Mousepox conjunctivitis: the role of Fas/FasL-mediated apoptosis of epithelial cells in virus dissemination

Malgorzata Krzyzowska1, Magdalena Polanczyk1,2, Monika Bas1, Joanna Cymerys1, Ada Schollenberger1, Francesca Chiodi3 and Marek Niemialtowski1

1 Immunology Laboratory, Division of Virology, Mycology and Immunology, Department of Preclinical Sciences, Faculty of Veterinary Medicine, Warsaw Agricultural University, Ciszewskiego 8, 02-786 Warsaw, Poland
2 Department of Neuroimmunology, School of Medicine, Oregon Health Sciences University, Portland, OR 97201, USA
3 Microbiology and Tumor Biology Center, Karolinska Institute, Nobels väg 16, S-17177 Stockholm, Sweden

Correspondence
Malgorzata Krzyzowska
krzyzowska{at}yahoo.com


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
BALB/c mice infected with the Moscow strain of Ectromelia virus (ECTV-MOS) show a large number of apoptotic cells, and an influx of lymphoid cells in the epithelium and substantia propria of conjunctivae, respectively. The presence of ECTV-MOS antigens in the epithelium of conjunctivae significantly upregulates Fas in the epithelial layer and FasL in the suprabasal layer of conjunctiva. Inhibition of FasL with blocking antibodies in cultures of conjunctival cells isolated from ECTV-MOS-infected BALB/c mice showed that the Fas/FasL pathway is important in apoptosis of ECTV-MOS-infected cells. The results also showed that the presence of cytokines, in particular interferon (IFN)-{gamma}, upregulated expression of Fas. Interleukin (IL) 2, 4, 10 and IFN-{gamma} were produced at the peak of conjunctivitis (at day 15 of infection) with a predominance of IFN-{gamma} and a small, but significant, production of IL4 and IL10 compared with non-infected animals. These results suggest that not only is Fas/FasL expression in conjunctiva involved in elimination of migrating Fas+ cells but also plays an important role in the turnover of conjunctival epithelium and thus may be crucial for ECTV spreading to the surrounding environment.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In the ocular environment, especially in tissues of the visual axis (corneal stroma, anterior chamber and subretinal space) there is an absolute requirement for clarity, and therefore any cellular infiltration is considered harmful (Streilein, 2003). Therefore, the eye is known as the site of immune privilege, where development of inflammation is consequently prevented by complex immunoregulatory mechanisms (Green & Ware, 1997; McKenna & Kapp, 2004). When the immune privilege fails an ocular disease ensues, as has been shown in experimental autoimmune uveitis (Gery & Streilein, 1994) and herpes stromal keratitis (Doymaz & Rouse, 1992; Niemialtowski & Rouse, 1992a, 1992b; Niemialtowski et al., 1994). Primary Human herpesvirus 1 (HHV-1) infection of the corneal epithelium leads to apoptosis of anterior keratocytes, which protects the visual axis (Wilson et al., 1997), and has been suggested to represent a defence mechanism against development of the viral infection. The immune privilege of the eye is also in part maintained by local expression of FasL, which acts by inducing apoptosis of Fas-bearing activated T cells (Streilein & Stein-Streilein, 2000; Ferguson et al., 2002). Apoptosis is defined as an active physiological process of cellular self-destruction, with specific morphological and biochemical changes. It can be induced by several different stimuli, including ligation of death receptors, specific cytokines, viral infection and growth factor deprivation (Granville et al., 1998; Marsden & Strasser, 2003). Fas antigen (CD95) is a cell surface death-receptor, which mediates apoptosis when bound to its ligand, FasL (Ashkenazi & Dixit, 1998). Fas is expressed on the surface of epithelial cells as a precursor for induction of apoptosis, cell death and shedding (Matsue et al., 1995). FasL interactions regulate a major pathway of apoptosis, which may play an important role in both mediating antiviral effects and pathogenesis of ocular infections. Brignole et al. (1998) have shown that human conjunctival epithelial cells normally express Fas antigen, and less constantly its ligand. Increased levels of Fas and FasL expression as well as the presence of apoptosis in the conjunctival epithelium have been shown to occur in patients with chronic conjunctivitis (Brignole et al., 2001) and the disease involving conjunctivae, the dry eye syndrome, keratoconjunctivitis sicca (Gao et al., 1998). Since the inflammatory process within the conjunctival epithelium poses a risk for the immune privileged cornea, conjunctivae and cornea may share common pathways of defence.

Ectromelia virus (ECTV; family Poxviridae, genus Orthopoxvirus) is a natural pathogen of laboratory mice that causes a generalized mousepox, with a high mortality rate and conjunctivitis occurring during natural infection (Fenner & Buller, 1997). Poxviruses, including ECTV, have been shown to encode many gene products that are able to suppress apoptosis, such as inhibitors of cytokine processing and proteolytic activation of caspases, soluble receptors that neutralize various cytokines, factors that inactivate interferon (IFN)-inducible antiviral enzyme activities and analogues of growth factors and hormones (Buller & Palumbo, 1991). During infection with ECTV several anti-apoptotic proteins, such as tumour necrosis factor receptor (TNFR) type II homologue (cytokine response modifier – crmD) and serpins SPI-1, SPI-2 and SPI-3, are produced as well as immunomodulatory proteins such as IFN-{gamma}-binding protein (Buller & Palumbo, 1991; Brick et al., 2000; Smith & Alcami, 2000; Smith et al., 2000; Turner et al., 2000; Saraiva & Alcami, 2001).

