Unit of Experimental Medicine1 and Unit of Cellular Genetics2, Institute for Cellular Pathology, Université Catholique de Louvain, UCL MEXP 7430, Av. Hippocrate 74, 1200 Bruxelles, Belgium
Author for correspondence: Jean-Paul Coutelier. Fax +32 2 764 7430. e-mail coutelier{at}mexp.ucl.ac.be
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Abstract |
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Introduction |
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In this report, we examined therefore whether LDV infection actually results in NK cell activation and in IFN- secretion by those cells. Our data indicate that the virus indeed triggers an enhancement of cytolysis and IFN-
expression by NK cells. However, neither the NK cell activation nor the IFN-
production results in control of virus replication, as found with a new and more accurate procedure for LDV titration.
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Methods |
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Virus.
Mice were infected by intraperitoneal injection of approximately 2x107 ID50 of LDV (Riley strain; ATCC) in 500 µl saline.
Antibodies.
B6507F4 and B6506A7 are IgG2a monoclonal antibodies (mAbs) recognizing the VP3 LDV protein (Coutelier et al., 1986 ). GK1.5 monoclonal anti-CD4 antibody was made available by F. W. Fitch and obtained through the courtesy of H. R. MacDonald (Dialynas et al., 1983
). 53/6.72 anti-CD8 monoclonal antibody was obtained from ATCC (Ledbetter & Herzenberg, 1979
). Both anti-CD4 and anti-CD8 antibodies were precipitated with ammonium sulfate. Their in vivo efficiency has been extensively reported (Coulie et al., 1985
; Coutelier, 1991
; El Azami El Idrissi et al., 1998
).
Purified asialoganglioside-GM1 (ASGM1) from bovine brain tissue (Sigma, reference G-3018) was dissolved by sonic dispersion in 0·5 ml of 0·9% NaCl with twice its amount of methylated bovine serum albumin (Sigma, reference A-1009). For the first immunization, 1·5 mg ASGM1 in complete Freund's adjuvant was injected intradermally into a rabbit, followed by 0·5 mg ASGM1 in incomplete Freunds adjuvant for the following immunizations (Kasai et al., 1980 ). The IgG fraction was obtained from the serum by 50% ammonium sulfate precipitation followed by resuspension in the same volume of PBS, pH 7·2. Different antibody amounts were tested in preliminary experiments of inhibition of IFN-
secretion. To inhibit NK cell activity, 300 µl antibody was needed and this volume was used in subsequent experiments.
LDV titration.
LDV titration was performed by a new sensitive particle-counting immunoassay based on the agglutination, by virus particles, of latex beads coated with two different anti-LDV mAbs. This assay greatly reduced the number of mice used for LDV titration; it was easier to perform than conventional methods of injection of sample sequential dilutions in groups of mice; and it allowed for more accurate determination of virus titres. It was developed as follows.
Preparation of latex.
Anti-LDV IgG2a mAbs (B6507F4 and B6506A7) (100 µg) were covalently coupled to 50 µl of carboxylated latex beads of 0·8 µm diameter (Estapor K150, Rhône Poulenc) by the carbodiimide method (Galanti et al., 1997 ). The coated particles were stored in aliquots at -20 °C. Before use, latex particles were sonicated for 10 s and diluted with glycine-buffered salinebovine serum albumin (GBSBSA) buffer (0·1 M glycine, 0·17 M NaCl, 10 mg/ml BSA, 6 mM NaN3, pH 9·2) to adjust the particle number.
Assay.
