1 Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Surrey GU24 0NF, UK
2 Biogen Idec Inc., Cambridge Center, Cambridge, MA 02142, USA
Correspondence
Terry Jackson
terry.jackson{at}bbsrc.ac.uk
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ABSTRACT |
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Present address: Danish Institute for Food and Veterinary Research, Department of Virology, Lindholm, DK-4771 Kalvehave, Denmark.
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INTRODUCTION |
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Several receptors for FMDV have been identified, these are the integrins (Jackson et al., 2003) and heparan sulfate proteoglycans (HSPGs) (Jackson et al., 1996
). Currently there is no convincing evidence of a role for HSPG in cell entry by field viruses (Baranowski et al., 1998
, 2000
; Escarmis et al., 1998
; Martinez et al., 1997
; Neff et al., 1998
; Sa-Carvalho et al., 1997
). Instead, field viruses depend on integrins for infection in vitro, and integrins are believed to be the receptors used in animals (McKenna et al., 1995
; Neff et al., 1998
).
Integrins are a family of integral membrane receptors that function as cell adhesion molecules (Hynes, 2002). Each integrin is a heterodimer formed by the non-covalent association of
- and
-subunits. In mammalian species, the integrin family consists of 24 different heterodimers, each of which has a distinct tissue distribution. A general property of integrins is that they exist in alternate active or inactive states (Hynes, 2002
). Conversion between these states is regulated by reversible changes in the conformation of the extracellular domains in a process termed integrin activation (Giancotti, 2003
; Hynes, 2002
; Kim et al., 2003
).
In cell culture, FMDV has been shown to use four integrins, v
1,
v
3,
v
6 and
v
8 as receptors to initiate infection (Berinstein et al., 1995
; Jackson et al., 2000
, 2002
, 2004
). Virus attachment to the integrin is mediated through an arginineglycineaspartic acid (RGD) tripeptide located on VP1 (Jackson et al., 2003
; Logan et al., 1993
). Several other integrins (
v
5,
5
1,
8
1 and
IIb
3) are known to bind this motif; however, despite having an RGD, FMDV appears unable to use all of these integrins to initiate infection and evidence for
v
5 and
5
1 as receptors has been consistently negative (Baranowski et al., 2000
; Duque & Baxt, 2003
; Jackson et al., 2000
; Mason et al., 1993
; Neff et al., 1998
).
The integrin receptors of FMDV have been studied extensively in cell culture. However, the role(s) of the various integrins in determining the tissue tropism and pathogenesis of FMDV have yet to be established. For an integrin to function as a virus receptor it would appear reasonable to postulate that it should be expressed on the cell types that are normally targeted. Here, we present analyses, using immunofluorescence confocal microscopy and real-time RT-PCR, of v
3 and
v
6 expression within the epithelia that are normally targeted by FMDV in cattle. These studies show that
v
6 but not
v
3 was expressed on the surfaces of the epithelial cells at the sites where FMDV is known to replicate at a high level during a natural infection. Expression of
v
6 on the cells targeted by FMDV within the interdigital-skin was confirmed by showing the presence of this integrin on the surfaces of infected cells. Together, these data suggest that, in epithelia normally targeted by FMDV in cattle, (i) integrin
v
6, but not
v
3, serves as the major receptor that determines virus tropism, and (ii),
v
6 is expressed constitutively at levels sufficient to allow initiation of infection.
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METHODS |
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Antibodies.
mAb 23C6 (anti-v
3) was from Serotec. mAb B3A (anti-
3), mAb HB1.1 (anti-
1) and mAb 10D5 (anti-
v
6) were from Chemicon. mAb 9EG7 was from Pharmingen. mAb 6.2A1 (anti-
v
6) was from Biogen.
mRNA extraction and real-time RT-PCR.
