Department of Medical Microbiology and Immunology, The Bartholin Building, University of Aarhus, DK-8000 Aarhus C, Denmark
Correspondence
Søren Paludan
srp{at}microbiology.au.dk
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ABSTRACT |
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MAIN TEXT |
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In this study, we present studies on the molecular mechanism governing expression of RANTES during HSV infection. To investigate the signalling pathways, we used chemical inhibitors of specific signalling molecules and cell lines stably transfected with various dominant-negative mutants.
The murine macrophage cells J774A.1 and murine fibroblast cells NIH 3T3 were grown and maintained in Dulbecco's modified Eagle's minimum essential medium supplemented with 5 and 10 % foetal bovine serum, respectively. Fibroblasts were serum-starved for 24 h before further treatment. Cells were infected with 1x106 p.f.u. (m.o.i. of 2 or 4) or 3x106 p.f.u. (m.o.i. of 6) of either HSV-1 (KOS) or HSV-2 (MS) ml-1. Where indicated, cells were treated with 3 µM N-tosyl-L-phenylalanine (TPCK; Sigma), or 10 µM SB203580 (Calbiochem) 20 min prior to infection. After the indicated periods of time, supernatants were harvested and RANTES levels were measured by ELISA (Peprotech). Alternatively, cells were lysed and luciferase activity was measured, or total RNA was harvested, reverse transcribed with oligo(dT) priming and analysed for the presence of RANTES, ICP27 and -actin by PCR. For transfections, lipofectamin (Invitrogen) was used. To avoid single-clone abnormalities, the stably transfected clones were pooled. Stable transfections were carried out with dominant-negative (dn) I
B kinase
(IKK
), IRF-3, p38, or the empty vector pcDNA3. Selection was performed using G418 at a concentration of 300 µg ml-1. Transient transfections were carried out with a luciferase-linked construct of the wild-type RANTES promoter (Casola et al., 2001
). Transfection efficiency and variation within plates was investigated using a GFP-vector (pEGFP-N1; Clontech), counting transfected cells by fluorescence microscopy. The variation of transfection efficiencies within plates was analysed by comparison of variation. Comparison of means was performed using the Student's t-test. P values <0·05 were considered significant.
When looking at RANTES production after HSV infection, we found that both HSV-1 and -2 induced RANTES expression in macrophages and fibroblasts and that protein was detectable from 8 h post-infection (p.i.) (Fig. 1AD). To examine whether the enhanced expression of RANTES was due to induced transcription, we performed a reporter gene assay using NIH 3T3 cells transfected with a luciferase-linked RANTES promoter construct. In parallel, the transfection efficiency and the possible variation within plates were investigated using a GFP vector. The variation of transfection efficiency between plates was shown not to be significant, with maximum efficiencies varying from 20 to 23 % (data not shown), and therefore subsequent experiments were performed in triplicate without further normalization. As seen in Fig. 1(E)
, RANTES transcription was significantly induced when cells were infected with HSV. Thus, expression of RANTES after HSV infection is regulated at the transcriptional level.
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The MAP kinase system and p38 are activated after infection with many viruses and have been connected to regulation of RANTES production (Kujime et al., 2000; Pazdrak et al., 2002
). Previous studies from this laboratory have shown that production of IL-6 and TNF-
in HSV-infected macrophages is dependent on p38 (Paludan, 2001
; Paludan et al., 2001
). To investigate the role of p38 in the expression of RANTES we used SB203580, a highly specific inhibitor of p38 MAP kinase. Treatment with SB203580 did not affect RANTES mRNA accumulation in HSV-infected macrophages and only slightly reduced mRNA levels in fibroblasts (Fig. 2
). A modest reduction in protein levels was observed in both cell types. For both cell lines this reduction was seen in repeated experiments, but only in NIH 3T3 cells did the reduction approach levels of significance, with P values just above 0·05 in some experiments and just below in others. These results suggest that p38 is not involved in HSV-induced RANTES transcription, but may play a minor role at the post-transcriptional level.
Given the above-described dependency on NF-B and the unsolved role of p38 in HSV-induced RANTES expression, we further investigated these factors using J774A.1 macrophages stably transfected with dominant-negative mutants of IKK
and p38. We also included dominant-negative IRF-3, known to be involved in expression of RANTES in response to some viruses (Genin et al., 2000
; tenOever et al., 2002
). The results showed that cells stably transfected with the empty vector (pcDNA3) retained the ability to produced RANTES mRNA and protein after HSV infection (Fig. 3
A, E). The p38 mutant cell line displayed only slightly reduced RANTES mRNA accumulation and protein secretion (Fig. 3D, F
). In contrast, the IRF-3 and IKK
mutant cell lines were strongly disabled in their capacity to produce RANTES following HSV infection (Fig. 3B, C, E
).
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Taken together, the results presented here show that cells respond to HSV infection by producing RANTES and that this proceeds through a mechanism dependent on NF-B and IRF-3. Production of RANTES and other chemokines by virus-infected cells may allow the organism to recruit leukocytes rapidly to the site of infection, thereby initiating an efficient antiviral response.
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ACKNOWLEDGEMENTS |
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Received 13 February 2003;
accepted 7 May 2003.