IOVS Heart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

(Investigative Ophthalmology and Visual Science. 2007;48:789-796.)
© 2007 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.06-0609
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow An erratum has been published
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Miles, D. H.
Right arrow Articles by Willcox, M. D. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Miles, D. H.
Right arrow Articles by Willcox, M. D. P.

The Induction and Suppression of the Apoptotic Response of HSV-1 in Human Corneal Epithelial Cells

David H. Miles,1 Archana Thakur,1 Nerida Cole,2,3 and Mark D. P. Willcox1,2,3

2From the Vision Cooperative Research Centre, Sydney, Australia; 1School of Optometry and Vision Science, The University of New South Wales, Sydney, Australia; and the 3Institute for Eye Research, Sydney, Australia.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. HSV-1 has been shown to block apoptosis in some cell lines when the cells are exposed to exogenous agents (e.g., sorbitol). The purpose of this study was to determine whether HSV-1 infection of human corneal epithelial (HCE) cells alone induces an early proapoptotic response and whether this response is subsequently downregulated during the infection.

METHODS. HCE cells were infected with HSV-1 or subjected to osmotic shock (sorbitol). Fluorescent staining for annexin V binding, mitochondrial membrane potential, and DNA condensation and assays for caspase 8, 9, and 3 activity and cytokeratin 18 cleavage were performed, to assess the apoptotic pathway.

RESULTS. HSV-1 infection of HCE cells induced a rapid proapoptotic response, characterized by translocation of phosphatidylserine to the external membrane, activation of caspases 8 and 3 within 2 hours, and cleavage of cytokeratin 18. However, the induced response was downregulated during the infection, and later stages of the apoptotic responses (e.g., DNA condensation) were not produced. Sorbitol treatment led to terminal apoptosis by 12 hours, as indicated by DNA condensation of treated cells and reduction in the number of viable cells.

CONCLUSIONS. HSV-1 can induce and subsequently suppress the apoptotic pathway in HCE. Suppression of apoptosis occurred only during HSV-1 infection and not after treatment with sorbitol, suggesting that the suppression of apoptosis may be a mechanism of viral survival.

Apoptosis is a complex, tightly regulated, and evolutionarily conserved mode of cell death. When cells are infected by certain viruses, apoptosis is triggered. However, many viruses are able to block apoptosis (reviewed by Miles et al.1 ). This mechanism is thought to be one by which a virus avoids the immune system, as cytotoxic T and natural killer cells actively target apoptotic cells—a process essential in viral clearance.1

The general molecular pathway of apoptosis is divided into an intrinsic and extrinsic arm.1 2 The intrinsic pathway is initiated in the mitochondria by triggers such as hypoxia or UV irradiation.1 This action stimulates cysteine aspartate protease (caspase) recruiting domains, leading to activation of caspase 9,3 usually followed by activation of downstream caspases, such as caspases 3, 6, and 7, resulting in cleavage of several death substrates4 5 and subsequent DNA condensation and fragmentation, indicating terminal apoptosis.

Initiation of the extrinsic apoptotic pathway begins with binding of ligands such as tumor necrosis factor-{alpha}, Fas ligand, and viruses to cell surface receptors.3 6 A subsequent tyrosine phosphorylation cascade stimulates various death domains in the cytoplasmic domains of these receptors.7 These trigger the activation of caspase 8.3 4 At this point, the two pathways merge and initiation of downstream caspases follows.4 Recent evidence has demonstrated that cells can be rescued from apoptosis induced by external agents in the presence7 8 9 10 11 12 13 14 or absence15 16 of herpes simplex viruses.

Herpes simplex virus (HSV)-1 infection of the cornea remains a major cause of vision loss worldwide, despite the availability of antiviral drugs.17 The effects of HSV-1 infection on apoptosis have been studied, mainly in liver and kidney epithelial cells, where results suggest that HSV-1 infection can prevent the induction of apoptosis when cells are exposed to exogenous proapoptotic agents (e.g., sorbitol).10 11 18 However, when protein synthesis in these cells is inhibited, cells infected with HSV-1 also undergo apoptosis. This suggests that HSV-1 proteins block apoptotic pathways.7 8 9 10 11 12 13 14 Not all cell types have the same apoptotic responses to HSV-1 infection. For example, HEp-2 cells initially have a proapoptotic response followed by blockage of the apoptotic pathway, whereas Vero cells (African green monkey kidney cells) show no or a reduced apoptotic response over 24 hours.7 In corneal epithelial cells, apoptosis has been shown to play a role in the pathogenesis of HSV keratitis; however, the apoptotic response of the cells to infection with this virus is not well characterized and cannot be inferred from data in other cell types.19 As such, we have recently demonstrated a rapid direct activation of elements of the proapoptotic pathway by HSV during infection in human corneal epithelial (HCE) cells via caspase 8.20

The purpose of the current investigation was to characterize the induction of early apoptotic events during infection of HCE cells by HSV-1 and to examine the ability of the virus to act subsequently to suppress the proapoptotic pathway.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cells tested were transformed HCE cells unless it is specifically stated that primary human corneal epithelial (PHCE) cells were used. All reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise specified. All assays were repeated on three separate occasions, and data are presented as the mean ± SD.

