JOURNAL OF VIROLOGY, Dec. 1991, p. 6743-6748
Vol. 65, No. 12
0022-538X/91/126743-06$02.00/0 Copyright © 1991, American Society for Microbiology
Early Signal Transduction in Measles Virus-Infected Lymphocytes Is Unaltered, but Second Messengers Activate Virus Replication RAIJA VAINIONPAA,1* TIMO HYYPIA,1 AND KARL E. 0. AKERMAN2 Department of Virology, University of Turku, SF-20520 Turku,' and Department of Biochemistry and Pharmacy, A bo Akademi, SF-20500 Turku,2 Finland Received 25 June 1991/Accepted 13 September 1991
In order to understand measles virus-lymphocyte interactions, we have started to analyze factors and events which regulate measles virus infection in peripheral blood mononuclear cells (PBMC). We analyzed the initiation of cell proliferation, induced by phytohemagglutinin, in infected and control PBMC by measuring intracellular free Ca2' by using fura-2. Measles virus-infected and control PBMC responded similarly with an increase in the amount of cytosolic free Ca2+, indicating that the early activation events are not affected and are not involved in immunosuppression. The activation signals, Ca2+ and protein kinase C, induced specifically and independently by Ca ionophore A23187 or 12-O-tetradecanoylphorbol-13-acetate (TPA), changed the restricted measles virus infection to a productive one. The combination of TPA and A23187 was the most potent activator of measles virus replication. TPA and A23187 operate through different activation mechanisms, and it is evident that measles virus replication depends on the activation of cellular signal pathways. Depletion of adherent cells enhanced virus replication, especially at the early stage of infection, indicating the inhibitory role of monocytes. Monocytes were strongly infected, but they supported complete measles virus replication only at a very low level, and virus replication could not be enhanced with TPA and/or A23187.
Measles virus (MV) infects immune cells and suppresses immune functions in vivo and in vitro (6, 7, 16, 19, 25, 29). Virus replication in nonstimulated lymphocytes is highly restricted (7), which may lead to a persistent MV infection. A very high proportion of peripheral blood mononuclear cells (PBMC) of patients with subacute sclerosing panencephalitis, a fatal neurological disease with MV etiology, has been reported to contain viral RNA (3). Phytohemagglutinin (PHA) stimulation of mononuclear cells in vitro activates MV infection in PBMC. Activation of cellular events has also been reported to alter other virus infections in PBMC, e.g., compounds which stimulate immune cells induce human immunodeficiency virus production from latently infected T lymphocytes (4, 30). The mechanisms responsible for the productive MV infection in activated immune cells are largely unknown. Binding of PHA to its receptor on the cell surface is known to activate a group of biochemical reactions associated with inositol phospholipid breakdown, Ca2+ mobilization and protein kinase C activation. These effects may be bypassed in vitro by 12-O-tetradecanoylphorbol-13-acetate (TPA), a tumor-promoting agent which activates protein kinase C, and Ca2+ ionophore A23187, causing an increase in the intracellular Ca2+ content. These compounds have been previously shown to induce activation of T lymphocytes through partially separate pathways (1, 8, 20, 27, 28). We report an analysis of interactions between lymphocyte activation and MV replication, an interesting model for studying factors and events regulating virus replication in PBMC. Our results show that although MV infection in lymphocytes is strongly immunosuppressive, transmembrane signal transduction is not affected by MV. Stimulation of second messenger-linked pathways induced by TPA and A23187 activate the restricted MV infection in lymphocytes. In contrast, activation is not observed in infected monocytes *
which themselves have an inhibitory function on MV replication. MATERIALS AND METHODS Virus and cells. Wild-type MV (24) was grown in Vero cells, and the virus stock was centrifuged at 8,000 x g for 5 min before use to remove cell debris. PBMC from six healthy immune adults were isolated in Ficoll-Isopaque density gradients (Ficoll-Paque; Pharmacia). For monocyte-depleted cultures (PBMC/d), adherent cells were separated from PBMC by incubation on petri dishes for 60 min at 37°C. PBMC enriched for monocytes (PBMC/e) were prepared by detaching the adherent cells with a rubber policeman and adding the cells to untreated cultures. For isolation of monocytes, adherent cells were collected after two repeated incubation cycles on petri dishes and vigorous washings. The proportional number of My4 (marker for monocytes)-positive cells in different cultures was assessed by using anti-My4 antibodies (Becton Dickinson) and fluorescent-conjugated anti-mouse antibodies in a fluorescenceactivated cell sorter (FACS) analysis flow cytometer. The cells were infected with MV and cultured as described earlier (7). Three days postinfection they were stimulated with TPA (Sigma; 10 nM), Ca ionophore A23187 (Sigma; 0.3 ,uM), a combination of TPA and A23187, or PHA (Difco; 10 jig/ml). Viability of the cells was estimated by the trypan blue exclusion method. Determination of intracellular free Ca2". Before being loaded with fura-2 (Molecular Probes Inc.), the cells were washed and resuspended into the experimental medium containing 137 mM NaCl, 5.0 mM KCI, 1.0 mM CaCl2, 1 mM MgCl2, 0.44 mM KH2PO4, 4.2 mM NaHCO3, and 10 mM glucose in N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) buffer (20 mM Tris, 10 mM NaCl, 0.1 mM EDTA [pH 7.4] at 37°C). Fura-2 was added at a concentration of 2 ,iM, and the suspension was incubated with constant agitation for 30 min. The cells were subsequently
Corresponding author. 6743
VAINIONPAA ET AL.
6744
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400 E200
.
410
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FIG. 1. PHA-induced increase in fura-2 fluorescence in control (a) and MV-infected (b) cells. See Materials and Methods for experimental details. The fluorescence at a cell concentration of 106 cells per ml was monitored in a constantly stirred cuvette. PHA (10 ,ug/ml) was added as indicated, and then 4 FLM ionomycin and 100 jiM MnSO4 were added for calibration. An increase in fluorescence denotes a rise in cytosolic free Ca2+.
washed. The fluorescence measurements were initiated by diluting the cells into the experimental medium containing 0.1% bovine serum albumin at 37°C, and the fluorescence was monitored with a Hitachi F4000 fluorescence spectrophotometer (340-nm excitation wavelength, 505-nm emission wavelength). At the end of the experiment 1 FiM ionomycin was added to obtain the fluorescence maximum, and then 10 ,uM MnSO4, which quenches the Ca2'-dependent fluorescence (5), was added. Calculation of free Ca2+ concentrations was performed by using the maximal and minimal fluorescence values according to the method of Tsien et al. (21). Lymphocyte proliferation assay. MV-infected and uninfected PBMC, either unfractionated or depleted or enriched for adherent cells, were incubated for 3 days postinfection and then stimulated with TPA, A23187, a combination of TPA and A23187, or PHA. Cells were grown in roundbottomed microtiter plates at a density of 105/100 pLI. 3Hthymidine (Amersham; 0.5 ,uCi per well) was added 18 h FL ! F I uc:r e;c6-eeir ce 1 FLLOPEL:EWE. E'TIC-)N = 4A ' T,E EvI;
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Vol. 65, No. 12
0022-538X/91/126743-06$02.00/0 Copyright © 1991, American Society for Microbiology
Early Signal Transduction in Measles Virus-Infected Lymphocytes Is Unaltered, but Second Messengers Activate Virus Replication RAIJA VAINIONPAA,1* TIMO HYYPIA,1 AND KARL E. 0. AKERMAN2 Department of Virology, University of Turku, SF-20520 Turku,' and Department of Biochemistry and Pharmacy, A bo Akademi, SF-20500 Turku,2 Finland Received 25 June 1991/Accepted 13 September 1991
In order to understand measles virus-lymphocyte interactions, we have started to analyze factors and events which regulate measles virus infection in peripheral blood mononuclear cells (PBMC). We analyzed the initiation of cell proliferation, induced by phytohemagglutinin, in infected and control PBMC by measuring intracellular free Ca2' by using fura-2. Measles virus-infected and control PBMC responded similarly with an increase in the amount of cytosolic free Ca2+, indicating that the early activation events are not affected and are not involved in immunosuppression. The activation signals, Ca2+ and protein kinase C, induced specifically and independently by Ca ionophore A23187 or 12-O-tetradecanoylphorbol-13-acetate (TPA), changed the restricted measles virus infection to a productive one. The combination of TPA and A23187 was the most potent activator of measles virus replication. TPA and A23187 operate through different activation mechanisms, and it is evident that measles virus replication depends on the activation of cellular signal pathways. Depletion of adherent cells enhanced virus replication, especially at the early stage of infection, indicating the inhibitory role of monocytes. Monocytes were strongly infected, but they supported complete measles virus replication only at a very low level, and virus replication could not be enhanced with TPA and/or A23187.
