendorf tubes, and a 50-p1 sample was immediately pipetted into 950 p1 of ice-cold PBS containing 0.1% paraformaldehyde (9, 24) to give the 0-min value. Aliquots of 50 RI of the remaining suspension were pipetted into Eppendorf tubes and incubated in a thermomixer (5436; Eppendorf) with shaking at 1,100 rpm. At the end of the desired incubation times, 950 p.l of ice-cold PBS with 0.1% paraformaldehyde was pipetted into the respective tubes, and the samples were kept on ice until analysis. Quantification of phagocytic killing. Unlabeled CRP samples were admixed with bacteria at the desired concentrations as described above, and 100-pl aliquots were pipetted into Eppendorf tubes. Paired samples were prepared for every time point; one was kept on ice as the control, and the other was incubated at 37°C. In several experiments, we ascertained that no bacterial multiplication occurred within 1 h at 37°C in the presence of cell-free plasma, so that the results for the iced controls were valid. After the given incubation periods, 100 RI of an aqueous 1% 3-[(3-cholami-

dopropyl)-dimethyl-ammonio]-l-propanesulfonate (CHAPS; Sigma, Munich, Germany) solution was added. After 5 min of incubation at 37°C and 1,100 rpm in the Eppendorf thermomixer, the samples received 0.8 ml of distilled water and were incubated for another 3 min at 37°C and 1,100 rpm. Thereafter, 50 pl of PBS containing 2% paraformaldehyde

added and the samples were kept on ice until analysis. To correlate this method with the standard procedure of colony counting, 20 ,ul of the samples was taken prior to fixation with paraformaldehyde, diluted 1:200 in saline, and plated out in duplicate. Flow cytometry. Flow cytometric analyses were performed with a FACScan flow cytometer (Becton and Dickinson) with computer-assisted evaluation of data (Lysis II software). The instrument settings for the flow cytometric phagocytosis assay were as follows. The sideward scatter (SSC) threshold was 220. The detector was set at E00, 406, 736, and 645 for forward scatter (FSC), SSC, fluorescence 1 (FL1), and fluorescence 2 (FL2), respectively. Log parameters were used for FSC, SSC, FL1, and FL2. Compensation

was

2247

for FL1 to FL2 was 2.3%, and compensation for FL2 to FL1 was 27.8%. A logarithmic amplification of all signals was essential in order to reveal simultaneously the relatively small staphylococci and the large phagocytes. RESULTS Analysis of the association of staphylococci with phagocytes. Figure 1 depicts the flow cytometric appearance of phagocytes and staphylococci in CRP. In Fig. 1A, the particles are displayed according to their size (FSC) and granularity (SSC). In Fig. 1B, the same particles are displayed according to granularity and orange fluorescence (FL2), and a region is set around the fluorescent particles (R1) in order to selectively detect the orange-labeled phagocytes. Analogously, staphylococci are selectively detected by displaying the particles according to granularity and green fluorescence (FL1), and a respective region is set around these particles (R2 in Fig. 1C). After these regions have been set, the respective fluorescing phagocytes and staphylococci are filtered out and selectively displayed. This was done by using the parameters FSC and SSC (Fig. 1D). Figure 1D can be superimposed on Fig. 1A, and it is apparent that phagocytes and staphylococci lie in the expected regions; i.e., the phagocytes are large and display high levels of granularity, whereas the staphylococci are small and exhibit low levels of granularity. It is equally apparent that the intended ratio of staphylococci to phagocytes of approximately 10:1 was attained. In Fig. 1E, the particles are displayed according to size and green fluorescence. This plot can be superimposed on Fig. 1C. The staphylococci are revealed as green fluorescent particles with low levels of granularity, whereas the granulocytes have high levels of granularity but are devoid of green fluorescence. This mode of presentation allows one to observe the association of bacteria with phagocytes. Figure 2 demonstrates these principles. Figure 2A to D shows the kinetics of opsonophagocytosis at a bacteria-to-phagocyte ratio of 1:1; Fig. 2E to H depicts an experiment conducted with 10 bacteria per phagocyte. The association between phagocytes and bacteria is well under way after 5 min, as revealed by the appearance of a green fluorescent phagocyte population. After 60 min, the majority of staphylococci have disappeared from the region set for free bacteria, and phagocytes are identifiable in the same plot as green fluorescent particles with high levels of granularity. Note that the green fluorescent phagocytes in Fig. 2H display a slight but significant shift to the right because of their increases in granularity after association with the staphylococci. By gating the phagocytes (counting only particles in R1; Fig. 1B) and analyzing their green fluorescence, it is possible to study their association with bacteria in a quantitative manner. As shown in Fig. 3A, approximately 6% of the phagocytes displayed weak fluorescence at time zero, whereas 77.2% exhibited marked green fluorescence after 30 min of incubation with labeled staphylococci at a ratio of 10 bacteria per cell. Figure 3B gives the results of three kinetic analyses performed at the two bacteria-to-phagocyte ratios. Most of the phagocytes that participated in opsonophagocytosis assumed the green fluorescence approximately 10 min after the samples were spiked with the bacteria. Thereafter, the percentage of phagocytosing granulocytes did not markedly increase. At the ratios of 1:1 and 10:1, approximately 36 and 76% of the phagocytes finally exhibited green fluorescence, respectively. Free staphylococci could be quantified by counting the particles that fell into the respective regions (R2; Fig. 1C)

2248

MARTIN AND BHAKDI

over a defined time period. Figure 4A described the idea underlying this principle. The upper panel of Fig. 4A shows the numbers of events counted over 15 s (thex axis gives the relative time scale) at the beginning of an experiment. Some 2,120 events were counted. After a phagocytosis period of 10 min, the number dropped to 1,562 events, corresponding to a decrease of approximately 22% of free staphylococci. After 60 min, the number was 86 events, corresponding to a decrease of over 95% of free staphylococci. Figure 4B depicts the results of three kinetic experiments performed with bacteria-to-phagocyte ratios of 1:1 and 10:1. It is apparent that the decrease in the numbers of free staphylococci occurred faster at a higher target cell-to-phagocyte ratio and that a 30- to 60-min incubation was required for almost all free staphylococci to disappear. Quantification of killing. To quantify killing, labeled bacteria were incubated with unlabeled CRP, and blood cells were subsequently lysed with CHAPS. Control experiments by both colony countings and flow cytometry showed that the CHAPS detergent did not reduce bacterial viability. In contrast, treatment with 0.5% deoxycholate (3 min) or 1 U of lysostaphin per ml (30 min) killed the bacteria, and this was accompanied by the total loss of fluorescence. After liberation from the phagocytes, bacteria that were not killed retained their green fluorescence, whereas nonviable cells did not reappear as fluorescent particles and were thus lost from the relevant region (Rl in Fig. 5). Killing could therefore be quantified in a straightforward fashion. The principle of this assay is shown in Fig. 5A, with the left pair of panels representing an experiment conducted at a bacteria-tophagocyte ratio of 1:1 and the right pair of panels representing an experiment conducted at a bacteria-to-phagocyte ratio of 10:1. Analysis for killing was performed after 1 h at 0 and 37°C. Note (i) the disappearance of all the large particles because of solubilization of blood cells with CHAPS (compare Fig. 5A with Fig. 1C) and (ii) the disappearance of green fluorescing particles in the lower panels because of phagocytic killing of the staphylococci. Figure SB shows the results obtained by counting the number of green fluorescent particles that fell into the set region Rl of Fig. 5A over a defined time period. Note that the numbers of events counted in the two controls (0°C) indeed corresponded to the 10-fold difference in bacterial loads. Thus, 488 events were counted over 30 s at the low (1:1) ratio, whereas 3,057 events were counted over 20 s at the high (10:1) ratio. After 1 h at 37°C, the numbers of events counted in the set region were 220 and 1,296, corresponding to approximately 55 and 60% killing, respectively. Parallel quantification of killing was performed by plating out the detergent-treated samples and determination of CFU. These control determinations unfailingly yielded values that were in excellent agreement with the flow cytometric data. In a total of 24 experiments, the values obtained by colony counting and flow cytometry