Cell Culture
SV40-immortalized HCE cells used in this study were obtained from the Riken Cell Bank (RCB/384; Ibaraki, Japan). Cells were maintained in supplemented hormone epithelial medium (SHEM; comprising minimum essential medium [MEM] and nutrient mixture F-12 1:1 vol/vol, epidermal growth factor 10 ng/mL, insulin 5 µg/mL, cholera toxin 0.1 µg/mL, fetal bovine serum [FBS] 5% vol/vol, dimethyl sulfoxide [DMSO] 0.5% vol/vol, and gentamicin 5 µg/mL). PHCE cells were derived from excised epithelial layers of donor corneas obtained from the Sydney Eye Hospital in accordance with the Declaration of Helsinki and explanted on tissue culture plates in medium with human corneal growth supplement (Epilife; Cascade Biologics, Inc., Portland, OR) and calcium chloride (4 mg/mL). The cells were seeded at 92,000/cm2 and incubated at 37°C in a 5% CO2-95% air incubator. Medium was exchanged on alternate days. Experiments were performed when cells were at 90% confluence. The number of cells was estimated by using trypan blue staining.

Virus and Titration Method
The HSV-1 virus strain MacIntyre (lot VR-539) was obtained from the American Type Culture Collection (ATCC, Manassas, VA). The McKrae HSV-1 strain and the laboratory-adapted KOS1.1 HSV-1 strain were the kind gift of James Hill (Louisiana State University Health and Science Center, New Orleans, LA). These HSV-1 strains were used to test whether viral strain specificity occurred in our system. All other experiments were performed with the MacIntyre HSV-1 strain. The plaque-forming assay method was used to titer the virus. Vero cells (Cascade Biologics, Inc. Portland, OR) were grown to confluence. Serial 10-fold dilutions of each virus were made in chilled maintenance medium (MEM; 1% vol/vol FBS). Culture medium (DMEM; 10% vol/vol FBS) was aspirated from the Vero cells, and 0.2 mL of viral inoculum was added. The infected cells were incubated for 1 hour at 37°C with intermittent rocking. The inoculum was removed and 1:1 vol/vol ratio of concentrated culture medium (2x) and carboxymethylcellulose (0.5% wt/vol solution) was added. A multiplicity of infection (MOI) of 2 was used in all experiments, as it was determined to be the viral titer that produced the greatest response (data not shown). Plates were incubated at 37°C in a 5% CO2-95% air incubator, and enumeration of plaques was performed with a Stuart colony counter (model S35; Barloworld Scientific, Stone, UK) on the fourth day.

HSV-1 Infection and Sorbitol Treatment of HCE Cells
Viruses were added to HCE in MEM with 1% vol/vol FBS at a ratio of 2:1 and allowed to attach to the cells for 30 minutes. Initial HSV-1 infection of HCE cells was confirmed by immunofluorescence using HSV-1-specific polyclonal antibodies to HSV-1 glycoproteins (Dako, Carpinteria, CA). Approximately 95% of transformed HCE cells were infected when an MOI of 2 was used with each of the HSV strains. After 30 minutes, unattached virus was aspirated and fresh SHEM was added. Uninfected (mock infected) HCE cells were used as a negative control. After the attachment period, cells were collected at 0.5, 1, 2, 4, 8, and 12 hours. These cells were suspended by the addition of trypsin. The trypsin was neutralized by the addition of SHEM (containing 5% vol/vol FBS) at a ratio of 2:1 media/trypsin volume, and HCE cells were collected by centrifugation at 300g for 5 minutes. The HCE cells were either stained with apoptotic fluorescent stains or lysed for caspase quantification.

Apoptosis by osmotic shock was induced by sorbitol, as previously described.21 Briefly, media were aspirated, and sorbitol (1.5 moles/L) in MEM was added to the cells and incubated for 1 hour at ambient temperature. The sorbitol was removed and fresh media were added. Cells were collected for assay as described earlier.

