Unité de Virologie et Immunologie Moléculaires, Institut National de la Recherche Agronomique, F-78352 Jouy-en-Josas cedex, France1
Unité dImmunophysiologie et Parasitisme Intracellulaire, Institut Pasteur, Paris, France2
Author for correspondence: Sabine Riffault. Fax +33 1 34 65 26 21. e-mail riffault{at}biotec.jouy.inra.fr
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Abstract |
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Introduction |
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Double-stranded RNA, formed as an intermediate during replication of RNA viruses, has been identified as a triggering signal for IFN-/
production by infected cells, whether or not they are leukocytes (Vil
ek & Sen, 1996
). Another signalling pathway has been identified for several enveloped viruses [e.g. herpes simplex virus type 1 (HSV-1), human immunodeficiency virus, human parainfluenza virus and porcine transmissible gastroenteritis coronavirus], which are able to trigger IFN-
synthesis in the absence of virus replication (Fitzgerald-Bocarsly, 1993
). A feature shared by these viruses is the presence of envelope glycoproteins capable of triggering IFN-
synthesis directly in the absence of virus replication (Ankel et al., 1994
, 1998
; Baudoux et al., 1998
; Ito et al., 1994
). The cells triggered to produce IFN-
have been named natural IFN-producing cells (NIPC) (Fitzgerald-Bocarsly, 1993
). According to studies in vitro on human or porcine blood cells, NIPCs belong to a very rare subset of leukocytes distinct from T cells, B cells, NK cells or monocytes (Fitzgerald-Bocarsly, 1993
) and express several markers of dendritic cells (Svensson et al., 1996
). Recent data generated in vitro suggest that a subset of dendritic cells (DC) called plasmacytoid monocytes (Cella et al., 1999
) or precursor of DC 2 (Siegal et al., 1999
) corresponds to the previously described NIPC and may be the main source of IFN-
among human mononuclear blood cells. The molecular motifs exposed on viral particles, the type of viral nucleic acid and the genetic programme that is expressed by the virus itself will therefore determine different IFN-
synthesis pathways. This implies that our understanding of the biological consequences of IFN-
production conjointly with other early immune mediators is not necessarily transposable from one virus-driven process to another and must be deciphered for each virus under study.
By using an experimental system that relies on intradermal (i.d.) injection of UV-inactivated HSV-1 (UV-HSV-1) in the ear of C57Bl/6 mice, we have previously described an in vivo counterpart of NIPC. We have reported that such an abortive virus input results in early and transient IFN-/
production in blood, with IFN-
/
-producing cells being detected mainly in the lymph node draining the site of virus delivery (Riffault et al., 1996
). This experimental model, which mimics natural sites of virus entry, allows a stepwise analysis of IFN-
/
effects independently of virus replication. We demonstrated previously that IFN-
/
production contributes to the rapid accumulation of leukocytes observed in the draining lymph node following i.d. UV-HSV-1 injection (Riffault et al., 1996
).
In the present study, we took advantage of our in vivo experimental model to address the question of the expression of another early immune mediator, IFN-, and of its potential regulation by IFN-
/
. Indeed, IFN-
production supported by NK cells characterizes the early process elicited by many viruses (Biron et al., 1999
; Hussell & Openshaw, 1998
). In mouse models, the early IFN-
synthesis by NK cells following virus infection is generally thought to be under the control of IL-12 (Biron, 1998
), yet IFN-
has also been reported to reduce IL-12 expression (Cousens et al., 1997
), while both IL-12 and IFN-
contribute to IFN-
synthesis by human T cells (Sareneva et al., 1998
; Sinigaglia et al., 1999
). By using a quantitative RTPCR technique (Colle et al., 1997
), we showed that IFN-
mRNA expression was rapidly and transiently upregulated in the lymph node draining the site of non-replicating UV-HSV-1 virus delivery. By using flow cytometric analysis, we showed that the IFN-
-producing cells belonged to the NK and, to a lesser extent, to the T lymphocyte lineages. We then analysed the IL-12p40 and IFN-
mRNA transcripts: a prompt transient increase of both transcripts was detected. Finally, the possible contribution of either mediator in the control of the early IFN-
burst was analysed using IFN-
/
-receptor knockout mice and in the presence of IL-12-neutralizing antibody delivered prior to UV-HSV-1 inoculation. We conclude that IL-12 played a major role and IFN-
/
a minor one, if any, in the signalling pathway(s) that leads to transient IFN-
production following the dermal delivery of UV-HSV-1.
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Methods |
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Mice were handled in accordance with institutional guidelines for animal care and use.
