Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Feb;86(2):178-88.
doi: 10.1016/j.exer.2007.10.008. Epub 2007 Dec 3.

Induction of interleukin-6 in human retinal epithelial cells by an attenuated Herpes simplex virus vector requires viral replication and NFkappaB activation

Suping Cai  1 , Curtis R Brandt
Affiliations

Induction of interleukin-6 in human retinal epithelial cells by an attenuated Herpes simplex virus vector requires viral replication and NFkappaB activation

Suping Cai et al. Exp Eye Res. 2008 Feb.

Abstract

Gene delivery has potential for treating ocular disease and a number of delivery systems have been tested in animal models. However, several viral vectors have been shown to trigger undesirable transient inflammatory responses in the eye. Previously, it was shown that an attenuated Herpes simplex virus vector (hrR3) transduced numerous cell types in the anterior and posterior segments of monkey eyes, but this was accompanied by inflammation. In the retina, retinal pigment epithelial cells were the predominant cell type transduced by hrR3. IL-6 is an important pro-inflammatory cytokine and may play a role in the response to the hrR3 vector. Infection of human ARPE-19 cells with hrR3 resulted in increased IL-6 expression and secretion 3-4h post-infection. In the presence of acyclovir (70 microM) or in cells infected with UV-inactivated hrR3, IL-6 was not up-regulated indicating viral replication was required. Expression of the HSV-1 alpha and beta genes may be necessary but was not sufficient for NF-kappaB activation and IL-6 up-regulation. The translocation of NF-kappaB into the nucleus also occurred between 3 and 4h post-infection, coincident with increased IL-6 expression. Inhibition of NF-kappaB translocation by an Adenovirus vector expressing a dominant negative IkappaB (AdIkappaBam) inhibited IL-6 up-regulation, indicating that NF-kappaB plays a role in increasing IL-6 expression in APRE-19 cells. The hrR3 virus lacks viral ribonucleotide reductase (RR) activity, thus RR is not required for NF-kappaB activation or IL-6 up-regulation in ARPE-19 cells.