Fluorescence Staining and Microscopy
Commercially available fluorescent stains were used to assess early (mitochondrial membrane potential, annexin V, and cytokeratin 18) and late (DNA condensation) apoptotic changes in total cell populations of sorbitol-treated, HSV-1-infected and uninfected cells over time. Stained cells were quantified using fluorescence microscopy (BH 2-RFC; Olympus, Tokyo, Japan) by a masked observer. For calculation of the percentage of cells showing apoptotic changes in all staining procedures, a minimum of 500 cells were counted at each time point in triplicate preparations.

Mitochondrial Transmembrane Potential Staining.
This stain detects the changes in the mitochondrial transmembrane potential of cells. Staining was performed according to the manufacturer’s protocol (BioVision, Firenze, Italy). Briefly, the cationic dye, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzamidazolo-carbocyanin iodide was added to the pelleted cell sample and allowed to incubate at ambient temperature for 15 minutes. Cells were washed with phosphate-buffered saline (PBS) twice and mounted on slides. A fluorescence microscope was used with a band-pass filter to detect FITC and rhodamine. The number of stained cells was counted and recorded as a percentage of the total cell population.

Annexin V Staining and Flow Cytometry.
This assay detects phosphatidylserine translocation from the inner to the outer leaflet of the cell membrane.22 23 Assays were performed according to the manufacturer’s protocol (MBL, Nagoya, Japan). Briefly, annexin V-FITC (binds to phosphatidylserine) and propidium iodide (PI, counterstain for DNA) were added to the pelleted HCE cells (approximately 106 cells per sample) and allowed to incubate at ambient temperature for 10 minutes. Any PI-positive cells were considered necrotic and not apoptotic. PHCE cells, stained with annexin V, were manually counted and the results recorded as the percentage of stained cells within the total cell population, whereas transformed HCE cells were analyzed by flow cytometry (Mo Flo MLS; Cytomation, Fort Collins, CO, with Cytomation Summit software version 3.1). For flow cytometry, cells were detected with FITC signal detector (excitation, 488 nm; emission, 530 nm) and PI staining by the phycoerythrin emission signal detector. Samples were gated by using a negative control (unstained viable cells in culture) and a positive control (HCE cells treated with ethanol for 1 hour, which stain with both FITC and PI).23

Cleavage of Cytokeratin 18.
The M30-fluorescein antibody recognizes cleavage of cytokeratin 18 (CK18) by caspases 3 and 7. Assays were performed according to the manufacturer’s protocol (Roche, Mannheim, Germany). Briefly, pelleted cells were fixed in ice-cold methanol for 30 minutes, then washed in PBS containing 0.1% vol/vol Tween 20 (PBS-T). Cells were then incubated with the M30 antibody for 30 minutes at room temperature, washed with PBS-T, and incubated with anti-mouse Ig-FITC-conjugated antibody and counted. The number of stained cells was counted and recorded as a percentage of the total cell population.

DNA Condensation.
DNA condensation was detected by using the intercalating agent bis-benzimide (Hoechst 33528; Hoechst, Frankfurt, Germany), from a method modified from that of Lam et al.24 Briefly, a stock solution (10 µg/mL) of bis-benzimide was prepared using ultrapure water (milli-Q; Millipore, Bedford, MA). Approximately 106 cells were collected at various time points, washed with PBS (pH 7.2), and fixed in 100 µL of 70% vol/vol ethanol. The cells were stained with bis-benzimide for 10 minutes at room temperature and counted. The number of stained cells was counted and recorded as a percentage of the total cell population.

Caspase Activity Assays
Assays for caspase 3, 8, and 9 were performed according to the manufacturer’s protocol using the recommended controls (R&D Systems, Minneapolis, MN). Data were normalized and expressed as percentage of activity of the cell population with the highest activity (i.e., for caspase 8 [Fig. 1 ], the activity in the HSV-1-infected cell population was taken as 100% and activity in other cell populations expressed relative to this population).


Figure 1
View larger version (18K):
[in this window]
[in a new window]

  		FIGURE 1. Relative amounts of caspase 8 activity in HCE cells infected with HSV-1 MacIntyre strain or treated with sorbitol or medium alone over 12 hours. *Significant differences between times or treatments (P < 0.05).

 
Caspase Inhibition Assays
Caspases 8 and 9 were inhibited by the amino acid sequences Z-IETD and Z-DEVD, respectively. These inhibitors were purchased from R&D Systems and used according to the manufacturer’s protocol. Briefly, the inhibitors were added to the cell cultures to a final concentration of 100 µM per well. Caspase inhibitors were present throughout the time course. The control was the absence of inhibitors. Caspase 3 activity in the cells was measured after 30 minutes, the time of peak activity during HSV-1 infection, or after 8 hours, the peak activity after sorbitol treatment.

