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CAVEAT LECTOR
PH Nibbering1, E Broug-Holub2, AC Bezemer1, R Jansen1, JGJ van de Winkel3, MF Geertsma1.
1 Dept. of Infectious Diseases, University Hospital, Leiden, The Netherlands.
2 Dept. of Cell Biology and Immunology, Free University, Amsterdam, The Netherlands.
3 Dept. of Experimental Immunology, University Hospital, Utrecht, The Netherlands.
Received 12/7/95; Accepted 12/29/95; On-line 1/1/96
Binding and phagocytosis of S. aureus by FcgammaRIIa-expressing fibroblasts:
Microbiological assessments revealed that heat-inactivated serum-opsonized S. aureus, but not non-opsonized S. aureus, were phagocytized by FcgammaRIIa-expressing cells (Figure 1).
Equal volumes of 5x107/ml of heat-inactivated serum-opsonized S.aureus (Black circles) or non-opsonized S. aureus (White circles) and 1x107/ml of FcgammaRIIa-expressing fibroblasts HBSS-gel were incubated at 37°C under slow rotation. At various intervals thereafter, samples of this suspension were removed and centrifuged to separate extracellular from cell-associated bacteria. Subsequently, the number of extracellular viable bacteria was determined microbiologically and the decrease in the number of extracellular bacteria, i.e. phagocytosis, was calculated.
Because this microbiological assay does not allow discrimination between cell-adherent and intracellular bacteria, phagocytosis was assessed by using FITC-labeled bacteria in combination with agents that modify the fluorescence of cell-adherent bacteria without affecting the fluorescence of intracellular bacteria. Incubation of FcgammaRIIa-expressing cells with different concentrations of FITC-labeled heat-inactivated serum-opsonized S. aureus for various intervals revealed that a cell-to-bacterium ratio of 1:5 and an incubation period of 90 min were optimal for quantitation of phagocytosis by FACS analysis (results not shown). All further experiments were performed under these conditions unless specified otherwise. Using trypan blue to quench the fluorescence of cell-adherent bacteria (17) it was found that FcgammaRIIa-expressing cells efficiently phagocytized opsonized S.aureus (Figure 2).
FcgammaRIIa-expressing cells were incubated for 90 min at 37°C with FITC-labeled heat-inactivated serum-opsonized S.aureus at a cell-to-bacterium ratio of 1:5. Free bacteria were removed and the cells resuspended in 0.02 M acetate buffer. Half of this cell-suspension was centrifuged and then resuspended in acetate buffer and the other half resuspended in acetate buffer containing 1 mg trypan blue/ml to quench the fluorescence of cell-adherent bacteria. Subsequently, the fluorescence of cells was measured on FACScan. Dark grey curves represent fluorescence from both cell-adherent and intracellular bacteria, light grey curves reveal fluorescence from intracellular bacteria and white curves indicate non-specific fluorescence of cells. Results are representative for three individual experiments.
Phagocytosis but not binding of bacteria to FcgammaRIIa-expressing cells was blocked when incubations were performed at 4°C; preincubation of 1x107 FcgammaRIIa-expressing cells with 10 µg/ml of the cytoskeleton inhibitor cytochalasin E for 5 min at 37°C led to a largely decreased phagocytosis of bacteria, although binding was not affected (results not shown).
For accurate determination of the percentage of intracellular bacteria, FcgammaRIIa-expressing cells that had been incubated with FITC-labeled serum-opsonized bacteria were examined by fluorescence microscopy using ethidium bromide to discriminate between intracellular and cell-adherent bacteria (Figure 3).
FcgammaRIIa-expressing cells were incubated for 90 min at 37°C with FITC-labeled heat-inactivated, serum-opsonized S.aureus. Subsequently, free bacteria were removed and a sample of the cell-suspension was mixed with ethidium bromide to stain the extracellular bacteria. Microscopical examination allowed identification of cell-adherent (orange) and intracellular (green) S. aureus.