In our previous paper (Krzyzowska et al., 2002), we showed that during the Moscow strain of Ectromelia virus (ECTV-MOS) infection of BALB/c mice, conjunctivitis accompanied by caspase-3-dependent apoptosis develops, which in turn leads to shedding of the epithelial cells of the conjunctivae. Since the Fas/FasL system is important for the development of immunological privilege and control of the immune response within the eye, we investigated the involvement of Fas/FasL interaction in apoptosis observed within conjunctiva as a result of ECTV-MOS infection. The specific immunological microenvironment that arises in the conjunctiva during ECTV-MOS infection and its role in epithelial cell shedding was also studied. We hypothesize that upregulation of Fas and FasL is an important mechanism for epithelial cell turnover exploited by ECTV to spread to the surrounding environment.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Virus.
The virulent ECTV-MOS (kindly provided by R. M. L. Buller, Saint Louis University Health Sciences Center, St. Louis, MO, USA) was used in all the experiments. The virus stock was 5x107 p.f.u. ml–1.

Animals and ECTV-MOS infection.
BALB/c (H-2d) mice (4–6 weeks old) were purchased from the Centre of Experimental Medicine in Warsaw, and were treated according to the Warsaw Agricultural University guidelines on the use and care of laboratory animals. Groups of eight to nine mice of both sexes were inoculated via the footpad with ECTV-MOS stock in PBS (0·001 LD50 per mouse, 1 LD50=5x103 p.f.u. ml–1) or PBS alone as a negative control. Mice were examined daily for the presence of clinical signs of mousepox. The severity of the disease was scored as follows: 0, no visible signs; 1, behavioural changes, hind footpads swelling; 2, ruffled fur, swollen eyelids, skin lesions on extremities and/or on the tail; 3, conjunctivitis and ulcerative blepharitis on lid margins, papules on ears, back, tail and footpad skin; 4, formation of gangrene or partial amputation of the tail. At 5, 10, 15 and 20 days post-infection (p.i.), mice were sacrificed and conjunctivae were isolated. Small sections were frozen immediately in liquid nitrogen or fixed in 10 % formalin. The remaining tissue was used for preparation of single cell suspensions, as described in our previous papers (Toka et al., 1996; Gierynska et al., 2000; Cespedes et al., 2001).

TUNEL assay.
The TUNEL (terminal deoxynucleotidyltransferase dUTP nick-end labelling) assay was performed on paraffin embedded sections with the ApopTag in situ kit (Intergen) according to the manufacturer's protocol as described elsewhere (Krzyzowska et al., 2002). Cells in suspension were TUNEL stained with the APO-BrdU kit (BD Biosciences) according to the manufacturer's manual and analysed further by FACS Vantage (BD Biosciences).

Assays for caspase-1, -3, -7, -8 and -9 activities.
Caspase-1, -3, -7 and -8 activities were measured according to the manufacturer's instructions and as described elsewhere (Krzyzowska et al., 2002). Substrates were as follows: Ac-YVAD-AMC [N-acetyl-Tyr-Val-Ala-Asp-AMC (7-amino-4-methylcoumarin)] substrate for caspase-1; Ac-DEVD-AMC [N-acetyl-Asp-Glu-Val-Asp-AMC (7-amino-4-methylcoumarin)] substrate for caspase-3 and -7; Ac-IETD-AFC [N-acetyl-Ile-Glu-Thr-Asp-AFC (7-amino-4-trifluormethylcoumarin)] substrate for caspase-8, (BD Biosciences). For caspase-9 activity, we used ‘Caspase-9 fluorometric assay’ (Oncogene) with LEHD-AMC [N-acetyl-Leu-Glu-His-Asp-AMC (7-amino-4-methylcoumarin)] as a caspase-9 substrate. Results for this assay are expressed as relative fluorescence unit (RFU) per 50 µg protein h–1 calculated from triplicate numerical data acquired from test and control samples on a Fluoroskan Neonate fluorometer by Transmit Software (LabSystems).

Cytokeratin 18-specific cleavage staining.
Antibody M30 CytoDEATH (Roche) recognizes a specific caspase-3 cleavage site within cytokeratin 18 that is not detectable in normal viable cells, and has been used for the detection of apoptotic cells in frozen sections of conjunctiva. Additionally, double staining of ECTV antigens was performed using ECTV-MOS-specific fluorescein isothiocyanate (FITC)-conjugated rabbit polyclonal antibodies. For both flow cytometry analysis and frozen sections the staining was done according to the manufacturer's manual, except that ECTV-MOS-specific FITC-conjugated rabbit polyclonal antibodies were added together with M30 antibody. Negative control staining was carried out using the appropriate isotype control antibodies for the primary antibodies. Flow cytometry analysis was done as described above. Sections were analysed in a Leica fluorescence microscope with a Hamatsu C4880 cold CCD camera.

Flow cytometry.
For flow cytometry analysis cells were stained with rat biotinylated anti-CD4 (clone RM4-6), anti-CD8 (clone 53-6.7) and anti-CD 19 (clone 1D3) IgG2a monoclonal antibodies (mAbs) (BD Biosciences) and in the second step with phycoerythrin (PE)-conjugated streptavidin (SAv–PE; BD Biosciences). Mac-3+ cells (mouse mononuclear phagocytes) were identified with the rat anti-Mac-3 IgG1 mAb (BD Biosciences) and then, in the second step, with rabbit anti-rat IgG1 PE-conjugated polyclonal antibody (BD Biosciences). For Fas and FasL detection, polyclonal rabbit anti-Fas (M-20; Santa Cruz Biotechnology) antibody and anti-Fas (clone 13; BD Biosciences) or anti-FasL (clone 33; BD Biosciences) mouse IgG1 mAbs were used, followed by FITC-conjugated bovine anti-rabbit polyclonal antibody (Santa Cruz Biotechnology) and goat anti-mouse IgG1 FITC-conjugated (BD Biosciences) or goat anti-mouse IgG1 PE-conjugated (BD Biosciences) polyclonal antibodies. For double staining of Fas/ECTV antigens and FasL/ECTV antigens, ECTV-MOS-specific FITC-conjugated rabbit polyclonal antibodies were used. All staining was done according to the manufacturer's protocols and as described by Krzyzowska et al. (2002). Negative control staining was carried out using appropriate isotype control antibodies for the primary antibodies. Cells were analysed in FACS Vantage (BD Biosciences). All antibodies were purchased from Pharmingen (BD Biosciences) if not otherwise stated.

Immunohistochemical stainings.
CD4+, CD8+, CD19+, Mac-3+, Fas+ and FasL+ cells were detected in the cryostat sections using the above-mentioned primary antibodies (dilution 1 : 100). In the second step, peroxidase–avidin–streptavidin (HRP–Sav) complex was used (dilution 1 : 200) for CD4+, CD8+ and CD19+, and for Mac-3+, Fas+ and FasL+, a biotinylated secondary goat anti-mouse or anti-rat antibody was applied (dilution 1 : 200) to the sections, followed by the HRP–Sav complex. Negative control staining was carried out using the appropriate isotype control antibodies for the primary antibodies. Sections were analysed with light microscopy and evaluated by counting positive cells in 10 high-power fields of three different slides divided by the number of all cells in the same high-power fields.

Immunofluorescence stainings.
Cryostat sections of conjunctivae were subjected to double immunostaining with anti-Fas (dilution 1 : 100), anti-FasL (dilution 1 : 100) and anti-ECTV-MOS (dilution 1 : 64) antibodies as described in the section flow cytometry above.

The presence of ECTV-MOS antigen(s) in the mouse conjunctivae was assayed by direct immunofluorescence with ECTV-MOS-specific FITC-conjugated rabbit polyclonal antibodies. Additionally, to investigate the expression of Fas in the context of IFN-{gamma} production we used rat biotinylated anti-mouse IFN-{gamma} antibody (XMG1.2; BD Biosciences) (dilution 1 : 100) together with anti-Fas (dilution 1 : 100), followed by incubation with SAv–PE (dilution 1 : 200). Cryostat sections were stained as described elsewhere (Krzyzowska et al., 2002). Nuclei were counterstained with Hoechst 33342 (1 µg ml–1). Negative control staining was carried out using the appropriate isotype control antibodies for the primary antibodies. Sections were analysed in the Leica fluorescence microscope with a Hamatsu C4880 cold CCD camera.

FasL-blocking assay.
Conjunctiva cell suspensions were prepared at 5, 10, 15 and 20 days p.i., as well as from control, uninfected mice, by pooling approximately 1x106 cells obtained from four mice and suspending in RPMI 1640 supplemented with 10 % (v/v) fetal bovine serum (FBS) and antibiotics (100 U penicillin ml–1 and 100 µg streptomycin ml–1). Cells (0·5x105 ml–1) were incubated overnight in the presence of 10 and 20 µg ml–1 FasL-blocking antibody (MFL-4; BD Biosciences) and subjected to TUNEL staining and flow cytometry analysis.

Cytokine ELISA.
For in vitro assay of cytokine secretion, the conjunctivae cell cultures were grown in: (i) serum-free RPMI 1640 medium (control for spontaneous cytokines secretion); (ii) RPMI 1640 medium supplemented with 5 µg concanavalin A (ConA) ml–1 (positive control for non-specific cytokine stimulation); (iii) RPMI 1640 medium with viral antigen [syngenic (H-2d) X-irradiated (250 Gy) BALB/c mice splenocytes stimulated with UV-inactivated ECTV-MOS]. Cell-free supernatants were harvested after 24, 48 and 72 h and stored frozen at –20 °C until cytokine assay. The amounts of IL2, IL4, IFN-{gamma} and IL10 were evaluated in triplicate using OptEIA ELISA kit (Pharmingen). The assays were performed using the protocols provided by the manufacturer.

Statistical analysis.
Differences in mean values were analysed using Student's t test or a non-parametric Wilcoxon test. All P values are two-tailed. Data in the text are expressed as mean±SEM.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
ECTV-MOS antigens in conjunctivae of infected mice
The presence of ECTV-MOS antigens in conjunctivae of infected mice was confirmed by direct immunofluorescence with rabbit anti-ECTV-MOS-FITC polyclonal antibodies (Fig. 1a). ECTV-MOS antigens were seen as early as 5 days p.i. (primary viraemia) mainly in the suprabasal layer of the conjunctivae, whereas at 10 and 15 days p.i. ECTV-MOS-infected cells were also identified in foci localized in the stromal layer of conjunctivae. After 20 days p.i., we could observe recovery from conjunctivitis as well as disappearance of ECTV antigens from epithelial layer and also the stromal layer.



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Fig. 1. (a) Simultaneous detection of ECTV-MOS antigens and apoptotic cells (M30+ cells) in cryostat sections of BALB/c mice conjunctivae by indirect immunofluorescence with ECTV-MOS-specific FITC-conjugated rabbit polyclonal antibodies (green) and mouse monoclonal M30 CytoDEATH antibody followed by subsequent incubation with anti-mouse IgG PE-conjugated antibody (red). The conjunctivae were isolated at 15 days p.i. (x1000). Arrows indicate orange double positive cells (M30/ECTV-MOS+). (b) ECTV-MOS antigen detection (green) in slide (a). (c) M30+ cells (apoptotic) (red) in slide (a). Cell nuclei were counterstained with Hoechst 33342 (blue). (d) TUNEL-positive cells in paraffin embedded sections of conjunctivae isolated at 15 days p.i. (x100). Arrows indicate positive cells. epi, Epithelium; sp, substantia propria; cor, cornea. The slides were prepared from conjunctivae isolated from six mice (12 eyes) in three separate experiments.

 
Apoptosis and detection of caspase activity
We have previously described apoptosis during mousepox conjunctivitis in BALB/c mice (Krzyzowska et al., 2002). Here, we confirm the presence of apoptotic, TUNEL-positive cells mainly in the epithelium and in the suprabasal layer of conjunctivae (Fig. 1d). Further study of caspase-1, -8 and -9 activities showed no statistically significant increase in activity of caspase-1 and caspase-9 during mousepox conjunctivitis. On the other hand, the increase of caspase-8 activity between 10 and 20 days p.i. in the infected animal was higher than any other caspase activity (Fig. 2) (82±2·8 RFU per 50 mg protein h–1 at 20 days p.i. versus 8·5±1·1 in control, P<=0·005). Furthermore, we examined whether the cells undergoing apoptosis were ECTV-MOS-infected or not. For this purpose, simultaneous detection of a specific caspase-3 cleavage site within cytokeratin 18 and ECTV-MOS antigens was performed with M30 antibody and specific anti-ECTV-MOS antibodies, respectively. Similar to the TUNEL staining results, flow cytometry analysis of M30+ cells revealed that the number of apoptotic cells increased significantly at 10 and 15 days p.i. to 32 and 19·1 %, respectively (P<=0·01) (Fig. 3), which was consistent with the increase in caspase-3 activity (Fig. 2).



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Fig. 2. Caspase-1, -3, -7, -8 and -9 activity assays in protein lysates of conjunctival cells isolated at 5, 10, 15 and 20 days p.i. Control, protein lysates of conjunctival cells isolated from uninfected mice. Each bar represents mean values obtained from three mice in three separate experiments±SEM. For details see Methods. RFU, Relative fluorescence units.

 


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Fig. 3. The percentage of ECTV-MOS-infected (ECTV+), apoptotic (M30+) cells and double positive (M30/ECTV+) cells in population of conjunctival cells isolated at 5, 10, 15 and 20 days p.i. as well as from control, uninfected mice. Each bar represents mean values obtained from a group of three mice in three separate experiments±SEM.

 
At the same time, the number of both apoptotic and ECTV-MOS-infected cells was also significant, but showed different patterns. During early onset of conjunctivitis (10 days p.i.) ECTV-MOS-infected cells consisted of approximately half of the M30+ population (Fig. 3, M30+ cells – 16·06 % and M30/ECTV-MOS+ cells – 31·98 %), whereas during fully-fledged conjunctivitis (15 days p.i.) virtually all cells undergoing apoptosis were ECTV-MOS-infected (M30+ cells – 19·5 % and M30/ECTV-MOS+ cells – 18·98 %). On the other hand, at 15 days p.i., the number of ECTV-MOS-infected cells was almost two times higher than the number of apoptotic cells, indicating that ECTV-MOS-infected cells are able to escape apoptosis (Fig. 3). However, at this time point we usually observed that ECTV-MOS-infected cells were also found in the foci of inflammation found in the stromal layer of conjunctiva (Krzyzowska et al., 2002). Immunofluorescence studies of frozen sections revealed that at 10 and 15 days p.i. (Fig. 1a) both infected and non-infected apoptotic cells were present in the epithelial layer. Interestingly, non-infected apoptotic (M30+) cells always underlay ECTV-MOS-infected cells in the subepithelial layer (Fig. 1a and c, thick arrows). ECTV-MOS-infected cells undergoing apoptosis were found usually at the uppermost layer of the epithelium (Fig. 1a and b, thin arrows). Apoptotic ECTV-MOS-infected cells were also identified in the inflammation foci of the stromal layer at later stages, at 20 days p.i.

Inflammatory lesion cell phenotype in mousepox conjunctivitis
Flow cytometry analysis of CD4 expression on conjunctival cells showed a significant increase in the number of positive cells at 10 (5·45±3·0 %; P<=0·03), 15 (5·97±1·0 %; P<=0·01) and 20 days p.i. (5·41±2·1 %; P<=0·04) as compared with the control group (2·01±2·0 %) (Table 1). Immunohistochemistry revealed the presence of CD4+ cells within foci infiltrating the substantia propria and the suprabasal layer of the palpebral conjunctivae (Table 1). The number of CD4+ cells in the substantia propria increased from 2·18±1·4 % in the control group to 5·48±1·9 % (P<=0·033) at 10 days p.i., 5·93±1·3 % (P<=0·009) at 15 days p.i. and 5·02±1·9 % at 20 days p.i. (P<=0·01). Additionally, the percentage of CD4+ cells increased significantly at 10 and 15 days p.i. (P<=0·05) in the epithelial layer of the conjunctivae, as compared with controls (Table 1).


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Table 1. Percentage of immunocompetent cells (CD4+, CD8+ and CD19+) in conjunctivae of BALB/c mice infected with ECTV-MOS measured by immunohistochemistry and flow cytometry analysis

For immunohistochemistry, the cells were counted separately in substantia propria and conjunctival epithelium whereas analysis with flow cytometry evaluated cells from both layers. IHC, Immunohistochemistry; FC, flow cytometry; s. prop., substantia propria; epi, epithelium; p.i., post-infection.

 
Similarly, CD8+ cells were found in foci localized in the lamina propria of palpebral conjunctivae and infiltrated the suprabasal layer (Table 1). Flow cytometry analysis showed a statistically significant (P<=0·001) increase of CD8+ cells between 10 (6·01±1·9 %) and 20 (5·07±2·0 %) days p.i. as compared with control mice (1·81±1·2 %) (Table 1).

As observed by immunohistochemistry, the percentage of CD8+ cells in the substantia propria increased between 10 and 20 days p.i. (5·57±1·0 % – 5 days p.i., 5·98±1·0 % – 10 days p.i. and 5·21±2·4 % – 15 days p.i.; P<=0·02) as compared with the values found in the control animals (1·81±1·2 %). In the epithelial layer of the conjunctivae the percentage of CD8+ cells increased significantly during mousepox at 10 and 15 days p.i. (7·6±0·2 % and 8·4±0·13 %, respectively) as compared with controls (2·1±0·1 %) (P<=0·05) (Table 1).

The percentage of B lymphocytes (CD19+ cells) increased significantly in the substantia propria only at 15 days p.i. (8·8±0·9 %) as compared with controls (5·0±1·0 %) (P=0·05) with a change in substantia propria : epithelium CD19+ cell from 5 : 1 to 3 : 1 (Table 1). In the epithelium, B lymphocytes were also found surrounding the goblet crypts.

As previously reported, flow cytometry results confirmed immunohistochemistry results (Table 1). Mac-3+ cell (tissue macrophages and dendritic cells) number increased both in the substantia propria and in the epithelium of palpebral conjunctivae at 5 and 10 days p.i. (6·23±3·5 and 7·51±2·3 %, P<=0·04 for 5 days p.i.; 1·3±0·03 and 1·4±0·08 %, P<=0·001 for 10 days p.i.; 5·50±3·0 and 1·0±0·05 % for the controls, respectively). Subsequently, the percentage of Mac-3+ cells in the substantia propria decreased below the count of positive cells observed in the control conjunctivae (Table 1). Flow cytometry results were similar with the percentage of Mac-3+ cells increasing to 7·55±2·0 % (P<=0·005) at 10 days p.i. and dropping to 3·81±1·0 and 2·73±2·09 % at 15 and 20 days p.i., respectively. The observed decrease was statistically significant (P<=0·05) when compared with results in control group (5·7±1·1 %) (Table 1).

Fas/FasL involvement in apoptosis during mousepox conjunctivitis
We investigated the influence of blocking FasL receptor on survival of conjunctival cells. MFL-4 antibody was used to block FasL on conjunctival cells prepared at 5, 10, 15 and 20 days p.i., as well as from uninfected mice. It was noted that 20 µg ml–1 of blocking antibody decreased apoptosis to a level comparable to that observed in the control (Fig. 4). However, this was only evident for cell cultures prepared at 10, 15 and 20 days p.i. A non-significant decrease in apoptosis was also notable at 10 µg ml–1. At 5 days p.i. the decrease of apoptotic cell numbers was not significant for all tested concentrations of the blocking antibody (Fig. 4).



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Fig. 4. Suppression of conjunctival apoptosis with the use of FasL-blocking antibody. Cultures of conjunctival cells isolated at 5, 10, 15 and 20 days p.i. as well as from control, uninfected mice were incubated with two different concentrations of FasL-blocking antibody (10 and 20 µg ml–1). Apoptosis was measured by the TUNEL method. Each bar represents mean values obtained from three mice in three separate experiments±SEM.

 
To investigate the role of Fas/FasL in vivo we stained frozen sections of conjunctivae taken at described time points. Double immunofluorescence staining for Fas and FasL revealed that double positive cells in the conjunctivae from the control mice resided mainly in the suprabasal layer and the cells of the whole epithelium had high levels of FasL (Fig. 5a). During infection, the majority of epithelial cells already showed enhanced Fas expression at 5 days p.i. The Fas staining gradually intensified and localized in the suprabasal layer by 10 days p.i. (Fig. 5b). At 15 days p.i., the number of double Fas/FasL+ cells increased, but at 20 days p.i. the pattern of expression reverted to that found in the control conjunctivae. Flow cytometry analysis confirmed the results described above. The increase in percentage of Fas+ cells was statistically significant at 5 days p.i. (P=0·009) and remained so until the end of the examined period (P<=0·001) (Fig. 5c). We noticed an increase in the percentage of FasL+ cells at the peak of conjunctivitis, namely between 10 and 20 days p.i. (P<=0·01) (Fig. 5c). The percentage of double Fas/FasL+ cells increased significantly from day 10 (27·2±2·0 % of total cells) to day 15 p.i. (38·4±1·6 %), corresponding to 65 and 80 % of Fas+ cells, respectively (Fig. 5c).



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Fig. 5. Fas and FasL expression in conjunctival epithelium by double immunofluorescence (a), (b) and flow cytometry analysis (c). Fas+ cells are stained with FITC (green) and FasL+ cells are stained with PE (red); double positive cells are from yellow to orange, depending on the intensity of staining. Cell nuclei were counterstained with Hoechst 33342 (blue). Conjunctivae from control, uninfected BALB/c mice (a) (x1000), ECTV-MOS-infected at 15 days p.i. (b) (x400). epi, Epithelium; sp, substantia propria; cor, cornea. The slides were prepared from conjunctivae isolated from six mice (12 eyes) in three separate experiments.

 
To examine the role of ECTV-MOS infection in upregulation of Fas/FasL expression on conjunctival cells we used double staining of frozen section and conjunctival cell suspensions for Fas/ECTV-MOS and FasL/ECTV-MOS antigens. Immunofluorescent staining of frozen sections from 15 days p.i. showed that Fas/ECTV-MOS+-expressing cells were identified mainly in the upper epithelial layer of conjunctiva (Fig. 6b, arrow), but non-infected cells also upregulated Fas (Fig. 6a). FasL/ECTV-MOS+ cells were identified at the subepithelial layer of conjunctiva (Fig. 6d, arrows). Simultaneous staining of Fas+ and IFN-{gamma}+ cells showed an increase of Fas expression in the close proximity of IFN-{gamma}-producing cells (Fig. 6f). Flow cytometry analysis showed that the numbers of Fas/ECTV-MOS and FasL/ECTV-MOS+ cells increased during the period of infection (Fig. 6g) with 26·67±1·9 and 33·5±2·0 % at 15 days p.i, respectively. At 20 days p.i. the levels of either Fas/ECTV-MOS+ or FasL/ECTV-MOS+ cells decreased and approached the levels comparable to those achieved at 5 days p.i. (Fig. 6g).



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Fig. 6. (a–d) Simultaneous expression of Fas (a), (b), FasL (c), (d) and ECTV antigens in conjunctival epithelium by double immunofluorescence. Fas+ or FasL+ cells are stained red and ECTV-MOS+ cells are stained green. (a) and (c) Conjunctivae were isolated from control, uninfected mice (x1000). (b) and (d) Conjuctivae were isolated at 15 days p.i. (x1000). (e) and (f) Simultaneous staining for Fas expression (green) and IFN-{gamma} production (red). (e) Conjunctivae were isolated from control, uninfected mice (x1000) and (f) conjuctivae were isolated at 15 days p.i. (x1000). Double positive cells are from yellow to orange, depending on the intensity of staining. Arrows indicate double positive cells. Cell nuclei were counterstained with Hoechst 33342 (blue). epi, Epithelium; sp, substantia propria; cor, cornea. The slides were prepared from conjunctivae isolated from six mice (12 eyes) in three separate experiments. (g) Flow cytometry analysis of ECTV-MOS-infected, Fas/ECTV+ and FasL/ECTV+ cells among conjunctival cells isolated at 5, 10, 15 and 20 days p.i. Each bar represents mean values obtained from three mice in three separate experiments±SEM.

 
Cytokine production
The levels of cytokine production in conjunctivae were investigated at 5 days p.i. (secondary viraemia) and at 15 days p.i. (the peak of conjunctivitis). There was no production of IFN-{gamma}, IL2 or IL10 (Fig. 7a, b and d) in either the infected or control conjunctivae prepared at 5 days p.i. (Fig. 7). However, a high level of IL4 (53·8 pg ml–1, P<=0·05) was detected at 5 days p.i. in infected mice compared with the controls (23·6 pg ml–1) (Fig. 7c). At 15 days p.i. all examined cytokines were produced by conjunctival cells both after antigen stimulation and non-specific stimulation with ConA. IFN-{gamma} production was the highest compared with other cytokines and reached 1221 pg ml–1 following ECTV-MOS-specific stimulation at 15 days p.i. (Fig. 7a; P<=0·001). The increase in IL2 production at 15 days p.i. was statistically significant when compared with controls and 5 days p.i. (Fig. 7b), but did not reach the values achieved for IFN-{gamma}. At 15 days p.i., the production of IL4 (254 pg ml–1, P<=0·05) and IL10 (204 pg ml–1, P<=0·05) by cells stimulated with ECTV-MOS was significantly higher as compared with the control animals and values reached at 5 days p.i. (Fig. 7c and d).



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Fig. 7. Production of IFN-{gamma} (a), IL2 (b), IL4 (c) and IL10 (d) by cells isolated from conjunctivae of control, uninfected BALB/c mice (con) and ECTV-MOS-infected BALB/c mice at 5 and 15 days p.i. in pg ml–1. Cultures for in vitro assay of cytokines secretion were grown in: (i) serum-free RPMI 1640 medium (control for spontaneous cytokine secretion); (ii) RPMI 1640 with viral antigens (see Methods); and (iii) RPMI 1640 supplemented with mitogen ConA at an optimal concentration of 5 µg ml–1 (positive control for non-specific cytokine stimulation). Each bar represents mean values obtained from three mice in three separate experiments. Asterisk, (*) results statistically important in comparison with control (P<=0·05). a*, Results statistically important in comparison with control and 5 days p.i. (P<=0·05) of results below detection threshold. b, Below detection.

 

   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The role of apoptosis in epithelial and mucosa turnover has been widely discussed (Sayama et al., 1994; Matsue et al., 1995). Fas is expressed on epithelial cells and together with FasL takes an active part in cell shedding (Matsue et al., 1995; Wilson et al., 1996). Brignole et al. (1998) demonstrated that human conjunctival epithelial layers normally express Fas, and to a lesser degree FasL. Our results show that mouse normal conjunctiva abundantly express FasL, but not Fas. However, as in human conjunctiva, in mice consecutive expression of Fas and FasL is mainly found in the suprabasal layer (Figs 5 and 6). In conjunctival diseases increased expression of Fas, CD40L and an apoptotic marker, APO 2.7, was shown in specimens from conjunctival impression cytology obtained from patients with keratoconjunctivitis sicca and various inflammatory ocular surface disorders (Brignole et al., 1998; Bourcier et al., 2000). In a well-known model of chronic ocular disease, such as HHV-1, infection leading to blepharitis, keratitis and conjunctivitis, constitutive expression of FasL in ocular tissue has been reported to play an important role in curtailing HHV-1-induced inflammatory responses (Griffith et al., 1996). However, there are no reports on the role of the Fas/FasL system in epithelial cell shedding during viral infection of conjunctiva and virus-induced inflammation. In this study, we show that during development of ECTV-MOS-induced conjunctivitis expression of both Fas and FasL increases, in particular the expression of Fas in the suprabasal layer of conjunctiva. Our previous work (Krzyzowska et al., 2002) has shown that ECTV-MOS infection involves whole epithelium (Fig. 1a), eventually leading to epithelial cell apoptosis as well as shedding of multiple layers of conjunctiva epithelium (Fig. 1b) that is dependent on caspase-3 and -8 activity (Fig. 2). Based on the evidence presented in this study, we propose that apoptosis of conjunctival epithelial cells results from consecutive upregulation of Fas and FasL expression (Figs 5 and 6) rendering cells more susceptible. Our study uses an ex vivo model to evaluate the role of Fas/FasL-induced apoptosis in conjunctivae. A pronounced effect of blocking conjunctival cell apoptosis with anti-FasL antibody was observed at the peak of ECTV-MOS-induced infection, i.e. at 10–20 days p.i. Interestingly, the FasL-blocking antibody effected cell survival in control cultures.

We therefore conclude that not only is the Fas/FasL paracrine or autocrine interaction important for the induction of apoptosis during in vitro ECTV-MOS infection of fibroblast cell lines, but it is also important for in vivo apoptosis of conjunctival cells. This finding is not surprising since it is well-known that ECTV-MOS replicates in skin and conjunctiva, and induces skin lesions as well as conjunctivitis leading to apoptosis of infected cells. Apoptotic cells that easily detach from the epithelial layer of conjunctiva are present in the tear fluid, excessively produced as a result of conjunctivitis. Excessive lacrimation may help to spread virus-containing apoptotic cells. Furthermore, during ECTV-MOS infection of BALB/c mice, pox skin lesions can also be found in close vicinity of eyelids (Fenner & Buller, 1997; Cespedes et al., 2001).

In contrast to the cornea, the conjunctiva is infiltrated with cells coming from blood vessels, and the blood-borne route is the common route for ECTV-MOS infection of conjunctivae. Several papers have discussed the role of macrophages and dendritic cells in ECTV-MOS infection spreading (Fenner & Buller, 1997; Cespedes et al., 2001). Histological examinations of conjunctivae revealed that during ECTV-MOS infection of the conjunctiva, inflammatory foci consisting of macrophages, neutrophils and T lymphocytes were found both in the suprabasal and the substantia propria layer, and they usually persisted late in infection (Krzyzowska et al., 2002). The presence of inflammation at the site of infection suggests that ECTV-MOS-induced inflammation can be the source of inflammatory factors stimulating upregulation of Fas and FasL. Indeed, production of cytokines at the peak of mousepox-induced conjunctivitis increased and involved IL2, IL4, IL10 and IFN-{gamma}. However, only IFN-{gamma} showed high levels of production, with IL4 and IL10 upregulated a small but significant amount.

A strong relationship between inflammation and apoptosis has been demonstrated in various experimental models using conjunctival epithelial cells. De Saint Jean et al. (1999) demonstrated that IFN-{gamma}-induced apoptosis of the human conjunctival Chang cells through a dose-dependent upregulation of Fas. Moreover, the same authors showed that the relative resistance of the human conjunctival Chang cells to Fas-induced apoptosis could be related to nuclear factor {kappa}B (NF-{kappa}B) activation. IFN-{gamma}-induced activation of transcription factor STAT1 thus counterbalanced NF-{kappa}B-anti-apoptotic actions (De Saint Jean et al., 2000). When comparing Fas and FasL expression levels during mousepox conjunctivitis, we observed that the increase in Fas expression was more consistent than the increase in FasL expression. This effect may be due to the upregulation of IFN-{gamma} production by ECTV-MOS-infected conjunctival cells (Fig. 7). What is more, the result of double staining for Fas and IFN-{gamma}-producing cells showed that Fas-expressing cells were found in close proximity to IFN-{gamma}-producing cells, which strongly supports the role of IFN-{gamma} in upregulation of Fas and regulation of conjunctival cell turnover.

Sustained production of cytokines during ECTV-MOS-infection seems to be due to a delayed virus clearance from the conjunctivae in spite of the presence of CD8+ and CD4+ cells.

These observations are consistent with our previous findings (Gierynska et al., 2000; Cespedes et al., 2001) showing lack of ECTV-MOS-specific cytotoxic activity during conjunctivitis in ECTV-MOS-infected BALB/c mice. The levels of IL4 and IL10 in conjunctivae during ECTV-MOS infection, although only increased a small extent, may help to predispose to a Th2-like local immune response and the absence of a cytotoxic T-lymphocyte reaction at the peak of conjunctivitis.

It is worth noting that inflammatory foci were found both in the suprabasal layer as well as in the substantia propria. From the high expression of FasL in the suprabasal layer of conjunctival epithelium, we suspected that FasL+ cells constitute the first barrier ensuring that any Fas+ inflammatory cells migrating from the stromal layer and representing a serious risk for inflammation within the epithelium undergo apoptosis. However, as shown previously (Cespedes et al., 2001), ECTV-MOS-infected cells easily passed this barrier and spread the viral infection. One possible explanation is that ECTV-MOS-infected cells migrating into the conjunctiva are relatively unsusceptible to apoptosis via the FasL-dependent pathway. In fact, ECTV-MOS-infected cells found in substantia propria (Fig. 6a) showed no significant, if any, Fas expression. On the other hand, ECTV-MOS has been shown to encode many genes, whose products are able to suppress apoptosis, such as TNFR type II homologue (crmD, crmE) and IFN-{gamma}-binding protein as well as serpins SPI-1, SPI-2 and SPI-3 (Buller & Palumbo, 1991; Brick et al., 2000; Smith & Alcami, 2000, 2002; Smith et al., 2000; Turner et al., 2000; Saraiva & Alcami, 2001; Smith & Alcami, 2002). Serpins SPI-1, SPI-2 and SPI-3 have been shown to protect cells in vitro from Fas-induced apoptosis (Mullbacher et al., 1999).

The conjunctival system of protection from inflammation through Fas/FasL-mediated apoptosis may not be exceptional for ocular tissues since simultaneous expression of Fas and FasL was shown for corneal epithelial and endothelial cells in healthy cornea. During corneal epithelial injury, soluble FasL expression was increased by IL1 released from corneal epithelium, thus triggering keratocyte apoptosis via autocrine Fas-mediated apoptosis (Mohan et al., 1997). Since the cornea does not possess any lymphatic drainage and the source of pathological events appears to be the outer epithelial layer as observed during mechanical injury or HHV-1 infection (Wilson et al., 1996), expression of FasL in the suprabasal layer does not serve to protect the tissue from invading Fas+ cells migrating from lymph and blood vessels situated in the substantia propria, as in the case of conjunctiva.

We therefore conclude that during ECTV-MOS infection various mechanisms of efficient immune evasion as well as Fas-dependent mechanisms of epithelial turnover are involved.

In this study, we investigated the consequences of orthopoxvirus infection for conjunctival cell destruction and also for viral spreading. To establish a first barrier against infiltration of the Fas+ lymphoid cells, the mouse conjunctiva relays on the expression of FasL in the subepithelial layer. ECTV-MOS interferes with apoptotic processes in the target cells and passes the existing FasL barrier to successfully replicate in the conjunctival epithelium. As a result of ECTV-MOS replication and sustained inflammation, epithelial cells upregulate Fas and undergo apoptosis, thus helping the virus to spread to the surrounding environment.


   ACKNOWLEDGEMENTS
 
This work was supported by the Polish National Committee for Scientific Research (grant no. 3 PO 4A 022 25) and by the Foundation for Polish Sciences [Professors of 2000 grants (no. 10/2000) for M. N.]. We thank Dr Felix N. Toka for critical reading of the manuscript.


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ABSTRACT
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RESULTS
DISCUSSION
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Received 21 October 2004; accepted 21 March 2005.



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