Statistical Analysis
Experiments were performed in triplicate. Data were analyzed for significance by one-way ANOVA (SPSS ver. 10; SPSS, Chicago, IL).


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Viral Strain Specificity
The HSV-1 viral strains McIntyre, KOS1.1, and McKrae were used to test whether viral strain specificity occurs in our system, as infection with all strains resulted in similar amounts of HSV-1 adsorbed to cells and similar peaks of annexin binding at 1 hour (data not shown). All subsequent experiments were performed using only the McIntyre HSV-1 strain. With regard to assessing the number of cells, there was approximately a 25% increase in cells over the 12-hour experimental period for cells infected with HSV-1, but a rapid decrease in cells (~55%) treated with sorbitol between 8 and 12 hours (Fig. 1) .

Quantification of Active Caspase 8 Protein Levels
Caspase 8 activity was measured over 12 hours (Fig. 1) . Within 30 minutes, active caspase 8 protein levels were significantly higher in HSV-1-infected HCE cells compared with sorbitol-treated or mock-treated HCE cells (P < 0.05). By 1 hour, active caspase 8 protein levels decreased significantly in HSV-1-infected HCE cells compared with levels at 30 minutes (P < 0.05). By 2 hours, active caspase 8 protein levels in sorbitol-treated cells had significantly increased compared with sorbitol-treated cells at 1 hour (P < 0.05), and HSV-1-infected, and mock-treated HCE cells at 2 hours (P < 0.05). After 2 hours, there were no significant differences among all samples.

Quantification of Active Caspase 9 Protein Levels
Active caspase 9 protein levels in HSV-1-infected cells were not significantly higher than levels in mock-treated cells over time (Fig. 2A) . Caspase 9 protein levels were significantly higher in sorbitol-treated cells than in HSV-1-infected or mock-treated cells at 8 hours (P < 0.05). A decrease was observed by 12 hours (P < 0.05).


Figure 2
View larger version (19K):
[in this window]
[in a new window]

  		FIGURE 2. (A) Relative amount of caspase 9 activity and (B) percentage of cells showing reduction in mitochondrial transmembrane potential in cells treated as indicated. *Significant differences between times or treatments (P < 0.05).

 
Mitochondrial Transmembrane Potential in HCE Cells
Cells infected with HSV-1 produced no change in mitochondrial membrane potential (Fig. 2B) . At 4 hours, the percentage of cells that lacked transmembrane potential significantly increased in sorbitol-treated HCE cells compared with the other samples (P < 0.05; Fig. 2B ). The percentage of cells lacking a transmembrane potential continued to increase in sorbitol-treated HCE cells compared with other samples at 8 (P < 0.05) and 12 (P < 0.05) hours.

Quantification of Active Caspase 3 Protein Levels
Caspase 3 activity was measured over 12 hours (Fig. 3) . Within 30 minutes, active caspase 3 protein levels were significantly higher in HSV-1-infected HCE cells than in sorbitol-treated or mock-treated HCE cells (P < 0.05). Active caspase 3 protein levels decreased significantly in HSV-1-infected HCE cells at 2 hours compared with levels at 30 minutes (P < 0.05). Sorbitol-treated HCE cells showed a significant peak of activity at 1 hour compared with that at 30 minutes (P < 0.05), and the level remained high at 2 hours compared with HSV-1-infected cells (P < 0.05). Between 4 and 12 hours, there were no significant differences among all samples at each time point.


Figure 3
View larger version (20K):
[in this window]
[in a new window]

  		FIGURE 3. Relative amount of caspase 3 activity in transformed and primary (inset) HCE cells infected over 12 hours as indicated. *Significant differences between times or treatments (P < 0.05).

 
Active caspase 3 protein levels in PHCE cells were significantly higher in HSV-1-infected PHCE cells than in sorbitol-treated or mock-treated PHCE cells (P < 0.05) after 30 minutes (Fig. 3 , inset). Active caspase 3 protein levels decreased significantly in HSV-1-infected PHCE cells at 1 hour compared with HSV-1-infected PHCE cells at 30 minutes (P < 0.05). Caspase 3 activity of sorbitol-treated PHCE cells increased significantly at 2 hours compared with that at 1 hour (P < 0.05) and compared with HSV-1-infected and mock-treated PHCE cells at 2 hours (P < 0.05).