PubMed Disclaimer

Figures

Figure 1
Figure 1. The structure of hrR3 genome
The top line shows the structure of the HSV-1 genome. The second line shows an enlargement of the region encoding the large subunit of ribonucleotide reductase (UL 39 gene; nucleotides 86,644 to 89,857) and the insertion of the lacZ gene in the UL39 locus at position 87,816 in the HSV-1 genome. TRL, terminal repeat long; UL, unique long; IRL, internal repeat long; IRS, internal repeat short; US, unique short; TRS, terminal repeat short; hrR3, herpes simplex virus type 1 ribonucleotide reductase mutant; lacZ, lacZ gene.
Figure 2
Figure 2. IL-6 expression in hrR3 infected ARPE-19 cells
Panel A. Immunoblotting of cell extracts. The ARPE-19 cells were grown to confluence in culture medium containing 10% FBS, and the cells were infected with hrR3 at a multiplicity of infection (MOI) of 1. Cell cultures were harvested at 4, 8 and 12 h post-infection. Cell lysates were electrophoresed and subjected to western blotting with polyclonal rabbit anti-human IL-6 antibody. The positions of protein molecular weight markers are shown on the right and IL-6 isoforms are indicated. This blot is representative of three separate experiments showing IL-6 is up-regulated in infected cells. Panel B. Effect of hrR3 infection on IL-6 secretion. ARPE-19 cell cultures grown to confluence were either mock-infected or infected with hrR3 at a MOI of 1. The culture supernatants were harvested at 3, 8, and 12 h post-infection and analyzed for IL-6 by ELISA. Cell numbers were counted at each time point with a hemacytometer. Results are presented as the mean ± standard error of the mean for three independent experiments. Filled bar: control. Shaded bar: hrR3.
Figure 3
Figure 3. hrR3 induced NF-κB p65 nuclear translocation in ARPE-19 cells at 30 min or 8 h after treatment
ARPE-19 cells, seeded in 4-well chamber slides, were infected with hrR3 at a MOI of 2, IL-1β (100 ng/ml), or with medium as a control. Thirty minutes (Panel A) or 8 h (Panel B) following the treatments the cells were immunostained with antisera specific for the p65 subunit of NF-κB. The nuclei were also stained with Hoechst dye and Hoechst images were pseudocolored green to facilitate image merger. All images were originally taken at 100x magnification. Panel C. The percentages of cells with nuclear NF-κB p65 at various time points post-treatment. □, IL-1β; Δ, hrR3; ○, IL-1 β control; ∇, hrR3 control.
Figure 4
Figure 4. (A) Immunoblot analysis of IκBaM expression in AdIκBaM transduced ARPE-19 cells
The ARPE-19 cells were grown to confluence and either mock-transduced or transduced with AdIκBaM at a MOI of 5 or 50. Cell lysates were then subjected to western blotting using mouse polyclonal anti-IκBa antibody. Actin staining was used to show the lanes were loaded equally. Panels B and C. IL-1 beta (B) or hrR3 (C) induced NF-κB p65 nuclear translocation in AdIκBaM or AdlacZ transduced ARPE-19 cells. ARPE cells were seeded in 4-well chamber slides. The following day the cells were transduced with the AdIκBaM or AdlacZ viruses for 24 h. The cells were then treated with IL-1 β for 1 h or hrR3 for 8 h. Following treatments, the cells were immunostained with antiserum specific for p65. Hoescht stained images were pseudocolored green to facilitate merging of the images. All images were originally taken at a magnification of 40x. (D). Immunoblot analysis of hrR3 induced IL-6 expression in AdIκBaM or AdlacZ transduced ARPE-19 cells. The ARPE-19 cells were grown to confluence and then transduced with AdIκBaM or AdlacZ. Twenty-four hours later the cells were then infected with hrR3 at a MOI of 1. At 8 h post-infection, cell lysates were subjected to western blotting with polyclonal rabbit anti-human anti IL-6. The blot was then stripped and re-probed with actin antibody as a loading control. Signals were detected using the ECL system. The positions of IL-6 isoforms are denoted with arrows.
Figure 5
Figure 5. Viral replication is required for IL-6 induction and NF-κB activation
(A). One-step growth curve of hrR3 and KOS in ARPE-19 cells. ARPE-19 cell cultures were infected with hrR3 or HSV-1 KOS at an MOI of 4. After a 1 h adsorption period, the monolayers were washed three times and refed. At the indicated times, the cultures were collected and the amount of total virus per sample was titered by plaque assay using Vero cells. Each data point represents the mean ± standard error of the mean titer from three independent experiments. ○, HSV-1 KOS; ●, hrR3. (B). IL-6 secretion in hrR3 or hrR3 plus ACV-infected ARPE-19 cells. Cell cultures were infected with hrR3 in the presence or absence of 70 nM ACV. After a 1 h adsorption period, the monolayers were washed three times and replenished with culture medium or culture medium with ACV. At the indicated times, culture supernatants were harvested, and the IL-6 concentration was determined by ELISA. The fold induction of IL-6 between hrR3 or hrR3 plus ACV-treated samples vs. control samples was calculated for each time point. The points represent the means +/− standard error of the means for triplicate samples. ■, hrR3 + ACV; formula image, hrR3. (C). hrR3 or UV-inactivated hrR3 induction of NF-κB p65 nuclear translocation in ARPE-19 cells. ARPE-19 cells were seeded in 4-well chamber slides the day before experiment. On the day of experiment, cells were infected with hrR3 or UV-inactivated hrR3 and eight hours later they were immunostained for NF-κB p65. Hoescht stained images were pseudocolored green to facilitate image merger. All images were originally taken at a magnification of 40x. (D). Immunoblot analysis of hrR3 or UV-inactivated hrR3 induced IL-6 expression in ARPE-19 cells. The ARPE-19 cells were grown to confluence and the cells were infected with hrR3 or UV-inactivated hrR3 at a MOI of 1 (the titer for UV-inactivated hrR3 was the titer prior to UV-irradiation). At 8 h post-infection cell lysates were subjected to western blotting with polyclonal rabbit anti-human IL-6. The blot was the stripped and re-probed with actin antibody as a loading control. Signals were detected using ECL system and the signal intensities were determined using Scion Image 4.0.3.2 (Scion Corp., Frederick, MD).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Amici C, Belardo G, Rossi A, Santoro MG. Activation of I kappa b kinase by herpes simplex virus type 1. A novel target for anti-herpetic therapy. J Biol Chem. 2001;276:28759–28766. - PubMed
    1. Amici C, Rossi A, Costanzo A, Ciafre S, Marinari B, Balsamo M, Levrero M, Santoro MG. Herpes simplex virus disrupts NF-kappaB regulation by blocking its recruitment on the IkappaBalpha promoter and directing the factor on viral genes. J Biol Chem. 2006;281:7110–7117. - PubMed
    1. Anderson KP, Frink RJ, Devi GB, Gaylord BH, Costa RH, Wagner EK. Detailed characterization of the mRNA mapping in the HindIII fragment K region of the herpes simplex virus type 1 genome. J Virol. 1981;37:1011–1027. - PMC - PubMed
    1. Bacchetti S, Evelegh MJ, Muirhead B. Identification and separation of the two subunits of the herpes simplex virus ribonucleotide reductase. J Virol. 1986;57:1177–1181. - PMC - PubMed
    1. Bacchetti S, Evelegh MJ, Muirhead B, Sartori CS, Huszar D. Immunological characterization of herpes simplex virus type 1 and 2 polypeptide(s) involved in viral ribonucleotide reductase activity. J Virol. 1984;49:591–593. - PMC - PubMed

Publication types