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Departments of
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Ophthalmology,
Internal Medicine, and
Laboratory Medicine and Pathology, Divisions of
Infection and
¶ Virology, Taipei Veterans General Hospital and National Yang-Ming University, Tapei, Taiwan, Republic of China;
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Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan, Republic of China;
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Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, Republic of China; and
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National Health Research Institutes, Taipei, Taiwan, Republic of China
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subfamily of herpesviruses, contains a dsDNA genome of
229,354 bp with a potential to encode >200 proteins (1).
HCMV infection usually develops asymptomatic lifelong infection in
healthy individuals, but can cause severe clinical complications such
as HCMV retinitis when reactivated in immunocompromised patients
(2, 3, 4). Although the detailed mechanisms of HCMV latency
and reactivation are not yet well understood, accumulating evidences
suggest that the virus can use a panel of viral proteins to escape from
cellular immune control and, thus, successfully survive and replicate
in host cells (5, 6, 7, 8). Cellular immune reactions and the associated inflammatory responses can be harmful to nearby tissues. Since minor inflammation can result in impaired vision or even blindness, the eye is naturally designed as an immune privileged site where infections usually do not lead to destructive immune reactions (9, 10). The underlying mechanism has been hypothesized to involve Fas ligand (FasL)-mediated programmed cell death (also called apoptosis) of Fas (CD95)-expressing T cells when attracted to the infection sites (9). In this case, activated T cells are eliminated through ligation of Fas by FasL and no serious immune reactions are induced. Thus, the damage to the eye is minimized. However, HCMV infection of human eyes is shown to cause large-scaled cell death and tremendous visual dysfunction (3, 4). Whether HCMV takes the advantage of the FasL-dependent immune evasion to exert its destructive effects remains an important issue. At least two studies argue for this hypothesis. First, in a mouse model of CMV retinitis, Bigger et al. (11) observed apoptosis of uninfected retinal cells in the lesions of retinitis. Second, FasL expression was found up-regulated upon HCMV infection of the human retinal pigment epithelial (HRPE) cells (12). Nevertheless, the correlation of the observed cell death in HCMV-infected human retina to FasL up-regulation is not clear, nor does the role of viral immediate-early (IE) proteins in stimulating the FasL synthesis.
In the present study, we present substantial evidences to show that FasL expression is indeed regulated by HCMV protein IE2 during the early stage of infection. In addition, elevated levels of FasL signals were detected together with HCMV IE Ags and apoptotic cells in the lesions of HCMV retinitis. These findings provide a potential mechanism for the pathogenesis of HCMV retinitis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HRPE cell line ARPE-19 and HCMV strain AD169 were obtained from American Type Culture Collection (Manassas, VA). HRPE cells were maintained in DMEM and Ham’s F-12 medium at a 1:1 ratio and supplemented with 10% FCS. HEL-299 cells were maintained in DMEM plus 10% FCS. Jurkat cells were maintained in RPMI 1640 medium plus 10% FCS. All of these cells were grown in a 37°C incubator supplied with 5% CO2. Cell-free virus stock was prepared from the supernatants of AD169-infected HEL-299 cells as described previously (13). One day before infection, subconfluent HRPE cells were switched to serum-free medium. HRPE cells were infected with AD169 at a multiplicity of infection (MOI) of 5. After virus absorption at 37°C for 2 h, cells were washed with PBS twice, fed with fresh culture medium, and grown in a 37°C incubator supplied with 5% CO2.
RNA analysis
Total RNA was extracted using the RNAeasy kit (Qiagen,
Chatsworth, CA). First-strand cDNA synthesis was conducted using
oligo(dT) primer and Moloney murine leukemia virus-reverse
transcriptase (Promega, Madison, WI). The derived cDNA products (5
µl) were PCR amplified in a 50-µl reaction mixture containing 0.2
mM dNTP, 2 mM MgCl2,10 pmol of each primer, and 1
U of Taq DNA polymerase. The primer pairs used were: hFasL
forward (5'-CAACTCAAGGTCCATGCCTC-3') and hFasL reverse
(5'-AGATTCCTCAAAATTGACCAG-3'); hFas forward
(5'-CTGCAGGAACGCCCCGGGACAC-3') and hFas reverse
(5'-GGTTGTTGAGCAATCCTCCGAAGT-3'); HCMV IE forward
(5'-CCAAGCGGCCTCTGATAACCAAGCC-3') and HCMV IE reverse
(5'-CAGCACCATCCTCCTCTTCCTCTGG-3'); -actin forward
(5'-TCCTGTGGCATCCACGAAACT-3') and -actin reverse
(5'-GAAGCATTTGCGGTGGACGAT-3'). PCR amplification was performed for
35 cycles at 94°C for 1 min, 52°C for 1 min, and 72°C for 2 min.