Caspase Inhibition Studies
Caspase 3 activity was measured in the presence of the caspase 8 and 9 inhibitor (Fig. 4) at the peak of caspase 3 activity in HSV-1-infected HCE cells (30 minutes) and sorbitol-treated HCE cells (8 hours). Caspase 3 activity in HSV-1-infected HCE cells in the presence of the caspase 8 inhibitor was significantly reduced compared with the activity in the absence of the inhibitor or in the presence of caspase 9 inhibitors (P < 0.05). In sorbitol-treated HCE cells, caspase 3 activity was not different in the absence or presence of either caspase 8 or 9 inhibitors at 8 hours (P = 0.45).


Figure 4
View larger version (20K):
[in this window]
[in a new window]

  		FIGURE 4. Relative amount of caspase 3 activity in the presence of caspase 8 inhibitor (100 µM), caspase 9 inhibitor (100 µM), or no inhibitor measured at the time point where maximum caspase 3 activity occurred in HCE cells infected with HSV-1 McIntyre strain, treated with sorbitol, or mock treated. *Significant differences between treatments (P < 0.05).

 
Detection of Caspase Cleavage of Cytokeratin 18
Part of the apoptotic pathway was evaluated by the active caspase 3 or 7-driven cleavage of CK18 (Fig. 5A) .25 Two peaks of caspase-driven CK18 cleavage were observed in HSV-1-infected HCE cells. The first peak was observed at 30 minutes where the percentage of positively stained cells in HSV-1-infected HCE cells was significantly higher than at 1 hour (P < 0.05) compared with the other conditions at 30 minutes (P < 0.05). The second peak was observed at 2 hours when the percentage of positively stained HSV-1-infected HCE cells was significantly higher than at 1 hour or 4 hours (P < 0.05), as well compared with other conditions at 2 hours (P < 0.05). There were significant (~10–15-fold) increases in the percentage of HCE cells treated with sorbitol that stained positively for CK18 at 4, 8, and 12 hours (P < 0.05) compared with the other treatments. The percentage of positively stained mock-treated HCE did not increase over time.


Figure 5
View larger version (19K):
[in this window]
[in a new window]

  		FIGURE 5. Percentage of cells showing (A) CK18 cleavage, (B) phosphatidylserine translocation, and (D) DNA condensation in transformed HCE cells and (C) phosphatidylserine translocation in primary HCE cells. *Significant differences between times or treatments (P < 0.05).

 
Detection of Phosphatidylserine Translocation
Initially, transformed HCE cells under various conditions were stained with annexin V and analyzed by flow cytometry over a short time course (0.5, 1, 2, and 4 hours; Fig. 5B ). Data are presented as a percentage of cells in the FITC positive, PI negative quadrant over total cells in all quadrants. The percentage of positively stained cells in HSV-1-infected HCE cells increased at 1 hour by approximately 10-fold, but significant differences were not observed at subsequent time points. The percentage of positively stained cells in sorbitol-treated HCE cells began an upward trend at 4 hours. The percentage of positively stained mock-treated HCE cells did not increase over time.

To determine whether these changes in the number of phosphatidylserine-positive cells also occurred in PHCE cells, the PHCE cells were tested under identical conditions over an extended time course (0, 0.5, 1, 2, 4, 8, and 12 hours; Fig. 5C ). The percentage of positively stained cells in HSV-1-infected PHCE cells increased significantly at 2 hours (P < 0.05) compared with other time points. At 2 hours, the percentage of positively stained cells in HSV-1-infected PHCE cells was significantly higher than the percentage of mock-treated PHCE cells (P < 0.05) but not sorbitol-treated PHCE cells (P = 0.08). At 4 hours, the percentage of positively stained cells in HSV-1-infected PHCE cells had significantly decreased compared with sorbitol-treated PHCE cells (P < 0.05). The percent of phosphatidylserine-positive cells treated with sorbitol at 12 hours was significantly higher than the percentage of positively stained cells treated with sorbitol at 4 or 8 hours (P < 0.05). In addition, the percentage of phosphatidylserine-positive cells treated with sorbitol was significantly higher than in cells under the other treatment conditions at 4, 8, and 12 hours (P < 0.05). The percentage of positively stained cells in mock-treated PHCE cells did not increase over time (P = 0.61, 0.99).

Detection of DNA Condensation by Hoechst 33528 Staining
Sorbitol-treated HCE cells showed a significant increase in the percentage of cells with condensed DNA compared with mock-treated and HSV-1-infected cells at 8 (P < 0.05) and 12 (P < 0.05) hours. Within the sorbitol-treated group, a significant increase was observed at 12 hours compared with 8 hours (P < 0.05; Fig. 5D ). A greater amount of DNA condensation was observed at 12 hours; however, a significant loss of cells (~50%, P < 0.05) was observed at this time point. HSV-1-infected cells showed no increase in DNA condensation over this 12 hours of the test period (Fig. 6) .


