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

Culture of FcgammaRIIa-expressing and parental 3T6 fibroblasts:

Mouse 3T6 fibroblasts were transfected with the pPW3 FcgammaRIIa cDNA (in pcDX vector) and pSV3_gpt using a Ca₃(PO4)₂ precipitation method and mycophenolic acid selection, as described (11). FcgammaRIIa-expressing 3T6 cells and parent 3T6 cells were cultured in RPMI 1640 medium (Gibco, Irvine, UK) supplemented with 5% heat-inactivated fetal calf serum (Gibco), 0.2 µg aminopterin/ml (Sigma Chemical Co., St. Louis, MO), 10 mg NaHCO₃/ml, 2.3 µg deoxycytidine/ml (Sigma), 15 µg hypoxanthine/ml (Sigma), 20 µg/ml mycophenolic acid/ml (Sigma), 2 mM pyruvate (Gibco), 5 µg thymidine/ml (Sigma), 10 µg xanthine/ml (Fluka, Buchs, Switzerland) and 50 µg gentamycine/ml in 5% CO₂-incubator at 37°C. Next, the cells were harvested with 0.05% (wt/vol) trypsin (Sigma) and 0.01% (wt/vol) EDTA in phosphate-buffered saline (PBS; pH 7.4). Before being investigated in the various assays, the cells were washed with RPMI 1640 medium.

Opsonization of bacteria:

Staphylococcus aureus (type 42D) were cultured overnight at 37°C in Nutrient Broth no.2 (Oxoid Ltd., Basingstoke, UK), harvested by centrifugation at 1,500 x g for 10 min and then washed three times with PBS. For opsonization, 1x10⁸ bacteria were incubated for 30 min at 37°C under slow rotation (4 rpm) in 1 ml HBSS supplemented with 0,1% (wt/vol) gelatin (HBSS-gel) and 15% (vol/vol) heat-inactivated human serum from the blood of healthy donors with bloodgroup AB. After two washes with ice-cold HBSS-gel, the bacteria were suspended in this medium at a concentration of 5x10⁷ bacteria/ml.

Microbiological assessment of phagocytosis of S. aureus by cells:

Phagocytosis of opsonized S. aureus by cells was determined as described before (16). In short, equal volumes of 5x10⁷ serum-opsonized S. aureus/ml and 1x10⁷ cells/ml HBSS-gel were incubated at 37°C and 4 rpm. At various intervals, ranging from 0 to 90 min, a sample of this suspension was removed, centrifuged for 4 min at 110 x g, and the number of bacteria in the supernatant was determined microbiologically. Phagocytosis is expressed as the percentage decrease in the number of extracellular bacteria.

FITC-labeling of S. aureus:

S. aureus were incubated at a concentration of 1x109/ml with 0.1-1.0 mg fluorescein isothiocyanate/ml (FITC, Sigma) in 50 mM NaHCO₃ in 100 mM NaCl (buffer, pH 9.0) for 20 min at room temperature in the dark. The bacteria were then washed twice with PBS to remove free FITC and resuspended in HBSS-gel to a concentration of 1x10⁸ bacteria/ml.

Phagocytosis of FITC-labeled serum-opsonized S. aureus by cells:

FACS analysis was used to quantitate phagocytosis of FITC-labeled opsonized S. aureus by cells. To determine the optimal ratio for phagocytosis, 5x10⁶ cells/ml HBSS-gel were incubated with various numbers of FITC-labeled serum opsonized S. aureus (ratio of cells:bacteria = 1:1, 1:2, 1:5, and 1:10) under slow rotation at 37°C for 90 min. Next, the non-cell-associated bacteria were removed by centrifugation at 500 x g at 4°C for 5 min and two washes with ice-cold PBS. The cells were resuspended in 0.02 M acetate buffer pH 5.8 at a concentration of 1x10⁷/ml. To distinguish between cell-adherent and intracellular bacteria, half of the cell-suspension was centrifuged, the pellet resuspended in an equal volume of 1 mg trypan blue/ml (Merck, Darmstadt, Ger) acetate buffer (17). As control, the other half of the cell-suspension in 0.02 M acetate buffer was kept on ice. Mean fluorescence intensity (MFI) was measured on a FACScan (Becton Dickinson, Mountain View, CA) equipped with an argon-ion laser (excitation wavelength at 488 nm, laser power 300 mW) and a band pass filter of 530 nm.

Incubation of cells that had phagocytized FITC-labeled opsonized bacteria with ethidium bromide and subsequent examination of these cells by fluorescence microscopy allowed distinction of cell-adherent and intracellular bacteria (18). Briefly, after removal of extracellular bacteria, FcgammaRIIa-expressing cells containing bacteria were resuspended in PBS. Next, a sample of this suspension was mixed with a solution containing 25 µg ethidium bromide/ml and a cytocentrifuge preparation was made for microscopical analysis with an Orthoplan fluorescence microscope (Leitz, Wetzlar, Germany). The numbers of cell-adherent (orange) and intracellular (green) bacteria per cell and the percentage of phagocytic cells were determined.