The expected sizes of PCR products for FasL, Fas, and -actin are
350, 266, and 314 bp, respectively.
Western blot assay
HCMV AD169-infected HRPE cells were harvested at days 1, 2, 3,
5, and 7 postinfection. The cell lysates were prepared as described.
Fifteen microliters of sample was boiled at 95°C for 5 min and
separated on a 10% SDS-PAGE. The proteins were transferred to
Hybond-ECL nitrocellulose paper (Amersham, Arlington Heights, IL) by
wet-transfer system. The mAbs used to identify protein products were
anti-FasL mAb (F37720/lot 3; BD Transduction Laboratories,
Lexington, KY), anti-Fas mAb (F22120; Transduction Laboratories),
anti-CMV IE mAb (MAB810; Chemicon International, Temecula, CA), and
anti--actin mAb (MAB81501; Chemicon International). The reactive
protein bands were detected by the ECL detection system (Amersham).
Transfection and dual-luciferase assay
The Lipofectamine 2000 system (Life Technologies, Grand Island, NY) was used for transfection of DNA into HRPE cells. The IE1 and IE2 plasmids had been described previously (14). The FasL promoter constructs (wild-type (wt) and deletion mutants) were kindly provided by Dr. C. Warrant (15). Briefly, 106 cells/well were seeded onto 6-well plates 24 h before transfection. DNA solution (250 µl of OPTI-MEM containing 3 µg of pGL2-hFasL reporter construct, 3 µg of p1R (IE1), 3 µg of pSIE2-wt (IE2) or mutant effector plasmids (IE2CD80), and 30 ng of pRL-tk Renilla normalizing luciferase vector) and reagent solution (5 µl of LF200 and 250 µl of OPTI-MEM) were separately preincubated at 25°C for 5 min. The transfections were then followed according to the manufacturer’s instructions. After 48 h of incubation, the cell lysates were prepared using the dual-luciferase reporter assay system (Promega). Both firefly and Renilla luciferase activities were monitored by illuminator (TD20/20; Turner Designs, Sunnyvale, CA).
ELISA test and flow cytometry
The concentration of soluble FasL (sFasL) in vitreous samples and in supernatants from infected HRPE cells was determined by ELISA kit (MBL, Nagova, Japan). The developed reaction was quantified by reading at A490 nm (MRX; Dynatech Laboratories, Chantilly, VA). Each individual sample was analyzed in triplicate. Apoptotic cells were determined by flow cytometry using propidium iodide and FITC-labeled annexin V (Immunotech, Luminy, France). Cells (106 cells/ml) were incubated with relevant Abs in PBS containing 1% BSA. The reaction was conducted at 4°C for 30 min in the dark. After incubation, the cells were washed twice with cold PBS, resuspended to 106 cells/ml in 250 µl of PBS, and analyzed by flow cytometry using a FACSCalibur apparatus (BD Biosciences, San Diego, CA). For blocking assays, the culture supernatants were pretreated with anti-FasL mAb (clone 4H9; MBL, Nagova, Japan) for 60 min at 37°C before the addition of Jurkat cells. Alternatively, Jurkat cells were pretreated with anti-Fas mAb (clone ZB4; MBL) for 60 min at 37°C before the addition of supernatants from AD169-infected HRPE cells.