Figure 6
View larger version (16K):
[in this window]
[in a new window]

  		FIGURE 6. Fluorescence micrographs of cell stained with Hoechst 33528.(A) Appearance of human corneal epithelial cells 8 hours after exposure to sorbitol. Arrows: cells showing DNA fragmentation and DNA condensation. (B) Appearance of human corneal epithelial cells 8 hours after infection with HSV-1 McIntyre strain. Arrows: cells showing no changes in the DNA. (C) Appearance of human corneal epithelial cells 8 hours after mock infection (control). Arrows: cells showing no changes in their DNA. Magnification x500.

 
A summary comparing the findings at 0.5 or 2 hours and at 8 hours for the presence of apoptosis markers in cells infected with HSV-1 compared with those challenged with sorbitol is shown in Table 1 . These time points were chosen, as they best illustrate the different response of the cells to the two apoptotic stimuli.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Summary of Data for the Significant Production of Apoptosis Markers

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
This study has demonstrated that HSV-1 infection can induce a proapoptotic response in HCE cells, as shown by activation of caspase 8 and 3, cleavage of cytokeratin 18, and translocation of phosphatidylserine to the outer membrane. The induced apoptotic response did not involve the intrinsic pathway, as there was no caspase 9 induction, nor was there a reduction in mitochondrial membrane potential. There was no terminal apoptotic response as there was no increase in DNA condensation. The response was not considered to be necrotic, as there was only minimal PI staining (in both HSV-1 infected and sorbitol-treated cells), and PI is a stain that can penetrate only cells without intact membrane structures. We have reported that HSV-1 infection of HCE can induce an initial increase (at 1 hour) and then subsequent suppression (4–8 hours) of mRNA levels for several apoptosis-associated genes such as caspase 8, TNFRp55, TRADD, FAS-associated protein factor, and FADD and that this occurred in HCE cells but not Vero cells.20

In the current study, we found that the induced proapoptotic response was subsequently downregulated by the virus. Downregulation of caspase 3 and phosphatidylserine translocation occurred in both primary and transformed corneal epithelial cells. This finding is important, as transformation of cells with the T antigen of SV40 can block apoptosis.26 It is possible that the small differences in the timing of caspase 3 downregulation between transformed and primary cell lines during HSV-1 infection (Fig. 3) may be due to differences in cell growth rates, which can affect metabolic events within the cell. The suppression of components during HSV-1 infection occurred on the same order as the stimulation. Caspase 8 activity was significantly reduced at 1 hour. Caspase 3 activity and phosphatidylserine translocation were significantly reduced at 2 hours. Finally, CK18 cleavage was suppressed at 4 hours. Stable mitochondrial transmembrane potential and caspase 9 activity were observed during HSV-1 infection (Fig. 2) .

This response is similar but not identical with that produced by HSV-1 infection in (HeLa)-HEp-2 cells, where induction of apoptosis occurred after infection with HSV-1 and in the presence of the protein synthesis inhibitor cycloheximide.27 28 HSV-1 appeared to trigger apoptosis independent of de novo protein synthesis before 3 hours after infection, but between 3 and 6 hours after infection, apoptosis was prevented.25 29 In our experiments using HCE cells, part of the apoptotic machinery is triggered as early as 30 minutes after infection, but either mammalian cell proteins or viral proteins subsequently shut off this initial apoptotic response. Differences in the apoptotic response triggered by HSV-1 between cell types has been noted previously.28 During infection of a human promyelocytic leukemia cell line, HSV-1 was unable to reverse or inhibit phosphatidylserine translocation.25 The exposure of phosphatidyl serine on the surface of apoptotic cells can trigger their specific recognition and removal by macrophages.22 The apparent reversal of phosphatidylserine translocation in HCE cells infected with HSV-1 may indicate that this effect rescues cells from potential phagocytosis. In a neuronal cell line, others have shown that a mutant HSV-1 activated the apoptotic machinery independent of caspase 3 via the mitochondrial membrane10 and a similar activation of the intrinsic pathway was seen with (HeLa)-HEp-2 cells.28 This activation of the intrinsic pathway is directly opposite to that in the current investigation where the extrinsic pathway only is activated. Indeed, Galvan et al.10 found no evidence that wild-type HSV-1 by itself could activate apoptosis in their neuronal cell line. However, in the (HeLa)-Hep-2 cell line, HSV-1 can induce caspase-dependent apoptosis, but primarily through the intrinsic pathway.11 These differences may result from the use of different cell lines and differing strains of HSV-1 mutants.