PepsinHCl reagent [25 µl; 0·3 mol HCl, 5 g porcine pepsin (Sigma) per litre, pH 1·5] was added to 25 µl of serial plasma dilutions and incubated for 10 min at room temperature. To quench the enzyme reaction, 25 µl of Tris (1 M, Merck)dextran T500 (4%, Pharmacia) reagent was added, together with 25 µl of the reconstituted particle reagent mix containing approximately 30000 latex bead un-agglutinated monomers. The resulting mixture was incubated at 37 °C for 1 h with continuous vortexing. The reaction was stopped by dilution with 500 µl of GBS. The un-agglutinated particles were counted in an optical counter able to discriminate the un-agglutinated from the agglutinated particles (Masson et al., 1981 ). The number of un-agglutinated particles was inversely proportional to the LDV concentration. A pool of plasma from NMRI mice infected for 18 h with LDV, with a virus titre of 1010·5 ID50/ml as measured by the classical end-point titration assay in mice (Rowson & Mahy, 1975
), was used as a standard and arbitrarily defined as containing 1000 agglutination units of LDV. A standard curve obtained with serial dilutions of this reference plasma pool is shown in Fig. 1
, with data obtained by using control latex beads coated with anti-carcinoembryonic mAbs (5905 and 5911, Medix Biochemica, Kauniainen, Finland) and results obtained by incubating pooled control plasma from uninfected NMRI mice with these two latex bead preparations.
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Cytolytic assay.
103 51Cr-labelled YAC-1 and TEPC.1033.C2 cells were incubated with effector cells, at different effector:target ratios in 200 µl Iscoves medium containing 10% FCS and supplemented with 0·24 mM L-asparagine, 0·55 mM L-arginine, 1·5 mM L-glutamine and 0·05 mM 2-mercaptoethanol in round-bottomed microplates for 4 h at 37 °C in 7% CO2. At the end of the incubation, radioactivity released from triplicate cultures was measured in a gamma counter. Results were expressed as percent lysis of the target cells according to the following formula: % lysis=(experimental 51Cr release-spontaneous 51Cr release)/(maximum 51Cr release-spontaneous 51Cr release)x100.
The sensitivity to NK-mediated lysis of YAC-1 and TEPC.1033 target cells was tested by using as effector cells spleen cells from BALB/c mice which had been incubated for 3 days with a mix of IL-12 and IL-18; these cytokines act in synergy to activate the lytic activity of NK cells (Lauwerys et al., 1999 ). We obtained, in a typical experiment, 20% of maximal specific lysis for TEPC.1033 and 60% for YAC-1. Thus, YAC-1 can be considered NK-sensitive target cells and TEPC.1033 NK-resistant target cells.
IFN-
assay.
IFN- sandwich ELISA was performed by using the Mouse IFN-
DuoSeT kit (Genzyme) or CytoSet (Biosource), according to the manufacturers instructions.
RNA extraction and PCR amplification.
Gene expression was analysed by RTPCR as described previously (El Azami El Idrissi et al., 1998 ). Cells were lysed in TRIzol reagent (Gibco-BRL). Total RNA was first extracted with chloroform and then precipitated with isopropanol, washed in ethanol and finally resuspended in 50100 µl water. Oligo(dT)-primed cDNA was prepared from approximately 5 µg RNA using 200 U M-MLV reverse transcriptase (Gibco-BRL) according to the manufacturers instructions. cDNA was amplified by PCR in a Thermal Reactor (Hybaid) with DyNAzyme DNA polymerase (Finnzymes) for actin and with a Gene Amp kit (Perkin Elmer Cetus) for IFN-
. The primers were as follows:
actin: 5' AGGCATTGTGATGGACTCC 3'
5' GCTGGAAGGTGGACAGTGAG 3'
IFN-: 5' GACAATCAGGCCATCAGCAAC 3'
5' CGCAATCACAGTCTTGGCTAA 3'
PCR products were analysed in 1% agarose gels containing ethidium bromide. Semi-quantitative results were obtained after blotting the PCR products on Zeta-Probe membranes (Bio-Rad) and hybridization overnight at 42 °C in Denhardts solution with internal probes labelled with 32P. The sequence of the probes was:
actin: 5' TATGAGCTGCCTGACGGCCA 3'
IFN-: 5' TCGCCTTGCTGTTGCTGA 3'.
Radioactivity was quantified with a PhosphorImager (Molecular Dynamics,) and the ratios between IFN- and actin messages were calculated after subtraction of non-specific background and shown as arbitrary units.