Immunohistochemistry and in situ hybridization
The research followed tenets of the Declaration of Helsinki. All samples were obtained after informed consent from the patients. Twenty-four eyeballs enucleated from dead AIDS patients were obtained at autopsy and immediately fixed with 4% paraformaldehyde. The 4-µm paraffin sections were deparaffinized in xylene, rehydrated in a series of graded alcohols, and immunostained with Abs against CMV IE Ag (MAB810; Chemicon), FasL (Transduction Laboratories), and HIV-p24 (clone Kal-1; DAKO, Glostrup, Denmark). Immunoreactive signals were detected with a mixture of biotinylated IgG Ab and peroxidase-conjugated streptavidin (LSAB2 system; DAKO). Apoptotic cells in tissue sections were identified by the TUNEL method (In Situ Cell Death Detection kit, POD; Boehringer Mannheim, Mannheim, Germany) as described previously (16). As a negative control, the TdT was omitted from the TdT buffer. Chromogenic development was then applied with 3-amino-9-ethyl-carbazole and slides were counterstained with methyl green. Positive staining was identified under the light microscope as red granules. The RNA in situ hybridization procedure was performed as described previously (17). The 4-µm deparaffinized slides were predigested with nuclease-free proteinase K (0.1 mg/ml; Boehringer Mannheim). One hundred microliters of FITC-conjugated antisense and sense probes was added onto each slide. The sequences of FasL probes used were: 5'-GAGCTTGAAACATCCCCAGGCCCAATCCTACCAAGGCAACC-3' (antisense primer, 461–500 nt) and 5'-GGGATGTTTCAGCTCTTCCACCTACAG-3' (sense primer, 486–512 nt). Hybridization products were visualized by alkaline phosphatase conjugated secondary Abs and chromogen nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Sigma, St. Louis, MO). Positive staining was identified as purple granules under the microscope.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Since constitutive expression of FasL is critical for maintaining
the immune privilege of the eye, we hypothesized that dysregulated FasL
expression by HCMV might be responsible for the pathogenesis of HCMV
retinitis. In the eye, HRPE cells are located at the blood-retina
barriers (13, 18). Infection of HRPE cells has been shown
to result in serious ocular manifestations and suspected to be critical
for the development of HCMV retinitis (13). In this study,
we first infected a HRPE cell line, ARPE-19, with HCMV AD169 and
monitored the expression of FasL. We found that the synthesis of FasL
protein in HCMV-infected HRPE cells was enhanced early after infection.
The up-regulation of FasL was initiated at day 1 after infection (Fig. 1
A). In contrast, the
expression level of Fas remained unchanged (Fig. 1A).
Consistent to the Western blot data, we observed an increase of FasL
mRNA (Fig. 1B). These experiments suggest that HCMV
induction of FasL is specific and is determined at the transcriptional
level. The early induction of FasL in infected cells further implicates
that viral IE genes might play a role in this event. This idea was
supported by the evidence that the mRNA level of IE genes was also
induced at day 1 after infection (Fig. 1B).
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Since IE2 is a potent transactivator (19, 20), we set
up to examine whether HCMV IE2 directly regulates FasL expression. We
found that IE2 induced the activity of FasL promoter efficiently in
HRPE cells. The activation was specific for IE2 because, first, another
HCMV IE protein, IE1, had no effect on the same promoter (Fig. 2A). Note that the inability
of IE1 in inducing FasL was not due to a low level of protein being
made. In fact, the IE1 protein was more abundant than IE2, based on the
Western blot results using a mAb (mAb810) against the common region
(exon 3) shared by IE1 and IE2 proteins. Second, removal of IE2
C-terminal 80 residues (IE2CD80), which are required for repressing the
transactivation activity of the tumor suppressor protein p53 (14),
greatly reduced the protein’s activation activity on FasL promoter
(Fig. 2B). Third, IE2 no longer activated a truncated
version of FasL promoter without its 5' region from -1147 to -370 nt
(Fig. 2C). Thus, we conclude that HCMV IE2 is able to
specifically regulate the FasL promoter, providing a molecular
mechanism linked to the pathogenesis of HCMV retinitis.
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Identification of the correlation between IE2 and FasL
up-regulation in cultured HRPE cells prompted us to examine whether the
increased FasL upon HCMV infection induces apoptosis of uninfected
cells as observed in HCMV-infected human retina (see below). Consistent
with previous data, the levels of sFasL in the cell-free supernatants
of infected HRPE cells were increased since day 1 after infection (Fig. 3A). To further investigate
whether secreted FasL from HCMV-infected HRPE cells causes
Fas-dependent apoptosis in vitro, we cultivated Jurkat cells in medium
supplemented with 50% of supernatants from mock or HCMV-infected cell
cultures, respectively. After 48 h of incubation, the percentage
of apoptotic cells in the cultures was determined by flow cytometry. We
found that cells exposed to HCMV-infected supernatants exhibited a
higher percentage of apoptosis as compared with those exposed to mock
supernatants (Fig. 3B). Furthermore, functional blocking of
Fas or FasL with respective mAbs prevented the HCMV-induced apoptosis
of uninfected Jurkat cells (Fig. 3B). Interestingly, we
found that neither anti-Fas nor anti-FasL could block the
apoptosis of 
8% of Jurkat cells when cultured with mock-infected
supernatants (Fig. 3B), suggesting that other
FasL/Fas-independent cell death pathways were also involved. Our
results provide evidences that HCMV-infected HRPE cells are able to
release large amounts of FasL, which, in turn, can induce apoptosis of
nearby cells, presumably through a Fas-mediated pathway.