In general, the apoptotic response of HCE cells during infection with HSV-1 was distinct to that induced by osmotic shock (sorbitol). Sorbitol treatment appeared to activate the apoptotic response through the intrinsic and extrinsic pathways. This activation of both the intrinsic and extrinsic pathways may explain why there was no effect of caspase 8 or 9 inhibitors on caspase 3 activation during sorbitol treatment. The suppression of caspase activity (caspase 8, 9, and 3) and CK 18 cleavage during sorbitol treatment may result from the many regulatory mechanisms that are present in the cell.29 This phenomenon appears to be independent of the apoptotic trigger. The reversal of phosphatidylserine translocation that occurred with HSV-1 infection but not osmotic shock could indicate a more specific downregulation of the apoptotic pathway by HSV-1. However, there can be a reversal of the translocation of phosphatidylserine in the absence of HSV-1 infection.14 The suppression of caspase 8, 9, and 3 activity occurred at a later time in sorbitol-treated HCE cells than in HSV-1-infected HCE cells. The reason for the apparent caspase 9 suppression observed at 12 hours could be the amount of cell death that occurred at this time point.

Viral entry could be the stimulator of the apoptotic pathway. However, Aubert et al.9 have reported that the initiation of apoptosis by the HSV-1 KOS1.1 strain in Hep-2 cells was not dependent on viral binding to the cells, although that had to induce apoptosis of the cells in the presence of the protein synthesis inhibitor cycloheximide. Further investigations are warranted to determine the molecular nature of the stimulator of HSV-1-induced apoptosis and the inhibitor(s) of the apoptotic response in HCE. It is possible that the cellular protein kinase B (PKB) is responsible for the inhibition of apoptosis. Phosphorylation of PKB occurs early during infection of Hep-2 cells and is responsible for preventing apoptosis in this cell line.30 Whatever the mechanism, the suppression of the apoptotic response appears to allow continued HCE cell survival and so may be a mechanism for ensuring a viable host cell for viral replication.


   Footnotes
 
Supported in part by an International Overseas Postgraduate Scholarship awarded by the University of New South Wales and the Australian Federal Government under the Cooperative Research Centres Program.

Submitted for publication June 5, 2006; revised October 17, 2006; accepted December 13, 2006.

