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. 1999 Dec;59(4):552-60.

Lassa and Mopeia virus replication in human monocytes/macrophages and in endothelial cells: different effects on IL-8 and TNF-alpha gene expression

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Lassa and Mopeia virus replication in human monocytes/macrophages and in endothelial cells: different effects on IL-8 and TNF-alpha gene expression

I S Lukashevich et al. J Med Virol. 1999 Dec.

Abstract

Cells of the mononuclear and endothelial lineages are targets for viruses which cause hemorrhagic fevers (HF) such as the filoviruses Marburg and Ebola, and the arenaviruses Lassa and Junin. A recent model of Marburg HF pathogenesis proposes that virus directly causes endothelial cell damage and macrophage release of TNF-alpha which increases the permeability of endothelial monolayers [Feldmann et al. , 1996]. We show that Lassa virus replicates in human monocytes/macrophages and endothelial cells without damaging them. Human endothelial cells (HUVEC) are highly susceptible to infection by both Lassa and Mopeia (a non-pathogenic Lassa-related arenavirus). Whereas monocytes must differentiate into macrophages before supporting even low level production of these viruses, the virus yields in the culture medium of infected HUVEC cells reach more than 7 log10 PFU/ml without cellular damage. In contrast to filovirus, Lassa virus replication in monocytes/macrophages fails to stimulate TNF-alpha gene expression and even down-regulates LPS-stimulated TNF-alpha mRNA synthesis. The expression of IL-8, a prototypic proinflammatory CXC chemokine, was also suppressed in Lassa virus infected monocytes/macrophages and HUVEC on both the protein and mRNA levels. This contrasts with Mopeia virus infection of HUVEC in which neither IL-8 mRNA nor protein are reduced. The cumulative down-regulation of TNF-alpha and IL-8 expression could explain the absence of inflammatory and effective immune responses in severe cases of Lassa HF.

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Figures

Fig. 1
Fig. 1
RT/PCR amplification of LAS GP1 and TNF-α mRNAs in MDM. RNAs from MDM infected with LAS (lanes 1–6) or MOP (lanes 8–11) were extracted at 12 (lanes 1, 3, 5, 8, 10) or 24 (lanes 2, 4, 6, 9, 11) hours p. i. and used in RT-PCR with LAS GP1, GAPDH or TNF-α primers. RNA from mock-infected cells treated with LPS was used as a positive control for natural TNF-α mRNA expression (lane 7, 382-bp amplicon). 2,000 copies of exogenous synthesized DNA was included in PCR as TNF-α internal calibration standard, ICS (lanes 5–8, 482-bp amplicon). M, positions of DNA markers: ICS, TNF standard; TNF, wild type TNF-α cDNA. Note that wild type TNF-α cDNA and GAPDH cDNA amplicons have the same size, 382 bp.
Fig. 2
Fig. 2
Quantitation of TNF-α mRNA expression in human LPS-stimulated and Lassa-infected MDM by competitive PCR. A. Gel-based analysis of TNF-α mRNA expression in LPS-MDM infected with Lassa virus. MDM grown in T25 flasks were stimulated with LPS, infected with Lassa virus (MOI = 5) and incubated for 12 and 24 hours. At 12 and 24 hours p. i. RNA was extracted, converted into cDNA and equivalent amounts of cDNA (based on GAPDH) were amplified. Quantitative PCR was performed with biotin-labeled TNF-α primers using different concentrations of cytokine template (1, 1:10, and 1:100 dilutions) and a constant amount (2,000 copies) of ICS (BioSource). 5 ul-aliquots were analyzed on 2% agarose. M, positions of DNA markers, 500 and 250 bp. B. Quantitation of PCR products. 25 ul from each PCR reaction was denatured under alkaline pH and serial dilutions (1:40–1:320) of TNF-α and ICS amplicons were made in hybridization buffer in ICS- or TNF-α-specific oligonucleotide capture wells. After hybridization the captured amplicons were detected by streptavidin conjugates. Numbers of TNF-α mRNA copies were calculated as follows: Total TNF-α Optical Density (OD)/Total ICS OD ×ばつ 2 ×ばつ Input copy number of ICS ×ばつ cDNA dilution.
Fig. 3
Fig. 3
IL-8 mRNA expression in human MDM infected with Lassa and Mopeia viruses. A. Total RNA was extracted from mock-infected cells (lanes 1, 2) and cells infected with Lassa (lanes 3, 4) and Mopeia (lanes 5, 6) viruses at 24 (lanes 3, 5) and 48 (lanes 1, 2, 4, 6) hours p. i. RNA was subjected by RT-PCR with IL-8 specific primers. Lane 2, RNA from cells stimulated with LPS, 10 ng/ml. Lane M denotes size markers. B. RT-PCR amplification of the same samples with GAPDH primers in parallel experiment.
Fig. 4
Fig. 4
Relative multiplex quantitative RT-PCR analysis of IL-8 mRNA expression in human LPS-stimulated MDM infected with Lassa virus. A. Gel-based analysis. cDNA from MDM RNA was synthesized and amplified with IL-8 and 18S rRNA/18S rRNA Compe-timers primer mix (2:8, Ambion) in replicates of three. 10 ul of each PCR was electrophoresed on a 2% agarose gel. Positions of size markers (200, 300, and 400 bp) as well as 18S rRNA cDNA and IL-8 cDNA are indicated. B. Semiquantitative Analysis. 32P-dCTP-labeled PCR samples were visualized with ethidium bromide as shown above in panel A. IL-8 and 18S specific bands were excised from the gel and incorporated radioactivities were measured by liquid scintillation counting. To standardize IL-8 expression, radioactivity in IL-8 PCR products was divided by the corresponding 18S rRNA cDNA radioactivity in each sample. Radioactivity of the non-template control was subtracted from experimental values. Results of three independent RT-PCR experiments are summarized as radioactivity (cpm) recovered from IL-8 amplicons and as a IL-8/18S cDNA normalized values.

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