In agreement with the results obtained by FACS analysis, binding of bacteria to FcgammaRIIa-expressing cells was observed after incubation at 37°C and 4°C and after preincubation of cells with cytochalasin E. Phagocytosis of bacteria was only observed when FcgammaRIIa-expressing cells were incubated with opsonized bacteria at 37°C (Table 1).
| Phagocytosis | Cells with bacteria (%) | Intracellular bacteria (number of bacteria/cell) | Cell-adherent bacteria (number of bacteria/cell) |
|---|---|---|---|
| at 37°C | 90±2 | 5.6±1.8 | 3.8±2 |
| at 4°C | 78±8 | 0.1±0.1 | 6.9±2 |
| Cytochalasin E pre-incubated cells* | 85±6 | 0.5±0.2 | 5.9±2 |
| FcgammaRIIa-expressing cells were incubated for 90 min at 37°C with
FITC-labeled heat-inactivated serum-opsonized S.aureus at a cell-to-bacterium
ratio of 1:10. Subsequently, free bacteria were removed and a
sample of the cell-suspension was mixed with ethidium bromide
to discriminate between intracellular and cell-adherent bacteria.
In each preparation, 50 cells were examined and the percentage
of cells with bacteria as well as the numbers of cell-adherent
and intracellular bacteria was determined by fluorescence microscopy.
Results are means ± SEM of four experiments.
* FcgammaRIIa-expressing cells were pre-incubated with 10 µg/ml of cytochalasin E for 5 min at 37°C. | |||
The percentage of phagocytizing cells was 90±2% (n=4), which is similar to the percentage cells expressing the human FcgammaRIIa (89±3%), as determined by FACS analysis. Together, these data indicate that FcgammaRIIa-expressing fibroblasts are able to phagocytize heat-inactivated, serum-opsonized bacteria.
Role of FcgammaRIIa in the phagocytosis of opsonized S. aureus:
To obtain more insight into the role of FcgammaRIIa in phagocytosis of opsonized bacteria by FcgammaRIIa-expressing cells, two sets of experiments were performed. First, phagocytosis of FITC-labeled, non-opsonized S. aureus by FcgammaRIIa-expressing cells was determined. The results revealed that about twenty-fold less non-opsonized than serum-opsonized bacteria were phagocytized by FcgammaRIIa-expressing cells, i.e. the mean number of intracellular non-opsonized bacteria amounted to 0.3±0.1/cell and serum-opsonized bacteria to 5.6±1.8/cell (n=4). Second, we compared phagocytosis of FITC-labeled serum-opsonized bacteria by FcgammaRIIa-expressing and parental fibroblasts. The results revealed that, in contrast to FcgammaRIIa-expressing cells, parental fibroblasts hardly phagocytized opsonized S. aureus, i.e. 5±1x104 bacteria/5x106 cells (n=4). Together, these data indicate the FcgammaRIIa mediates the phagocytosis of opsonized bacteria by FcgammaRIIa-expressing cells.
Involvement of PTK in the phagocytosis of serum-opsonized S. aureus by FcgammaRIIa-expressing cells:
Since activation of protein tyrosine kinases (PTK) is implicated in FcgammaRIIa-mediated phagocytosis by phagocytic cells (9, 25-27), the effect of inhibitors of PTK on the phagocytosis of serum-opsonized S. aureus by FcgammaRIIa-expressing cells was investigated. The results revealed that the PTK-inhibitor tyrphostin-47, but not its inactive analog tyrphostin-1, inhibited (p<0.05) phagocytosis of S. aureus by FcgammaRIIa-expressing cells (Figure 4a). Neither of tyrphostins (p>0.1) affected binding of the bacteria to these cells (data not shown).
To find out whether FcgammaRIIa cross-linking results in tyrosine phosphorylation of cellular proteins, the pattern of tyrosine phosphorylated proteins after stimulation of cross-linking of FcgammaRIIa on cells was determined. The results showed that FcgammaRIIa cross-linking induced within 30 sec an increase in the tyrosine phosphorylation of muliple proteins, which became dephosphorylated after 2 min of stimulation (Figure 4b). Incubation of cells with tyrphostin-47 reduced the tyrosine phosphorylation of the various proteins by FcgammaRIIa cross-linking (results not shown), as reported previously (9). Furthermore, incubation of cells with mAb LeuM3 (anti-human CD14 antibody, Becton Dickinson (San Jose, CA), serving as an isotype-matched control) followed by bridging antibody did not induce an increase in tyrosine phophorylation of proteins (Figure 4b, control).
a. Effect of tyrphostin-47 on phagocytosis of S. aureus by FcgammaRIIa-expressing cells.
FcgammaRIIa-expressing cells were pre-incubated for 30 min at 37°C with 10 µM tyrphostin-47 (Black circles), tyrphostin-1 (Black squares) or PBS (White circles) and then incubated with FITC-labeled heat-inactivated, serum-opsonized S. aureus. Next, free bacteria were removed and a sample of the cell-suspension was mixed with ethidium bromide and the number of intracellular (green) and cell-adherent (orange) bacteria was determined in 50 cells by microscopic examination. Results are mean number of intracellular bacteria/FcgammaRIIa-expressing cell ± SEM.
b. Effects of FcgammaRIIa cross-linking on tyrosine phosphorylation of proteins in cells.
FcgammaRIIa-expressing cells were incubated for 3 min with PBS or with 2µg/ml of anti-FcgammaRIIa antibody IV-3 and then 25µg bridging secondary antibody was added to achieve cross-linking of FcgammaRIIa. At indicated intervals, the reaction was stopped by addition of 2 x concentrated SDS sample buffer at 100°C, and the the lysates were subjected to 7.5% SDS-PAGE, followed by Western blot analysis with using-phosphotyrosine mAb 4G10 and 125I-labeled protein A. The results of one experiment quantified on a PhosphorImager are respresentative of three individual experiments are given.
Intracellular killing of S. aureus by FcgammaRIIa-expressing cells:
To find out whether FcgammaRIIa-expressing cells were capable of intracellular killing of S. aureus, cells that had ingested opsonized S. aureus were incubated with serum. It is known that serum is obligatory for obtaining maximal killing of ingested S. aureus by human monocytes and mouse macrophages (8, 21). The results revealed that serum efficiently stimulated the intracellular killing of
S. aureus by FcgammaRIIa-expressing cells (Figure 5).
FcgammaRIIa-expressing fibroblasts were incubated with serum-opsonized S. aureus for 90 min. Then, extracellular bacteria were removed by washing and killing of the internalized bacteria was initiated by the following stimuli: normal human serum (Black squares), serum from patients with agammaglobulinemia (White squares), heat-inactivated serum (Black circles) or, as control HBSS (White circles). At indicated intervals, a sample was taken and the number of viable bacteria was determined. Results are means ± SEM of 5-8 experiments.
To determine whether oxygen-dependent microbicidal mechanisms are involved in the intracellular killing of opsonized S. aureus by FcgammaRIIa-expressing fibroblasts, cells containing S. aureus were incubated with the NADPH oxidase-inhibitor DPI before stimulation with normal human serum. The results showed that DPI did not affect (p>0.1) the killing process, i.e. intracellular killing by DPI-treated cells and control cells at 90 min was respectively 94±2% and 96±2%, (n=3). In agreement with these observations, FcgammaRIIa-expressing cells did not (p>0.1) produce H2O2 upon PMA stimulation or after addition of 100-fold excess of opsonized S. aureus. Upon stimulation of the cells with 100-fold excess serum opsonized-bacteria, no NO2 - -production was observed (n=3). The combination of 10 µg/ml of lipopolysaccharide, 100 units/ml of recombinant rat interferon-gamma and 100 units/ml of recombinant mouse tumor necrosis factor-alpha stimulated NO2- production by FcgammaRIIa-expressing cells. This amount was 1±1 µmol NO2 - /mg cell protein for non-stimulated cells and 34±4 µmol NO2 - /mg cell protein for stimulated cells (n=3).
The possible role of FcgammaRIIa in this killing process was investigated by incubating FcgammaRIIa-expressing cells that had ingested bacteria with heat-inactivated serum, purified IgG, anti-FcgammaRII mAb and bridging secondary antibody. The results revealed that heat-inactivated serum (Fig. 5) as well as the other FcgammaR-specific stimuli (results not shown) did not trigger intracellular killing of S. aureus by these cells. Contrary, serum from patients with from agammaglobulinemia stimulated the killing process (Figure 5). These data indicate that heat-labile serum factor(s) stimulate the intracellular killing of S. aureus by FcgammaRIIa-expressing cells.