Disclosure: D.M Miles, None; A. Thakur, None; N. Cole, None; M.D.P. Willcox, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Mark D. P. Willcox, Vision Cooperative Research Centre, Level 4, Rupert Myers Building, Gate 14, Barker Street, UNSW, Sydney NSW 2052, Australia; m.willcox{at}ier.org.au.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Miles DH, Willcox MDP, Athmanathan S. Ocular and neuronal cell apoptosis during HSV-1 infection: a review. Curr Eye Res. 2004;29:79–90.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  2. Hengartner MO. The biochemistry of apoptosis. Nature. 2000;407:770–776.[CrossRef][Medline][Order article via Infotrieve]
  3. Ashkenazi A, Dixit VM. Death receptors: signaling and modulation. Science. 1998;281:1305–1308.[Abstract/Free Full Text]
  4. Thornberry NA, Lazebnik Y. Caspases: enemies within. Science. 1998;281:1312–1316.[Abstract/Free Full Text]
  5. Li W, Kuszak J, Dunn K, et al. Lens epithelial cell apoptosis appears to be a common cellular basis for non-congenital cataract development in humans and animals. J Cell Biol. 1995;130:169–180.[Abstract/Free Full Text]
  6. Stennicke HR, Deveraux QL, Humke E, et al. Caspase-9 can be activated without proteolytic processing. J Biol Chem. 1999;274:8359–8362.[Abstract/Free Full Text]
  7. Wang S, Wang S, Miura M, et al. Murine caspase-11, an ICE-interacting protease, is essential for the activation of ICE. Cell. 1998;92:501–509.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  8. Aubert M, Blaho JA. The herpes simplex virus type 1 regulatory protein ICP27 is required for the prevention of apoptosis in infected human cells. J Virol. 1999;73:2803–2813.[Abstract/Free Full Text]
  9. Aubert M, O’Toole J, Blaho JA. Induction and prevention of apoptosis in human HEp-2 cells by herpes simplex virus type 1. J Virol. 1999;73:10359–10370.[Abstract/Free Full Text]
  10. Galvan V, Brandimarti R, Roizman B. Herpes simplex virus 1 blocks caspase-3-independent and caspase-dependent pathways to cell death. J Virol. 1999;73:3219–3226.[Abstract/Free Full Text]
  11. Galvan V, Brandimarti R, Munger JG, Roizman B. Bcl-2 blocks a caspase-dependent pathway of apoptosis activated by Herpes simplex virus 1 infected in HEp-2 cells. J Virol. 2000;74:1931–1938.[Abstract/Free Full Text]
  12. Koyama AH, Miwa Y. Suppression of apoptotic DNA fragmentation in Herpes simplex virus type 1-infected cells. J Virol. 1997;71:2567–2571.[Abstract]
  13. Leopardi R, Roizman B. The herpes simplex virus major regulatory protein ICP4 block apoptosis induced by the virus. Proc Natl Acad Sci USA. 1996;94:9583–9587.
  14. Leopardi R, Van Sant C, Roizman B. The herpes simplex virus 1 protein kinase US3 is required for protection from apoptosis induced by the virus. Proc Natl Acad Sci USA. 1997;93:7891–7896.
  15. Fadok VA, Bratton DL, Rose DM, Pearson A, Ezekewitz RA, Henson PM. A receptor for phosphatidylserine-specific clearance of apoptotic cells. Nature. 2000;405:85–90.[CrossRef][Medline][Order article via Infotrieve]
  16. Maeno E, Ishizaki Y, Kanaseki T, Hazama A, Okada Y. Normotonic cell shrinkage because of disordered volume regulation is an early prerequisite to apoptosis. Proc Nat Acad Sci USA. 2000;97:9487–9492.[Abstract/Free Full Text]
  17. Brandt CR. The role of viral and host genes in corneal infection with herpes simplex virus type 1. Exp Eye Res. 2005;80:607–621.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  18. Galvan V, Roizman B. Herpes simplex virus 1 induces and blocks apoptosis at multiple steps during infection and protects cells from exogenous inducers in a cell-type-dependent manner. Proc Natl Acad Sci USA. 1998;95:3931–3936.[Abstract/Free Full Text]
  19. Heiligenhaus A, Mrzyk S, Bauer D, Steuhl KP. Apoptosis in human non-necrotizing stromal herpes simplex keratitis. Klin Monatsbl Augenheilk. 2000;217:178–182.[CrossRef][Medline][Order article via Infotrieve]
  20. Miles D, Athmanathan S, Thakur A, Willcox MDP. A novel apoptotic interaction between HSV-1 and human corneal epithelial cells. Curr Eye Res. 2003;26:165–174.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  21. Izawa M, Teramachi K. Down-regulation of protein kinase C activity by sorbitol rapidly induces apoptosis in human gastric cancer cell lines. Apoptosis. 2001;6:353–358.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  22. Fadok VA, Voelker DR, Campbell PA, et al. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Virol. 1992;148:2207–2215.
  23. Martin S, Reutelingsperger C, McGahon A, et al. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J Exp Med. 1995;182:1545–1556.[Abstract/Free Full Text]
  24. Lam TT, Abler AS, Tso MO. Apoptosis and caspases after ischaemia-reperfusion injury in rat retina. Invest Ophthalmol Vis Sci. 1999;40:967–975.[Abstract/Free Full Text]
  25. Jerome KR, Tait JF, Koelle DM, Corey L. Herpes simplex virus type 1 renders infected cells resistant to cytotoxic T-lymphocyte-induced apoptosis. J Virol. 1998;72:436–441.[Abstract/Free Full Text]
  26. McCarthy SA, Symonds HS, Van Dyke T. Regulation of apoptosis in transgenic mice by simian virus 40 T antigen-mediated inactivation of p53. Proc Natl Acad Sci USA. 1994;91:3979–3983.[Abstract/Free Full Text]
  27. Koyama AH, Adachi A. Induction of apoptosis by herpes simplex virus type 1. J Gen Virol. 1997;78:2909–2912.[Abstract]
  28. Nguyen ML, Kraft RM, Blaho JA. African green monkey kidney Vero cells require de novo protein synthesis for efficient herpes simplex virus 1-dependent apoptosis. Virol. 2005;336:271–290.
  29. LeBlanc AC. Natural cellular inhibitors of apoptosis. Prog Neuropsychopharmacol Biol Psychiatry. 2003;27:215–229.[CrossRef][Medline][Order article via Infotrieve]
  30. Bennetti L, Roizman B. Protein kinase B/Akt is present in activated form throughout the entire replicative cycle of {Delta}US3 mutant virus but only at early times after infection with wild-type herpes simplex virus 1. J Virol. 2006;80:3341–3348.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow An erratum has been published
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Miles, D. H.
Right arrow Articles by Willcox, M. D. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Miles, D. H.
Right arrow Articles by Willcox, M. D. P.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS