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
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ABSTRACT |
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INTRODUCTION |
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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-
-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.
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METHODS |
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Animals and ECTV-MOS infection.
BALB/c (H-2d) mice (46 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. ml1) 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 h1 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 (SAvPE; 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, peroxidaseavidinstreptavidin (HRPSav) 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 HRPSav 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- production we used rat biotinylated anti-mouse IFN-
antibody (XMG1.2; BD Biosciences) (dilution 1 : 100) together with anti-Fas (dilution 1 : 100), followed by incubation with SAvPE (dilution 1 : 200). Cryostat sections were stained as described elsewhere (Krzyzowska et al., 2002
). Nuclei were counterstained with Hoechst 33342 (1 µg ml1). 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 ml1 and 100 µg streptomycin ml1). Cells (0·5x105 ml1) were incubated overnight in the presence of 10 and 20 µg ml1 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) ml1 (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- 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.
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RESULTS |
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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 %; P0·03), 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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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.; P0·02) 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 ml1 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 ml1. 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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DISCUSSION |
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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-
. However, only IFN-
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-
-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
B (NF-
B) activation. IFN-
-induced activation of transcription factor STAT1 thus counterbalanced NF-
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-
production by ECTV-MOS-infected conjunctival cells (Fig. 7
). What is more, the result of double staining for Fas and IFN-
-producing cells showed that Fas-expressing cells were found in close proximity to IFN-
-producing cells, which strongly supports the role of IFN-
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-
-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.
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ACKNOWLEDGEMENTS |
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Received 21 October 2004;
accepted 21 March 2005.
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