The details of the RNA extraction procedure, cDNA synthesis and real-time RT-PCR have been described previously (Alexandersen et al., 2002c; Oleksiewicz et al., 2001
; Reid et al., 2002
, 2003
; Zhang & Alexandersen, 2003
). Briefly, total cellular mRNA was extracted using the MagNa Pure LC mRNA Extraction kit II (Roche) with an automated nucleic acid robotic workstation (Roche). RNA was reverse transcribed using the MultiScribe reverse transcriptase kit with random hexamers (Applied Biosystems). cDNA was then added to a TaqMan PCR mix (Applied Biosystems) containing 0·9 pmol each primer µl1 and 0·2 pmol FAM-labelled probe µl1. PCR amplification was carried out as described previously (Zhang & Alexandersen, 2004
). The sequences of the primers and probes for integrin amplification were:
6 forward primer, 5'-GAAGAATGTGTTGACAAATGCAAAC-3';
6 reverse primer, 5'-CAGAGAACAGGAAACAAAGCTATCC-3';
6 probe, 5'-ACCATCAATGAAGAAGAA-3';
3 forward primer, 5'-GAGTGCGGCAGCTGTGTCT-3';
3 reverse primer, 5'-GCAGGTGGGACACTTCTCACA-3';
3 probe, 5'-TGTCCCCATAGGAGCC-3'. Using
3 and
6 cDNA templates, the PCRs were shown to be specific for their target sequences as the
6 and
3 primer/probe sets were negative for the
3 and
6 templates, respectively. Similarly, control reactions carried out using mRNA extracted from the tissues and not reverse transcribed were also negative in the real-time PCRs.
Immunofluorescence microscopy.
Tissues were collected at post-mortem and dissected into 0·5 cm3 blocks, fixed in 4 % paraformaldehyde for 1 h at room temperature and transferred to PBS at 4 °C. Sections (70 µm) were prepared using a Leica vibrating microtome (Monaghan et al., 2001
). For softer tissues (e.g. soft palate), sections were cut using a razor blade. Tissue sections were permeabilized in 0·1 % Triton X-100 for 1 h at room temperature and blocked overnight in block buffer [PBS supplemented with 1 mM CaCl2, 0·5 mM MgCl2, 0·05 % (w/v) sodium azide and 0·5 % (w/v) BSA]. Sections were incubated with the primary antibody (10 µg ml1) for 1·5 h at 37 °C. Control sections were incubated either in the absence of the primary antibody or with an irrelevant matched isotype antibody. The sections were washed in PBS containing 1 mM CaCl2, 0·5 mM MgCl2 and incubated with a species-specific IgG conjugated to Alexa dyes (Molecular Probes) for 1·5 h at 37 °C. After washing, the sections were incubated with 4,6-diamidino-2-phenylindole (DAPI; Molecular Probes) for 30 min. The sections were then washed with water and mounted onto glass slides using Vectashield Mounting Medium for fluorescence (Vector Labs). Hand-cut tissues were mounted using Hardset Vectashield Mounting Medium (Vector Labs). Sections were viewed with a Leica TCS/SP2 confocal microscope.
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RESULTS |
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The 1 chain.
The 1 chain pairs with at least 12 different
chains (Hynes, 2002
) and epithelial cells normally express at least three different
1 integrins,
2
1,
3
1 and
9
1. Expression of a fourth
1 integrin,
5
1 is rapidly induced by injury in most epithelia (Cavani et al., 1993
; Juhasz et al., 1993
; Larjava et al., 1993
).
Fig. 1(ac) shows tongue epithelium (UU66) labelled with a
1-specific antibody. The pattern of
1 expression was consistent with that reported for
1 integrins on normal mammalian epithelia.
1 was detected at the highest level on the cells of the basal layer and at the edge of the papillae. Towards the outer keratinized layer, the level of integrin expression was reduced (Fig. 1a and b
). Fig. 1(c)
shows a higher magnification image of the cells of the stratum spinosum that lie adjacent to the basal cells. On these cells,
1 was located primarily at the cell surfaces with little or no
1 detected in the cytoplasm. The
1 chain was also detected on the vasculature including the large blood vessels of the dermis and on the capillaries located inside the papillae (Fig. 1b
). The labelling shown in Fig. 1
was specific as a matched isotype antibody was negative (data not shown).