Measles virus (MV) infects immune cells and suppresses immune functions in vivo and in vitro (6, 7, 16, 19, 25, 29). Virus replication in nonstimulated lymphocytes is highly restricted (7), which may lead to a persistent MV infection. A very high proportion of peripheral blood mononuclear cells (PBMC) of patients with subacute sclerosing panencephalitis, a fatal neurological disease with MV etiology, has been reported to contain viral RNA (3). Phytohemagglutinin (PHA) stimulation of mononuclear cells in vitro activates MV infection in PBMC. Activation of cellular events has also been reported to alter other virus infections in PBMC, e.g., compounds which stimulate immune cells induce human immunodeficiency virus production from latently infected T lymphocytes (4, 30). The mechanisms responsible for the productive MV infection in activated immune cells are largely unknown. Binding of PHA to its receptor on the cell surface is known to activate a group of biochemical reactions associated with inositol phospholipid breakdown, Ca2+ mobilization and protein kinase C activation. These effects may be bypassed in vitro by 12-O-tetradecanoylphorbol-13-acetate (TPA), a tumor-promoting agent which activates protein kinase C, and Ca2+ ionophore A23187, causing an increase in the intracellular Ca2+ content. These compounds have been previously shown to induce activation of T lymphocytes through partially separate pathways (1, 8, 20, 27, 28). We report an analysis of interactions between lymphocyte activation and MV replication, an interesting model for studying factors and events regulating virus replication in PBMC. Our results show that although MV infection in lymphocytes is strongly immunosuppressive, transmembrane signal transduction is not affected by MV. Stimulation of second messenger-linked pathways induced by TPA and A23187 activate the restricted MV infection in lymphocytes. In contrast, activation is not observed in infected monocytes *
which themselves have an inhibitory function on MV replication. MATERIALS AND METHODS Virus and cells. Wild-type MV (24) was grown in Vero cells, and the virus stock was centrifuged at 8,000 x g for 5 min before use to remove cell debris. PBMC from six healthy immune adults were isolated in Ficoll-Isopaque density gradients (Ficoll-Paque; Pharmacia). For monocyte-depleted cultures (PBMC/d), adherent cells were separated from PBMC by incubation on petri dishes for 60 min at 37°C. PBMC enriched for monocytes (PBMC/e) were prepared by detaching the adherent cells with a rubber policeman and adding the cells to untreated cultures. For isolation of monocytes, adherent cells were collected after two repeated incubation cycles on petri dishes and vigorous washings. The proportional number of My4 (marker for monocytes)-positive cells in different cultures was assessed by using anti-My4 antibodies (Becton Dickinson) and fluorescent-conjugated anti-mouse antibodies in a fluorescenceactivated cell sorter (FACS) analysis flow cytometer. The cells were infected with MV and cultured as described earlier (7). Three days postinfection they were stimulated with TPA (Sigma; 10 nM), Ca ionophore A23187 (Sigma; 0.3 ,uM), a combination of TPA and A23187, or PHA (Difco; 10 jig/ml). Viability of the cells was estimated by the trypan blue exclusion method. Determination of intracellular free Ca2". Before being loaded with fura-2 (Molecular Probes Inc.), the cells were washed and resuspended into the experimental medium containing 137 mM NaCl, 5.0 mM KCI, 1.0 mM CaCl2, 1 mM MgCl2, 0.44 mM KH2PO4, 4.2 mM NaHCO3, and 10 mM glucose in N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) buffer (20 mM Tris, 10 mM NaCl, 0.1 mM EDTA [pH 7.4] at 37°C). Fura-2 was added at a concentration of 2 ,iM, and the suspension was incubated with constant agitation for 30 min. The cells were subsequently
Corresponding author. 6743
VAINIONPAA ET AL.
6744
|. '-T~
Cai /nM a)
400 E200
.
410
[ 230
b)t pha 1 min
FIG. 1. PHA-induced increase in fura-2 fluorescence in control (a) and MV-infected (b) cells. See Materials and Methods for experimental details. The fluorescence at a cell concentration of 106 cells per ml was monitored in a constantly stirred cuvette. PHA (10 ,ug/ml) was added as indicated, and then 4 FLM ionomycin and 100 jiM MnSO4 were added for calibration. An increase in fluorescence denotes a rise in cytosolic free Ca2+.
washed. The fluorescence measurements were initiated by diluting the cells into the experimental medium containing 0.1% bovine serum albumin at 37°C, and the fluorescence was monitored with a Hitachi F4000 fluorescence spectrophotometer (340-nm excitation wavelength, 505-nm emission wavelength). At the end of the experiment 1 FiM ionomycin was added to obtain the fluorescence maximum, and then 10 ,uM MnSO4, which quenches the Ca2'-dependent fluorescence (5), was added. Calculation of free Ca2+ concentrations was performed by using the maximal and minimal fluorescence values according to the method of Tsien et al. (21). Lymphocyte proliferation assay. MV-infected and uninfected PBMC, either unfractionated or depleted or enriched for adherent cells, were incubated for 3 days postinfection and then stimulated with TPA, A23187, a combination of TPA and A23187, or PHA. Cells were grown in roundbottomed microtiter plates at a density of 105/100 pLI. 3Hthymidine (Amersham; 0.5 ,uCi per well) was added 18 h FL ! F I uc:r e;c6-eeir ce 1 FLLOPEL:EWE. E'TIC-)N = 4A ' T,E EvI;
-t
T.-, ,I. "
l
,
a
I
!4:,