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To examine whether our previous observation is physiologically
relevant, we investigated whether sFasL could be released from
HCMV-infected retina into a vitreous body. Diagnosis of HCMV retinitis
was based on the clinical results of indirect ophthalmoscopy (Fig. 4A) and fluorescein
angiography (data not shown). As presented in Table I, before anti-HCMV treatment, the
averaged sFasL level of vitreous fluids among the eight AIDS patients
with HCMV retinitis was 440 pg/ml, with a SD of 36 pg/ml. In contrast,
after complete ganciclovir treatment and the appearance of regressed
lesions in HCMV-infected retina, the sFasL levels of vitreous fluids in
these eight patients declined dramatically (Table I). To exclude the
potential effects of HIV infection on the release of sFasL, seven AIDS
patients without HCMV retinitis were also examined. We found that the
sFasL levels in these patients were all below the detection limit
(Table I).
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E) as
compared with that obtained from AIDS patients without HCMV retinitis
(Fig. 4B). To examine whether apoptosis occurs in these
regions, TUNEL assay was performed. We observed many apoptotic cells in
the foci of HCMV-infected retina (Fig. 4F). In contrast, no
apoptotic cells were identified in HCMV IE-negative samples (Fig. 4C). Consistent with our observation that HCMV IE2
up-regulates the FasL expression, we detected higher levels of FasL in
HCMV IE-positive specimens (Fig. 4, G and J) as
compared with the basal level of FasL in AIDS patients without HCMV
retinitis (Fig. 4D). Dual immunohistostaining for FasL and
HCMV IE Ag was also conducted to determine whether increased expression
of FasL does correlate with HCMV infection (Fig. 4, H and
I). We found that the FasL and HCMV IE Ags were colocalized
in infected neuroretina and HRPE cells of HCMV-infected retina (Fig. 4I). A number of HRPE cells with intranuclear and typically
cytoplasmic inclusions in the lesions of HCMV-infected retina were also
observed under the microscope (Fig. 4I). Arguing against the
involvement of HIV in the lesions of HCMV-infected retina,
immunostaining with HIV Ag-specific Abs was performed on samples
collected from AIDS patients with or without HCMV retinitis. We found
that no HIV-1 p24 Ag was present in HRPE cells among all specimens
examined (data not shown). The rarely detected HIV-1 p24 Ag was found
to localize at few neuroretinal cells (Müll cells; data not
shown). Therefore, HIV might not have substantial influence in this
instance if there is any. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This report provides the first evidence that HCMV infection can induce
apoptosis of Fas-expressing cells via IE2 induction of FasL expression
in virally infected human retina. Nevertheless, whether IE2 activation
of FasL promoter is modulated directly through DNA binding of IE2 to a
specific site on the FasL promoter or represents a result of indirect
effects mediated by other proteins is largely unknown. As shown in Fig. 2C, a truncated version of the FasL promoter (-370) that
loses at least a putative NF-
B binding site responded to IE2
activation to a smaller degree. Note that HCMV IE2 induction of NF-B
promoter has been reported previously in many studies
(22). Whether NF-B is involved in IE2-mediated
up-regulation of FasL is currently under investigation.
Because we also observed the basal expression of Fas receptor on the
surface of HCMV-infected cells (Fig. 1A), one can argue that
cis-ligation of Fas by FasL might induce cell death of the
virally infected cells. To answer this question, we examined whether
apoptosis occurs in HCMV-infected HRPE cells. Using the TUNEL assay, we
did not observe any apoptotic cells within 3 days after infection (data
not shown). Although the underlying mechanisms are not completely
understood, several groups have argued against the death of virally
infected cells at least in the early stage of infection. It seems that
many viruses have evolved unique strategies to avoid its own death
while eliminating nearby uninfected cells, e.g., HIV virus. HIV
infection also stimulates FasL expression. However, death of
HIV-infected cells is inhibited, even Fas receptors are usually
abundant on the surfaces of infected cells. This mystery was not
resolved until recently by Geleziunas et al. (23). Based
on their study, HIV can inhibit the ASK1-dependent signaling pathway,
which is downstream of the Fas/FasL pathway. Thus, by impairing
functions of downstream regulators, HIV-1 can protect the infected host
cells from undergoing apoptosis. As for HCMV, at least two strategies
have been reported to overcome apoptosis. First, our laboratory has
demonstrated that HCMV IE2 can down-regulate p53 activity, which is
critical for the induction of cell apoptosis and is located even more
downstream of ASK1 along the cell death signaling pathway
(14). Second, a HCMV-encoded cell death suppressor, viral
inhibitor of caspase-8-induced apoptosis, has been newly
identified (24). It was shown to inhibit Fas/FasL-mediated
apoptosis by binding to and subsequently inactivating caspase-8.
Therefore, it is possible that HCMV can use different approaches to
prevent its own death, even cis-ligation of overexpressed
FasL and Fas occurs on the surfaces of HCMV-infected cells.