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Fig. 1(d) shows tongue epithelium (UU66) labelled with an antibody (9EG7) that recognizes
1 integrins in their activated state. Expression of activated
1 was detected on the basal surfaces of the basal cells, and to a lesser extent on blood vessels. This pattern for activated
1 integrins was observed on all of the epithelia investigated.
The 3 chain.
The 3 chain associates with two
chains,
v and
IIb, forming
v
3 and
IIb
3, respectively (Hynes, 2002
). The integrin
IIb
3 is expressed exclusively on platelets, whereas
v
3 is normally expressed on endothelial cells and has been detected on certain blood vessels for several mammalian species (Damjanovich et al., 1992
; Kubo et al., 2001
; Mette et al., 1993
; Singh et al., 2000
, 2001
).
Fig. 1(e and f) shows sections of tongue epithelium (UU66 and UX10) labelled with a
3-specific antibody.
3 expression was detected primarily on the large blood vessels of the dermis (Fig. 1e
) and to a lesser extent on the capillaries located inside the papillae (Fig. 1f
).
3 expression was not detected on the epithelial cells of the stratum spinosum. Fig. 1(e)
shows that the
3 chain may be present on the basal cells of the tongue epithelium; however, the amount of
3 detected was low compared with the vasculature. Furthermore,
3 labelling on the basal cells was not detected on other tongue sections prepared from animal UU66, nor was it detected on the basal cells of the tongue epithelia of three other animals investigated. Similarly,
3 was not detected on the epithelial cells at the other sites investigated, including the interdigital-skin (UX10 and UX11), coronary band (UX10, UX11, VA16, VD38 and VD39), flank-skin (UU66, UX10 and UX11), and ventral and dorsal soft-palate (UU66, UX10, UX11, VA16, VD38 and VD39). We also failed to detect expression of
v
3 on several of these epithelia using an
v
3-specific antibody (mAb 23C6; data not shown).
Labelling for the 3 chain on the vasculature was predominantly weak, which may indicate low levels of protein expression. However, it is possible that the low level of
3 detection results from the anti-integrin antibodies being poorly cross-reactive for bovine integrins or from poor recognition of the integrin in paraformaldehyde-fixed tissues. Therefore, we labelled primary bovine thyroid cells with the antibodies used to detect
v
3 (mAb 23C6) and the
3 chain (mAb B3A), following the protocols that were used to prepare the tissue for confocal microscopy. These experiments showed that both antibodies readily detected
v
3 on the surfaces of paraformaldehyde-fixed cells (data not shown). This observation suggests that the failure to detect
3 expression on epithelial cells in fixed tissues most likely results from a low level of
v
3 expression rather than from an artefact of tissue preparation.
The integrin v
6.
The 6 chain pairs only with
v forming
v
6 (Hynes, 2002
). Fig. 2
(ac) shows tongue epithelium (VA16) labelled with an anti-
v
6 antibody (mAb 10D5). In the tongue,
v
6 was detected only on the epithelial cells and not on the vasculature, or the cells located in the dermis below the basement membrane. In contrast to the
1 chain,
v
6 was expressed more strongly on the cells of the stratum spinosum than on the cells of the basal layer. Fig. 2(c)
shows a higher magnification image of the cells in the stratum spinosum. As with the
1 chain, expression of
v
6 was largely confined to the cell surfaces with little evidence of integrin expression in the cytoplasm. The same pattern of
v
6 expression was observed on the tongue epithelium of the other animals investigated (UU66, UX10 and UX11). The labelling shown in Fig. 2
was specific, as labelling with a matched isotype antibody was negative for this tissue (data not shown).
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The integrin v
6 was also detected on the surfaces of the epithelial cells in the coronary band (Fig. 3
ac). The level of
v
6 expression of the epithelial cells in the coronary band appeared to be lower compared with the tongue and interdigital-skin (compare Fig. 2
with Fig. 3
) and furthermore,
v
6 appeared to be expressed at similar levels on the cells of the stratum spinosum and basal layer. In addition, at this site,
v
6 expression showed greater variation between animals and was detected within the coronary band of animals VD39, UX10 and VA16 (Fig. 3
), but not within the coronary band of animal VD38. Expression of
v
6 was also detected on the epithelial cells of the ventral soft-palate and its expression was also variable between animals. Integrin
v
6 was detected within the ventral soft-palate of animals VA16 (Fig. 3d
) and VD39 (Fig. 3e
), but not within this tissue of animals UU66, UX10, UX11 and VD38 (Fig. 3f
). In contrast to the above epithelia,
v
6 was not detected within the dorsal soft-palate or flank-skin of any of the animals investigated (data not shown).
|
This analysis showed that the high levels of v
6 protein (compared with
v
3) detected in the tongue and interdigital-skin by immunofluorescence confocal microscopy was supported by the PCR data as, at these sites,
6 mRNA was more abundantly expressed than
3 [
Ct=+4·9±1·8 (mean±SD, n=8) where + indicates
6 as the most abundant mRNA species]. In contrast, at sites where little or no integrins (
v
3 and
v
6) were detected (the ventral and dorsal soft-palate),
3 and
6 mRNAs were present at similar levels to each other (
Ct=+0·3±1·2, n=10).
Integrin expression in FMDV-infected epithelia
The above studies show that, in cattle, v
6 is expressed on the epithelial cells normally targeted by FMDV. To confirm that the cells infected with FMDV express this integrin, we investigated
v
6 expression within infected animals. Preliminary investigations indicated that the morphological structure of the tissue was not well preserved and the detection of cellular antigens was poor in advanced lesions (Monaghan et al., 2005
). Therefore, we used early lesion material collected from the interdigital-skin of cattle inoculated by injection in the tongue, as this route is the most effective way to establish infection in this species. This strategy enabled the appearance of early lesions on the feet to be predicted with greater accuracy.
To investigate v
6 expression in uninfected cattle, we used mAb 10D5. This mAb blocks the binding of natural ligands of
v
6 and consequently also inhibits the binding of FMDV (Jackson et al., 2000
). Thus, we reasoned that, in animals with high viraemia, virus bound to the integrin might inhibit the binding of mAb 10D5, thereby preventing integrin detection. Therefore, to study integrin expression within infected tissues, we used a non-blocking anti-
v
6 mAb 6.2A1 (Weinreb et al., 2004
).
Fig. 4 shows expression of
v
6 within infected interdigital-skin epithelia. Fig. 4(a)
shows the edge of an early lesion collected from animal VI31. The cells are stained with DAPI (blue) and Alexa-488-conjugated phalloidin (green) to visualize the cell nuclei and actin cytoskeleton, respectively. The cells infected with FMDV (red) were detected using a rabbit polyclonal serum against type O FMDV, which recognizes viral capsid proteins. Infected cells were abundant throughout the stratum spinosum and the intensity of virus labelling was greatest towards the middle of the lesion compared with its edge. These observations suggest that the cells in the middle of the lesion may be at a more advanced stage of infection. It is worth recalling that in the interdigital-skin, the cells of the stratum spinosum showed the highest level of
v
6 expression in uninfected animals. Fig. 4(c and d)
shows a similar region of the early lesion from the same animal labelled for FMDV (red) and
v
6 (green). At the edge of the lesion,
v
6 protein was observed on the surfaces of both uninfected and infected cells. Towards the middle of the lesion, the infected cells appeared to express little or no
v
6. At present we cannot be certain why these cells appear to express less surface
v
6; however, it is likely that they are at a more advanced stage of infection than the cells at the edge of the lesion and the reduction in
v
6 expression results from a combination of the poor morphology within infected samples, and the inhibition of both host-cell protein synthesis and protein secretory pathways that are characteristic of cells infected with FMDV in vitro (Belsham et al., 2000
; Moffat et al., 2005
).
