Human major group rhinoviruses downmodulate the accessory function of monocytes by inducing IL-10 (original) (raw)
Media, reagents, and chemicals. The cell culture medium RPMI-1640 (GIBCO BRL, Grand Island, New York, USA) supplemented with 2 mM L-glutamine, 10% FCS, 100 U/mL penicillin, and 100 μg/mL streptomycin was used in this study. The HRV-blocking reagent WIN 52035-2 (25) was a kind gift from the Sterling-Winthrop Research Institute (Rensselaer, New York) and was used at a final concentration of 5 μg/mL. The superantigens staphylococcal enterotoxin A (SEA) and B (SEB) from Staphylococcus aureus were obtained from Serva (Heidelberg, Germany) and were used at a concentration of 10 ng/mL. Tetanus toxoid and purified protein derivative of Mycobacterium tuberculosis (PPD) were purchased from Connaught Laboratories (Willowdale, Ontario, Canada) and used at a concentration of 1 μg/mL. LPS from Escherichia coli (serotype 0127-B8) and polymyxin B were obtained from Sigma Chemie GmbH (Deisenhofen, Germany). Recombinant human GM-CSF and IL-4 were kindly provided by Novartis Research Institute (Vienna, Austria). IFN-γ was a gift from G.R. Adolf (Ernst Boehringer Institut für Arzneimittelforschung, Vienna, Austria). IL-10 was purchased from R&D Systems Inc. (Minneapolis, Minnesota, USA).
Antibodies used in this study. The following murine mAb’s were generated in our laboratory: negative control mAb VIAP (calf intestine alkaline phosphatase–specific), 6B6 (CD11a), VIM13 (CD14), 4D3 (CD33), and 1/47 (MHC class II). Hybridomas producing mAb W6/32 (MHC class I), G28-5 (CD40), and TS2/9 (CD58) were obtained from American Tissue Culture Collection (ATCC; Rockville, Maryland, USA). UCHT-1 (CD3), MEM18 (CD14), and UCHL1 (CD45R0) were kindly provided by An der Grub (Bio Forschungs GmbH, Kaumberg, Austria). OKT3 (CD3) was obtained from Ortho Diagnostics (Raritan, New Jersey, USA). The mAb HD37 (CD19) was provided by G. Moldenhauer (Heidelberg, Germany). MAb RR1/1 (CD54) was a gift from Bender AG (Vienna, Austria). The mAb L307 (CD80) was from Becton Dickinson Immunocytometry Systems (San Jose, California, USA). IT2.2 (CD86) and the PE-labeled mAb C11.5 (anti–IL-12) were purchased from PharMingen (San Diego, California, USA). MAb 3G8 (CD16), the FITC-labeled mAb MP9-20A4 (anti–TNF-α), and PE-labeled mAb JES3-9D7 (anti–IL-10) for cytoplasmic staining of IL-10 were obtained from Caltag Laboratories Inc. (Burlingame, California, USA). FITC-labeled FIB-3 mAb (anti–IL-1β) was from Dianova (Hamburg, Germany). The neutralizing polyclonal anti–IL-10 antibody (PAL-hIL10) was obtained from Strahtmann Biotech (Hannover, Germany).
Rhinovirus preparation. HRV-14 was obtained from ATCC and routinely grown in suspension cultures of HeLa cells (strain Ohio; Flow Laboratories, McLean, Virginia, USA). Cells were cultivated in S-MEM medium (Joklik modification; catalog no. 22300-107; Life Technologies Inc., Rockville, Maryland, USA) supplemented with 7% heat-inactivated horse serum (catalog number 01051-M; Diagnostic Products GesmbLT, Wiener Neudorf, Austria), 1% penicillin/streptomycin, 1% glutamine, 1% pluronic F68 (catalog no. P1300; Sigma Chemical Co., St. Louis, Missouri, USA), and 1% nonessential amino acids. The same medium was used during infections, except that serum was reduced to 2% and 1 mM MgCl2 was added. Cells were infected at a ratio of ten 50% tissue culture infectious doses (TCID)50 per cell. HRV-14 was harvested 40 hours after infection. Cell debris was pelleted by low-speed centrifugation. HRV was prepared from cell culture supernatant by polyethanolglycol (PEG) precipitation (7% PEG 6000, 3% NaCl). Virus was resuspended from the PEG pellet in PBS, aliquoted, and stored at –70°C. Working dilutions of used HRV-14 stock preparations contained less than 10 pg LPS/mL. No biologic effects of these very low LPS concentrations were detectable. The preparations did not induce significant TNF-α or IL-6 production in monocytes (see Figure 5). Moreover, the capacities of HRV-14 preparations to induce IL-10 production, inhibit T-cell proliferation, and downregulate MHC class II expression could not be inhibited by the LPS inhibitor polymyxin B (10 μg/mL) (data not shown).