Virus.
HSV-1 strain Shelley (generously provided by Professor P. Lebon, Hospital St Vincent de Paul, Paris) was propagated in Vero cells. Supernatant, collected 2 days post-infection, was centrifuged at 2000 g to remove cellular debris. Virions were concentrated further by ultracentrifugation at 100000 g (rotor TI45) through a cushion of 20% sucrose. Semi-purified HSV-1 virions were resuspended in PBS, irradiated with 1·5 J/cm2 UV light and stored at -80 °C. Virus titre (TCID50) was determined on Vero cells before and after UV irradiation to check that the inactivation was complete. Endotoxin activity in virus inoculum was quantified by using the Limulus amaebocyte lysate assay (QCL1000, BioWhittaker).
I.d. injection and tissue processing.
UV-HSV-1 (5x107 TCID50 before UV inactivation) was injected i.d. in a volume of 50 µl in the right ear of each mouse. The left ears received the same volume of PBS as a control. In some experiments, a rat IgG MAb directed against the mouse IL-12p70 chain (clone C17.8, kindly provided by Dr G. Trinchieri, Wistar Institute, Philadelphia, PA, USA, and Shering Plough, Dardilly, France) was used to neutralize IL-12 function in vivo (Wysocka et al., 1995 ). Partially purified C17.8 antibody or control rat Ig (1 mg of 40% ammonium sulphate precipitate from ascites or serum preparation, respectively) were injected intraperitoneally (i.p.) 15 h and 1 h before i.d. UV-HSV-1 injection.
Mice were killed and auricular lymph nodes collected at different time-points after UV-HSV-1 injection. Lymph nodes were disrupted between two sheets of nylon net (Blutex quality monofilament; Tissages Tissus Techniques, Combles, France) in Eagles minimal essential medium (MEM) supplemented with 100 U/ml penicillin and 0·1 mg/ml streptomycin (MEM-PS), as described previously (Riffault et al., 1996 ).
Immunolabelling and flow cytometry.
The draining and contralateral lymph nodes were collected 10 h after i.d. UV-HSV-1 injection. Our preliminary studies showed that staining of IFN--positive cells was better when lymph nodes were kept overnight on ice in MEM-PS, and that cultivating the dissociated lymph node cells for a few additional hours at 37 °C in the presence of Brefeldin A did not result in enhanced IFN-
staining. Thus, in further experiments, lymph nodes were kept on ice overnight before being processed and cells were stained directly.
Lymph node cells (12x106) were incubated for 15 min on ice in 96-well flat-bottomed microtitre plates (Falcon 3072) with the following biotin-conjugated MAbs (PharmingenBecton Dickinson) diluted in cold PBS: anti CD3 (clone 500A2, 15 µg/ml), anti pan-NK cells (clone DX5, 20 µg/ml) and matching isotype controls. After washing, the cells were incubated for 15 min at 4 °C with FITCstreptavidin diluted in cold PBS (15 µg/ml) (PharmingenBecton Dickinson). Lymph node cells were then fixed at room temperature with 2% paraformaldehyde in PBS and permeabilized with 0·1% saponin in PBS before being labelled for 15 min at room temperature with PE-conjugated MAb anti-IFN-
(clone XMG1.2, 2 µg/ml) (Caltag) diluted in PBS with 0·1% saponin or with the matching isotype control (Caltag). Flow cytometry was performed with a FACScan (Becton-Dickinson). Data were collected on 3000080000 cells (Lysis software).
RNA extraction and reverse transcription.
Total RNA isolated from lymph node cells according to the method of Chomczynski & Sacchi (1987) was treated with 10 U DNase I (Boehringer Mannheim) in 0·1 M sodium acetate, 5 mM MgSO4, pH 5, with 60 U ribonuclease inhibitor (RNaseOUT, Gibco BRL) for 1 h at 37 °C. DNase I was eliminated by phenolchloroform extraction. The amount of total RNA was quantified at 260 nm. Of each RNA sample, 5 µg was reverse transcribed by using 400 U M-MLV reverse transcriptase, RNase H- (Promega) with 7 µM random hexanucleotide primers [pd(N)6, Pharmacia Biotech], 10 mM of each dNTP and 60 U RNaseOUT.
Quantitative PCR.
IFN-, IL-12p40 and
-actin cDNA were quantified by using a PCR method involving co-amplification with an internal standard, as described previously (Colle et al., 1997
). To quantify IFN-
mRNA expression, we set up a similar competitive PCR assay. The IFN-
DNA internal standard was generated by mutation of a unique BfaI restriction endonuclease site into a SmaI site. A BfaI site is present in the 10 murine IFN-
sequences available from GenBank (accession numbers X01974, M13660, K01238, X01973, X01971, X01972, M13710, D00460, M68944, M28587), whereas SmaI sites are absent from these IFN-
sequences. Mutagenesis was performed on the IFN-
1 coding sequence cloned into a pGEM4 vector (van der Korput et al., 1985
) by means of the QuikChange site-directed mutagenesis kit (Stratagene). Consensus PCR primers were designed in order to amplify the different murine IFN-
sequences: sense primer, 5' CTCATAACCTCAGGAACAAGAGAGCCT 3'; antisense primer, 5' GCATCAGACAGGCTTGCAGGTCATT 3'. The resulting IFN-
PCR products were 288 bp long and endonuclease restriction by both BfaI and SmaI generated products of 229 bp and 59 bp. PCRs were performed in a final volume of 50 µl in 200 µl microtubes (MicroAmp, Perkin Elmer). The mixture contained the cDNA template and the internal DNA standard (105, 104 or 103 copies) diluted in 10 mM TrisHCl (pH 9 at 25 °C), 50 mM KCl with 100 pmol of each primer, 300 µM of each dNTP and 2·5 U Taq polymerase (Promega). Amplification required 3540 cycles on a Perkin Elmer 2400 thermocycler as follows: 30 s at 94 °C, 30 s at 61 °C and 1 min at 72 °C. Two aliquots of each PCR were subjected to digestion with one of the restriction enzymes specific for the wild-type template or the standard template (listed in Colle et al., 1997
). Digestions were carried out in 20 µl containing 10 µl of the PCR products, 2 µl 10x concentrated enzyme buffer and 5 U of the appropriate restriction enzyme. The digested PCR products were analysed by electrophoresis in an ethidium bromide-stained, 2% agarose gel.
Statistical analysis.
Results are expressed as means ± SD. Data were collected from at least four distinct experiments, in which lymph nodes from at least four animals were pooled. The statistical significance of differences between test groups was analysed by Students t-test (P<0·01).
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Results |
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Taking into account the kinetics of IFN- mRNA accumulation, the percentage and the phenotype of the IFN-
-positive lymph node cells were analysed by flow cytometry 10 h after UV-HSV-1 injection. The percentage of IFN-
-positive cells was significantly higher (P<0·01) in cell populations from the lymph node draining the UV-HSV-1-loaded ear (0·53±0·24%) than from the contralateral lymph node (0·15±0·06%) (Fig. 2
). As we reported previously (Riffault et al., 1996
), the total number of nucleated cells in the lymph node draining the UV-HSV-1 loaded ear was 3-fold higher than in the contralateral lymph node. Thus, the absolute number of IFN-
-producing cells was about 10-fold higher in the draining lymph node than in the contralateral lymph node.
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The very prompt and prominent expression of IFN- mRNA suggested that this cytokine could be the triggering signal of the early IFN-
synthesis. To address this point, we used IFN-
/
receptor-knockout 129 mice (IFNAR1-/- 129) (Muller et al., 1994
). The levels of IFN-
mRNA within the lymph node of knockout and wild-type 129 mice were quantified at different time-points after i.d. UV-HSV-1 injection. A burst of IFN-
mRNA that peaked at 12 h was detectable in the lymph nodes of mice, whether the IFNAR1 gene was present or not (Fig. 4a
b). In addition, quantification of the IL-12p40 mRNA showed a similar upregulation of this transcript whether the IFNAR1 gene was present or not (data not shown).
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Discussion |
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The IFN--producing T cells may correspond to the memory phenotype CD44hi T lymphocytes that accumulate and proliferate in lymphoid tissues in response to IFN-
/
, irrespective of their clonotypic receptor (Tough et al., 1996
). We have demonstrated in a previous study that IFN-
/
synthesis is induced rapidly in the lymph node draining the UV-HSV-1-loaded ear (Riffault et al., 1996
).
We have shown that both IFN- and IL-12p40 transcripts are upregulated rapidly and transiently in the draining lymph node. Therefore, we investigated whether the early T- and NK-dependent IFN-
production was under the control of IFN-
/
and/or IL-12. The use of IFNAR1-/- mice as well as IL-12-neutralizing antibody allowed us to establish the following points: (i) whether the IFN-
/
receptor-signalling pathway is functional or not, the transient overexpression of IL-12 and IFN-
transcripts is maintained, and (ii) IL-12 is required for IFN-
transcript accumulation. Interestingly, the lower amount of IL-12p40 or IL-12p70 in serum of mice collected 48 h after parenteral injection of LCMV suggests downregulation of their synthesis by IFN-
/
(Cousens et al., 1997
). Depending on the virus system, e.g. replicating versus non-replicating virus, route of delivery, temporal window of analysis, loco-regional versus systemic immune response, different regulatory pathways may be transiently switched on or off. Under our experimental conditions, which involve delivery of non-replicating virus particles into a dermal site, we showed previously that IFN-
/
contributes to the rapid leukocyte accumulation we detected in the lymph node that drains the UV-HSV-1-loaded site (Riffault et al., 1996
). In the present analysis, we observed a significant 50% decrease (P<0·01) in leukocyte accumulation in the IFNAR1-/- 129 mice compared with wild-type mice (3·17±0·84x106 versus 6·56±1·50x106 lymph node cells at 6 h, mean±SD of four experiments). The number of cells in the contralateral lymph node remained at a control level (corresponding to time zero) and was not statistically significantly different between knockout and wild-type mice (data not shown). Therefore, the data obtained from knockout and wild-type 129 mice suggest that IFN-
/
contributes to leukocyte recruitment in the lymph node and that IL-12 provides additional signals supporting T and NK cell IFN-
synthesis.
Studies in situ of the timing and location of IFN-/
and IL-12 production will provide a basis for understanding the dynamic transient processes triggered by the delivery of UV-HSV-1 within the dermis, a site otherwise tractable to analysis of leukocyte trafficking (Belkaid et al., 1996
). Indeed, the candidate leukocytes that are expected to produce these cytokines within the lymph node are (i) subcapsular resident siglec-1+ mononuclear phagocytes, (ii) leukocytes entering through the afferent lymphatic vessels and (iii) leukocytes entering through the high endothelial venules (HEV) (Girard & Springer, 1995
; Kraal & Mebius, 1997
; Cyster, 1999
). Relevant markers are available for many of these leukocytes and these will be helpful in distinguishing the subcapsular macrophages, neutrophils, monocytes and dendritic cells emigrating from the epidermis/dermis or dendritic cells entering through the HEV (Salomon et al., 1998
; Cyster, 1999
). The early neutrophil recruitment we have observed previously in the lymph node following UV-HSV-1 delivery in the C57BL/6 ear dermis (Riffault et al., 1996
) is consistent with a contribution by neutrophils in IL-12 synthesis at this site (Romani et al., 1997
). As mentioned above, different teams have recently attempted to characterize the leukocytes that transcribe and produce IFN-
/
under steady-state conditions or following exposure to UV-inactivated HSV (Siegal et al., 1999
), influenza virus (Cella et al., 1999
) or infectious enveloped RNA and DNA viruses (Milone & Fitzgerald-Bocarsly, 1998
). Leukocytes of the dendritic cell lineage(s) do produce and/or transcribe IFN-
/
depending on the experimental conditions, which have varied from one team to another. They are: (i) CD3- CD14- CD16- CD19- HLADR+ mannose receptor (MR)+ so-called dendritic cells (Milone & Fitzgerald-Bocarsly, 1998
), (ii) CD3- CD11c- CD4+ preDC2, otherwise known to depend on IL-3 as an anti-apoptotic factor (Siegal et al., 1999
), or (iii) CD11c- CD4+ HLADR+ IL-3R+ MR- so-called plasmacytoid monocytes (Cella et al., 1999
). The latter subset can also produce a small amount of IL-12 on exposure to LPS and CD40L (Cella et al., 1999
).
The pathways by which HSV-1 triggers human NIPC to synthesize IFN- involve interaction between its membrane glycoprotein gD and chemokine receptors (Ankel et al., 1998
) and/or the mannose receptor (Milone & Fitzgerald-Bocarsly, 1998
). Whether the same pathways can trigger IL-12 expression is not yet known. NIPC are therefore a component of the host immune response that can be activated very rapidly upon infection, even before the virus undergoes replication in host cells. The local cytokine environment elicited in this way could contribute both to limiting virus spread downstream of the lymph node draining the site of virus delivery and to stimulation of virus-reactive T and B lymphocytes.
The IFN--producing cells that we have detected in the regional lymph node could be the mouse counterparts of the human NIPC. One further question to which an answer is needed is the following: among the cells of the dendritic lineage(s), will a subset of DC at a given stage of differentiation be directly or indirectly reactive to UV-HSV-1 by simultaneous and/or sequential synthesis/release of IFN-
/
and of bioactive heterodimeric IL-12?
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Acknowledgments |
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Received 8 March 2000;
accepted 30 June 2000.