J. CLIN. MICROBIOL.

never varied from each other by more than 7%. The results of a total of 31 experiments conducted at a bacteria-tophagocyte ratio of 10:1 are shown in Fig. 6. Killing rates of 66% ± 12.5% and 79% ± 7.5% were observed after 1 and 2 h of incubation, respectively, whereby most of the killing occurred during the first 30 min. At the lower bacteria-tophagocyte ratio, killing rates did not differ significantly (data not shown). Additional experiments were performed. In those experiments, cells were lysed with 10 volumes of distilled water rather than with CHAPS, with or without subsequent brief sonication. In those cases, however, we invariably noted that lysis of the granulocytes was incomplete, and some bacteria thus remained associated with the blood cells. The killing rates after 60 min were significantly higher (85 to 90%). However, because of incomplete liberation of the bacteria, these data were deemed unreliable.

DISCUSSION Several novel elements distinguish the procedure described here from earlier flow cytometric assays for phagocytosis. BCECF-AM was introduced as a vital fluorescent dye which does not impair bacterial viability. By applying logarithmic amplification of all signals, both phagocytes and free staphylococci could be detected simultaneously. The method of counting particles within a set region over time made it possible to quantify the disappearance of free staphylococci for analysis of both opsonophagocytosis and killing. This procedure required the use of an appropriate software program (Lysis II; Becton and Dickinson Laboratories). Finally, an optimal method had to be devised to liberate the bacteria from the phagocytes. CHAPS was identified as an ideal solubilizing agent, whereas other detergents such as deoxycholate (used in our previous work with C. albicans [32]) or octylglucoside damaged viable staphylococci and caused loss of their green fluorescence. Phagocytic killing was found to be accompanied by the disappearance of green fluorescent particles in the detergent-treated samples. This may have been due to frank dissolution of the bacteria within the phagocytes, to the egress of the marker from damaged cells, or to bleaching effects because of myeloperoxidase-mediated chlorination of fluorescein (20). Together, the innovations described above led to the development of a phagocytosis test that is easy to perform and that requires neither special skills nor handling of hazardous materials. The assay provides quantitative data on opsonophagocytic function at the individual cell level and permits the extent of target cell killing to be quantified. Manipulation of blood samples is minimal. Hence, artificial impairment of phagocyte function because of isolation procedures can probably be excluded. Any defect in the phagocytosis system, whether the defect is derived from opsonic

FIG. 1. Region setting for analyzing opsonophagocytosis of BCECF-AM-stained, green fluorescent staphylococci by monoclonal antibody My7-labelled, orange fluorescent phagocytes. Some 10,000 events were acquired. Logarithmic amplifications of size and granularity were essential in order for both the phagocytes and the relatively small bacteria to be detected simultaneously. Nonfluorescent blood cells and undefined material could be excluded from the analysis by gating the fluorescent particles. (A) The relative sizes (FSC) and the relative granularities (SSC) of all cells are shown. (B) To detect phagocytes, events are shown according to their granularities (SSC) and their relative orange fluorescences (FL2). The phagocytes display a pronounced granularity with orange fluorescence because of the monoclonal antibody My7 and are seen as a distinct population (Rl). (C) To detect the staphylococci, events are shown according to their granularities (SSC) and their relative green fluorescences (FL1). The bacteria exhibit minimal granularities and a bright green fluorescence and are similarly visible as a distinct population (R2). (D) Phagocytes (Rl) and staphylococci (R2) of the same sample are shown according to their relative sizes (FSC) and granularities (SSC), whereas all other particles were excluded from the analysis. (E) Same as panel D, but the parameters selected were granularity (SSC) and green fluorescence (FL1).