Induction of IL-10 production in monocytes by HRV-14. (a) Supernatants of monocytes (1 × 106/mL) cultured in the presence of HRV-14 (10 TCID50 per cell), LPS (100 ng/mL), or medium for 2 days were analyzed for IL-10, IL-1β, and TNF-α production by ELISA. The figure shows mean values ± SEM of 3 experiments. (b) In separate experiments, detection of cytokine production in monocytes was performed by cytoplasmic staining with specific mAb’s. Monocytes (1 × 106/mL) were cultured for 24 hours in the presence of HRV-14 (10 TCID50 per cell) or LPS (100 ng/mL). After 12 hours of culture, monensin (5 μM) was added in all instances. The cells were harvested, fixed, permeabilized, and subsequently stained with PE-labeled anti–IL-10 mAb. For determination of IL-1β or TNF-α production by cytoplasmic staining, monocytes were incubated with the indicated stimuli for 6 hours in the presence of monensin (5 μM). Cytoplasmic cytokine expression was analyzed by flow cytometry. Overlay histograms show expression in mock-treated cells (open histograms) and monocytes stimulated with HRV-14 (gray histograms) or LPS (open histograms, thick line). The results shown are representative of 3 experiments.
Preparation of purified HRV. Virus stock preparations were further purified using sucrose gradients. Virus was pelleted by centrifugation at 100,000 g for 2 hours and resuspended in a small volume of “virus buffer” (50 mM Tris [pH 7.5], 2 mM MgCl2). It was then treated with DNase I (5 mg/mL; catalog no. 104169; Boehringer Mannheim, Mannheim, Germany) and RNase A (5 mg/mL; catalog no. 104159; Boehringer Mannheim) for 10 minutes at room temperature and further digested with trypsin (0.5 mg/mL; catalog no. 01104-H; Dipro) for 5 minutes at 37°C. After addition of _N_-laurylsarcosin (0.1%; catalog no. LA083; Schuchardt, Munich, Germany) digestion was continued at 4°C overnight. After removal of unsoluble material by low-speed centrifugation, the sample was centrifuged on a sucrose gradient (7.5–45% sucrose in virus buffer) for 2 hours at 155,000 g. Virus-containing fractions are visible as a turbid band in the middle of the gradient and were collected with a syringe. Virus was concentrated by pelleting and resuspension in virus buffer.
Ultraviolet inactivation of virus. Ultraviolet-inactivated (UV-inactivated) virus was prepared by irradiating virus suspensions in a 24-well tissue culture dish (200 μL/well) on ice for 10 minutes with a 75 W UV source (254 nm) at a distance of 5 cm. Treatment resulted in complete loss of infectious titer.
Cell preparation. PBMCs were isolated from heparinized whole blood of normal healthy donors by standard density gradient centrifugation with Ficoll-Paque (Pharmacia, Uppsala, Sweden). Subsequently, T cells and monocytes were separated by magnetic sorting using the MACS technique (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) as described previously (26). Purified T cells were obtained through negative depletion of CD11b, CD14, CD16, CD19, CD33, and MHC class II–positive cells with the respective mAb’s. Monocytes were enriched by using the biotinylated CD14 mAb VIM13 (purity >95%) as described previously (26).