Treatment of cells with a protein tyrosine kinase (PTK) inhibitor:

To investigate whether PTK activity was essential for FcgammaRIIa-mediated phagocytosis of opsonized S. aureus, FcgammaRIIa-expressing cells were incubated for 30 min at 37°C with 10 µg tyrphostin-47 (Calbiochem, La Jolla, CA), a competitive inhibitor of the binding of tyrosine to PTK (19), prior to addition of opsonized bacteria. As a control, cells were incubated with 10 µg/ml of tyrphostin-1 (19), an inactive analog of tyrphostin-47.

Assessment of tyrosine phosphorylation of cellular proteins:

Tyrosine phosphorylation of cellular proteins in FcgammaRIIa-expressing cells after FcgammaRIIa cross-linking was determined by the method of Connelly et al. (20) with minor modifications. In brief, 5x10⁷ cells/ml HBSS were stimulated at 37°C for the indicated intervals by FcgammaRIIa cross-linking; the reaction was terminated by mixing an 80-ml aliquot of this mixture with 100 ml of 2 x concentrated sample buffer (20% sodium dodecyl sulfate, 0.1 M dithiotreitol, 10% 2-mercaptoethanol, 10% glycerol and 0.005% bromophenol blue in 10 mM Tris buffer pH 7.0), followed by heating at 100°C for 5 min. Cell lysates were subjected to electrophoresis in 7.5% SDS-PAGE, and proteins were electrophoretically transferred to nitrocellulose (Whatmann, Maidstone, UK). After overnight exposure to 2% BSA blots were incubated for 2 hours with 1 µg/ml antiphosphotyrosine antibody 4G10 (Upstate Biotechnology Inc, Lake Placid, NY) in Tris buffer (pH 8.0). After binding of mAb 4G10 to tyrosine phosphorylated proteins, the blot was incubated with 1 µCi/m of ¹²⁵I-labeled protein A (Amersham, Bucks, UK)/ ml Tris buffer. The blot was analyzed with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Intracellular killing assay:

Intracellular killing of S. aureus by FcgammaRIIa-expressing cells was determined as described (21) with minor modifications. In short, equal volumes of 1x10⁷ cells/ml and 5x10⁷ opsonized bacteria/ml of HBSS-gel were mixed and then incubated for 90 minutes at 37°C under slow rotation. Phagocytosis was stopped by shaking the tubes in crushed ice. Non-ingested bacteria were removed by differential centrifugation and two washes at 4°C. A suspension of 5x10⁶ FcgammaRIIa-expressing cells that had ingested serum opsonized S. aureus per ml of HBSS-gel was reincubated at 37°C and 4 rpm for various intervals ranging from 0 to 90 min, with one of the following stimuli: 1) 10% (vol/vol) human serum, 2) 500 µg/ml IgG, which was isolated from pooled normal human serum by ammonium sulfate precipitation and anion exchange chromatography on DEAE-Sephacel (Sigma) as described (21), 3) FcgammaRIIa-cross-linking by mAb IV-3 (anti-FcgammaRII, 27 µg/ml of murine IgG_2b; ATCC, Rockville, MD) followed by 25 µg/ml of F(ab')₂ goat anti-mouse Ig (Cappel, Durham, NC) as described (8-10), and 4) 10% (vol/vol) serum of a patient with agammaglobulinemia (Bloodbank 89/9650; less than 0.1 mg Ig/ml). As control, cells were reincubated in HBSS-gel. At various intervals, intracellular killing was terminated by transferring the tubes to crushed ice. Subsequently, the cells were disrupted to release the internalized bacteria. The number of viable bacteria was then determined microbiologically. The percentage of intracellular killing of S. aureus by FcgammaRIIa-expressing cells was calculated using the following formula:

Intracellular killing (%) = (N₀-N_t)/N₀ x 100%

in which N₀ is the number of viable cell-associated bacteria at time-point 0 and N_t is the number of viable cell-associated bacteria at time t.

Incubation of cells with diphenylene iodonium:

To suppress oxygen-dependent killing mechanisms, 1x10⁷ cells/ml were incubated with 5 µM of the NADPH oxidase inhibitor, diphenylene iodonium bisulfate (DPI, 22; a generous gift from Dr. A.R. Cross, Dept. Biochemistry, University of Bristol, Bristol, UK), for 15 min at 37°C. As control, cells were incubated with 0.5% DMSO, the diluent of DPI.

Measurement of H₂O₂ production by cells:

H₂O₂ production by FcgammaRIIa-expressing cells without a stimulus and upon stimulation with 100 ng phorbol myristate acetate/ml was assayed by the horseradish peroxidase-mediated H₂O₂-dependent oxidation of homovanillic acid (23) and the results are expressed as nmol H₂O₂/(1x10⁶ cells x 60 min).

Measurement of NO₂^- production by cells:

The amount of NO₂^- produced by FcgammaRIIa-expressing cells was determined by the Griess method (24). Briefly, 50 µl of the culture medium was mixed with 50 µl of Griess reagents consisting of 1% sulphanylamide, 0.1% naphtylethylenediamide-dihydrochloride, and 2.5% H₃PO₄. Ten min later, the absorbance by the reaction product was read at 550 nm on the Titertek Multiscan Plus. The number of cells was estimated by quantitation of the amount of cellular proteins. The results are expressed as µmol NO₂^- /mg cell protein.

Statistical analysis:

All data represent means ± SEM of at least three experiments. The significance of differences was analyzed by Mann-Whitney U test.