In summary, our results indicate that Fas/FasL-mediated apoptosis previously thought to be important in the maintenance of immune surveillance within human retina (9, 10, 18) might turn out to be destructive once manipulated by HCMV.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprints requests to Dr. Cheng-Wen Wu, National Health Research Institutes, 128 Yen-Chiu-Yuan Road, Taipei 11529, Taiwan, Republic of China. E-mail address: ken{at}nhri.org.tw
3 Abbreviations used in this paper: HCMV, human CMV; FasL, Fas ligand; HRPE, human retina pigment epithelium; sFasL, soluble FasL; IE, immediate early; MOI, multiplicity of infection; hFasL, human FasL; wt, wild type.
Received for publication May 10, 2001. Accepted for publication August 2, 2001.
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References |
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-induced indoleamine 2,3 deoxygenase and inducible nitric oxide synthase in the replication of human cytomegalovirus in retinal pigment epithelial cells. J. Immunol. 162:957.This article has been cited by other articles:
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  M. C.-J. Cheeran, J. R. Lokensgard, and M. R. Schleiss Neuropathogenesis of Congenital Cytomegalovirus Infection: Disease Mechanisms and Prospects for Intervention Clin. Microbiol. Rev., January 1, 2009; 22(1): 99 - 126. [Abstract] [Full Text] [PDF]
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 R. L. Sanders, C. L. Clark, C. S. Morello, and D. H. Spector Development of Cell Lines That Provide Tightly Controlled Temporal Translation of the Human Cytomegalovirus IE2 Proteins for Complementation and Functional Analyses of Growth-Impaired and Nonviable IE2 Mutant Viruses J. Virol., July 15, 2008; 82(14): 7059 - 7077. [Abstract] [Full Text] [PDF]
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 M. Zhang, B. Marshall, and S. S. Atherton Murine Cytomegalovirus Infection and Apoptosis in Organotypic Retinal Cultures Invest. Ophthalmol. Vis. Sci., January 1, 2008; 49(1): 295 - 303. [Abstract] [Full Text] [PDF]
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 S.-H. Chiou, Y.-P. Yang, J.-C. Lin, C.-H. Hsu, H.-C. Jhang, Y.-T. Yang, C.-H. Lee, L. L. T. Ho, W.-M. Hsu, H.-H. Ku, et al. The Immediate Early 2 Protein of Human Cytomegalovirus (HCMV) Mediates the Apoptotic Control in HCMV Retinitis through Up-Regulation of the Cellular FLICE-Inhibitory Protein Expression J. Immunol., November 1, 2006; 177(9): 6199 - 6206. [Abstract] [Full Text] [PDF]
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M. Shlapobersky, R. Sanders, C. Clark, and D. H. Spector Repression of HMGA2 Gene Expression by Human Cytomegalovirus Involves the IE2 86-Kilodalton Protein and Is Necessary for Efficient Viral Replication and Inhibition of Cyclin A Transcription. J. Virol., October 1, 2006; 80(20): 9951 - 9961. [Abstract] [Full Text] [PDF]
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M. Michaelis, T. Suhan, A. Reinisch, A. Reisenauer, C. Fleckenstein, D. Eikel, H. Gumbel, H. W. Doerr, H. Nau, and J. Cinatl Jr Increased Replication of Human Cytomegalovirus in Retinal Pigment Epithelial Cells by Valproic Acid Depends on Histone Deacetylase Inhibition Invest. Ophthalmol. Vis. Sci., September 1, 2005; 46(9): 3451 - 3457. [Abstract] [Full Text] [PDF]
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M. Zhang, H. Xin, P. Roon, and S. S. Atherton Infection of Retinal Neurons during Murine Cytomegalovirus Retinitis Invest. Ophthalmol. Vis. Sci., June 1, 2005; 46(6): 2047 - 2055. [Abstract] [Full Text] [PDF]
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 Y.-G. Yoo and M.-O. Lee Hepatitis B Virus X Protein Induces Expression of Fas Ligand Gene through Enhancing Transcriptional Activity of Early Growth Response Factor J. Biol. Chem., August 27, 2004; 279(35): 36242 - 36249. [Abstract] [Full Text] [PDF]
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Y. Momma, C. N. Nagineni, M. S. Chin, K. Srinivasan, B. Detrick, and J. J. Hooks Differential Expression of Chemokines by Human Retinal Pigment Epithelial Cells Infected with Cytomegalovirus Invest. Ophthalmol. Vis. Sci., May 1, 2003; 44(5): 2026 - 2033. [Abstract] [Full Text] [PDF]
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