Cultivation of monocytes. Monocytes (1 × 106/mL) were cultured in 24-well plates (Corning-Costar Europe, Badhoevedorp, the Netherlands) in the presence of HRV-14 (10 TCID50 per cell) or aliquot amounts of supernatants from uninfected HeLa cells at 37°C. After 2 days, cells were harvested, washed in PBS, and analyzed by flow cytometry. Monocyte-derived dendritic cells (md-DCs) were generated by culturing purified blood monocytes for 8 days with a combination of GM-CSF (50 ng/mL) and IL-4 (100 U/mL) as described previously (26).
T-cell proliferation assays. For the allogeneic mixed lymphocyte reaction (MLR), allogeneic, purified T cells (105) were incubated with graded numbers of irradiated (30 Gy, 137Cs source) freshly isolated monocytes, md-DCs, or Epstein Barr virus–transformed (EBV-transformed) B-lymphoblastoid cells (B-LCLs) for the indicated periods. HRV (10 TCID50 per cell) or respective aliquots of mock-cultured HeLa supernatants were added at the beginning of the MLR. In some experiments, supernatants (100 μL) of monocytes cultured in the presence of HRV-14 or HeLa supernatants were added. Experiments were performed in 96-well cell culture plates in RPMI-1640 medium supplemented with 5% human AB-serum (PAA Laboratories, Munich, Germany). Proliferation of T cells was monitored by measuring (methyl-3H)-TdR (ICN Pharmaceuticals Inc., Irvine, California, USA) incorporation on day 5 of culture. Cells were harvested 18 hours later, and radioactivity was determined on a microplate scintillation counter (Packard, Meriden, Connecticut).
Antigen stimulation assays were performed with fresh PBMCs (105 per well). The cultures were set up in the presence of tetanus toxoid (1 μg/mL), PPD (1 μg/mL), SEA or SEB (10 ng/mL), or OKT3 (1 μg/mL). Proliferation was measured by (methyl-3H)-TdR incorporation on day 3 (SEA, SEB, OKT3) and day 5 (tetanus toxoid, PPD) of culture.
Immunofluorescence analysis. For membrane staining, cells (5 × 105) were incubated for 30 minutes at 0–4°C with unconjugated mAb. After washing twice with PBS, Oregon Green–conjugated goat anti-mouse antibody from Molecular Probes Inc. (Eugene, Oregon, USA) was used as a second-step reagent. Flow cytometric analysis was performed using a FACScan flow cytometer (Becton Dickinson).
Determination of cytokine production. Monocytes (1 × 106/mL) were cultured either mock treated or stimulated with LPS (100 ng/mL), with or without pretreatment for 24 hours with IFN-γ (300 U/mL), or in the presence of HRV (10 TCID50/monocyte) in 24-well plates (Corning-Costar Europe). After 24 hours, the supernatants were harvested and analyzed by ELISA or used in T-cell proliferation assays. For cytoplasmic staining, monensin (5 μM) was added during the last 12 hours. The cells were harvested and fixed for 20 minutes at room temperature by adding 100 μL of FIX solution (An der Grub). Subsequently, cells were washed once with 4 mL of PBS, resuspended in 100 μL of PBS, and permeabilized by the addition of 100 μL of PERM solution (An der Grub). Immediately, the indicated PE-conjugated anti-cytokine mAb’s were added and incubated for 20 minutes at room temperature. The cells were then washed twice, resuspended in PBS (200 μL), and analyzed by flow cytometry.
Cytokines were measured by sandwich ELISAs using matched-pair antibodies. Capture and detection antibodies for human IL-1β were obtained from Genzyme Pharmaceuticals (Cambridge, Massachusetts, USA); for IL-10 and IL-12 p70, from R&D Systems Inc.; and for TNF-α, from PharMingen. Standards consisted of human recombinant material from R&D Systems Inc. Assays were performed in duplicate according to the recommendations of the manufacturers. The lower limit of detection was 10 pg/mL for IL-1β and 20 pg/mL for IL-10, IL-12, and TNF-α.