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Results |
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Discussion |
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Our results indicate different kinetics for the activation of diverse NK cell functions elicited by LDV infection, with a very early IFN- production, rapidly followed by an increase in cytolytic activity and finally a slightly delayed accumulation of CD49b+ cells. This might be explained by a sequential activation of these functions that may be controlled by distinct cytokines (reviewed by Biron et al., 1999
). The enhancement of NK cell-mediated cytolytic activity of spleen and peritoneal cells after LDV infection may be due to secretion of IFN-
/
by infected macrophages (Plagemann et al., 1995
; Koi et al., 1981
; Leclercq et al., 1987
; Heremans et al., 1987
). In addition, other cytokines expressed early after LDV inoculation, such as IL-12 (Coutelier et al., 1995
), may also be involved in NK cell activation, especially in regard to their cytokine production (Biron et al., 1999
). Moreover, IL-15, a growth factor for NK cells secreted by macrophages (Doherty et al., 1996
; Biron et al., 1999
) that is also produced in response to LDV infection (data not shown), may be responsible for the increase in the proportion of this cell population observed in the spleen of infected animals. Alternatively, we can speculate that distinct cell sub-populations are responsible for the various NK cell functions. For instance, preliminary results obtained in SCID and CD1-deficient mice suggest that the NK T cell sub-population may be involved in LDV-induced IFN-
production, in addition to the classical NK cells (data not shown).
NK cells have been shown to play a role in the host defence against viruses, such as MCMV, HSV and influenza virus, but not against LCMV (reviewed by Biron, 1997 ; Biron et al., 1999
). Although it is recognized that LDV viraemia persists despite the development of a T and B cell-mediated anti-viral immune response (Plagemann et al., 1995
; Even et al., 1995
; van den Broek et al., 1997
), little is known about the effects of NK cells on the replication of this virus. Our data, obtained in mice treated with anti-ASGM1 antibody that depletes the NK cell population, indicate that the early and rapid replication of LDV is not controlled by these cells. Moreover, the inability of G129 mice to respond to IFN-
did not modify virus titres. This result confirms previous observations that, although IFN-
may protect mice against LDV-induced polioencephalomyelitis (Cafruny et al., 1997
), it does not reduce the ability of the virus to replicate in vivo (Cafruny et al., 1999
). Together, our results thus indicate that neither NK cells nor the cytokine they produce interfere significantly with the replication of LDV.
Although in most mouse strains infection with LDV does not induce overt pathology, this virus greatly affects the immune system, triggering enhanced humoral responses, with a strong IgG2a-restriction of both anti-viral and non-anti-viral antibodies that are secreted in infected mice (Coutelier & Van Snick, 1985 ; Coutelier et al., 1986
). We recently showed that the production of IgG2a-restricted anti-LDV antibodies, but not of total IgG2a, is controlled by IFN-
(Markine-Goriaynoff et al., 2000
). In addition, LDV modulates the differentiation of T helper lymphocytes by inhibiting Th2 cytokine production (Monteyne et al., 1993
, 1997
b). LDV also activates macrophages (Meite et al., 2000
). LDV-triggered activation of NK cells, and the resulting IFN-
secretion, may explain some of these effects of the virus on B and T lymphocytes, as well as on macrophages. Activated macrophages will then secrete cytokines such as IL-6, IL-12, IL-15 or IL-18 (Coutelier et al., 1995
; Markine-Goriaynoff et al., 2001
; unpublished data) that may in turn participate in NK cell activation, leading to a reinforcement of the inflammatory characteristics of the immune response developing in the infected animals. Activation of cells in the innate immune system, and especially of NK cells in the early stages after infection may thus play a pivotal role in the immune alterations triggered by this virus.
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Acknowledgments |
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Footnotes |
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b Present address: Control Engineering and System Analysis Department, Université Libre de Bruxelles, 1050 Bruxelles, Belgium.
c Present address: GlaxoSmithKline Biologicals, 1330 Rixensart, Belgium.
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References |
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Received 6 February 2002;
accepted 10 June 2002.