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FIG. 5. To analyze the killing of staphylococci, blood cells were lysed with the detergent CHAPS. (A) A total of 4,000 particles were counted and depicted according to their relative granularities (SSC) and relative green fluorescences (FL1). Note the absence of particles with high granularities in contrast to Fig. 1, which indicates successful lysis of the blood cells. A narrow region (Rl) was set around the viable staphylococci, which exhibited green fluorescence (FL1) and minimal granularity (SSC). Examples are given for a 1-h incubation on ice and at 37°C at bacteria-to-phagocyte ratios of 1:1 (left panels) and 10:1 (right panels). The decrease in the numbers of events in Rl directly reflects the extent of killing. (B) To determine the exact killing rate, events that fell into region Rl were counted over a defined time period (x axis). The numbers of events that fell into region Rl are displayed on they axis and are listed at the bottom of each graph. The graphs on the left and right depict results of incubations at 0 and 37°C, respectively. The total duration of the selected time period and the ratio of bacteria to phagocytes are given at the upper left of each panel.

entiation is required, it may be achievable by using a third fluorescent marker, e.g., a labeled antibody directed against a cell wall component of the bacteria (35, 46), or by quenching with an appropriate agent (6, 9, 13, 19). Although the major thrust of the study described here was to develop a new technology, one significant finding that deserves special mention surfaced. The mean extent of phagocytic killing of S. aureus observed by use of our method was significantly lower than the values given in the majority of published reports. It is almost dogma that 85 to 95% killing of staphylococci occurs within 30 to 60 min of their uptake by granulocytes (2, 27, 29, 31, 38, 40, 48, 50). This high efficiency of killing occurred only sporadically in our experiments. Since our flow cytometric analyses were all verified by colony countings, we needed to search for a basic methodological flaw in either the present protocol or in those of past reports. This search led us to identify the cell lysis step as a major source of error. By flow cytometry, the method of lysing cells with distilled water with or without short sonication was found to be inefficient. When we used the water lysis technique in combination with colony countings, we observed apparent killing rates of 80 to 90%. However, the values dropped to 60 to 70% when parallel determinations were performed with detergent solubilization. We then became aware of a short but important note by Gargan et al. (15), who reported similar observations. Those investigators discovered that water lysis is only effective at pH 11 and that the killing rates for S. aureus Wood 46 100

assessed under such optimized conditions fall from over 80% to approximately 70% because of the true liberation of the bacteria from the phagocytes. Our numbers presented here agree excellently with those recent findings. In a related context, we found that an increase in the bacteria-to-phagocyte ratio to 100:1 generated artifacts because the phagocytes aggregated and were not totally solubilized even with the detergent. Apparent killing rates of over 90% were then observed. This finding cautions against the use of bacterial loads that are too high. In sum, the true extent of killing of different bacteria by phagocytes probably requires careful reinvestigation. It is also not clear whether there is really a significant difference between the capacity of neutrophils to kill staphylococci as opposed to their ability to kill C. albicans (10, 25, 26, 28, 32). The majority of phagocytosing cells became distinguishable as a greenly fluorescing population approximately 10 min after the addition of bacteria to CRP. In contrast, the disappearance of free bacteria displayed retarded kinetics. At the higher bacteria-to-phagocyte ratio, 20 to 30 min elapsed before >90% of the staphylococci became associated with the phagocytes. At the low bacterial loads, uptake kinetics appeared to be even slower, with over 90% disappearance of free particles occurring after 60 min. Our working hypothesis is that initiation of the phagocytic process in a cell augments its activity. Once committed, the respective cell population is therefore preferentially engaged in the

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2254

MARTIN AND BHAKDI

ongoing phagocytic process. Thus, phagocytosis of staphylococci may not occur in a simple, random fashion. The flow cytometric technique described here permits opsonophagocytosis of staphylococci to be studied with exquisite precision. In addition to its potential usefulness in the routine clinical laboratory, the method will facilitate further studies on unsolved and partially controversial aspects regarding the host factors that are involved in phagocytic defense and on the bacterial determinants that counteract these processes. In the case of staphylococci, such factors include bacterial capsules (22, 49) and protein A (39). The method should be extendable to any microorganism that takes up BCECF-AM or a similar dye, if one can find a suitable detergent that solubilizes blood cells to allow subsequent differentiation between killed and viable bacteria.

ACKNOWLEDGMENTS This work was supported by the Deutsche Forschungsgemeinschaft (SFB 311) and the Verband der Chemischen Industrie. We thank Monika O'Malley for excellent secretarial assistance. 1.

2.

3.

4.

5.

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