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The above experiments confirm that, in infected cattle, v
6 is expressed on the surfaces of the epithelial cells targeted by FMDV in the interdigital-skin. In contrast, using the anti-
3 mAb we did not detect expression of the
3 chain on the infected cells of the interdigital-skin of either animal investigated. Similarly, the
3 chain was not detected on the uninfected cells immediately adjacent to the edge of the lesion.
We also investigated the activation status of the 1 integrins in infected interdigital-skin epithelium. Fig. 4(b)
shows that the labelling for activated
1 integrins within the infected tissue was similar to that observed for uninfected animals. Labelling of activated
1 integrins was detected primarily on blood vessels within the papillae and on the basal surfaces of the cells in contact with the basement membrane.
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DISCUSSION |
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In general, v
6 expression showed a good correlation with both the abundance of
6 mRNA and the sites of lesions in cattle. The tongue and interdigital-skin are sites where FMDV replicates to a high level and lesions normally form (Alexandersen et al., 2003
). At these sites,
v
6 protein was readily detected in the tissue and
6 mRNA was more abundantly expressed than that of
3. Within these epithelia, highest levels of
v
6 were present on the surfaces of epithelial cells of the stratum spinosum. These are the cells targeted by FMDV in cattle (Fig. 4
). Similarly, these cells are targeted in the tongue of FMDV-infected pigs (Monaghan et al., 2005
).
The integrin v
6 was also detected on surfaces of epithelial cells within the coronary band and ventral soft-palate. However, at these sites,
v
6 showed greater variation between animals and was detected on three of four and two of six animals for the coronary band and ventral soft-palate, respectively. These observations were reflected in the real-time RT-PCR data, as within the ventral soft-palate,
3 and
6 mRNAs were present at similar levels to each other. The variable expression of
v
6 at these sites could result from differential integrin expression between cattle or, alternatively, from non-uniform expression of
v
6 within the epithelium of individual animals. This latter scenario could result from upregulation of
v
6 at sites of inflammation (see below). The variable expression of
v
6 protein within the coronary band and ventral soft-palate is, however, consistent with the frequency of lesions at these sites. Lesions are often but not always present on the coronary band and rarely seen on the ventral soft-palate (S. Alexandersen, unpublished observation). Although it is likely that FMDV infection (and hence lesion formation) requires a number of cellular factors, it is interesting to speculate that lesion formation at these sites is dependent on expression of
v
6.
The dorsal soft-palate and flank-skin are sites where lesions do not normally occur in FMDV-infected cattle, and at these sites v
6 protein was not detected. Consistent with these observations,
3 and
6 mRNAs appeared to be present at similar levels to each other within the dorsal soft-palate. However, despite being a site where lesions do not normally form, the dorsal soft-palate has attracted special interest because the epithelial cells have been shown to harbour viral RNA at early times post-infection (Alexandersen et al., 2003
). Despite this evidence that the epithelial cells of the dorsal soft-palate are probably targeted by FMDV, it is puzzling why lesions do not develop at this site (Alexandersen et al., 2003
). The mechanisms that underlie this apparent lack of cytopathology are unknown, but may be linked to the ability of FMDV to establish a persistent infection, as epithelial cells of the dorsal soft-palate have also been shown to harbour viral RNA in persistently infected cattle (Alexandersen et al., 2003
; Prato Murphy et al., 1999
; Zhang & Alexandersen, 2004
; Zhang & Kitching, 2001
). Although we cannot rule out the possibility that
v
6 is expressed at low level at this site, our data suggest that infection within the dorsal soft-palate is not mediated by
v
6 receptors.
Our studies confirmed that v
6 is expressed on the surfaces of cells infected with FMDV within the interdigital-skin. At the edge of the lesion,
v
6 was seen on the surfaces of both uninfected and infected cells. Towards the middle of the lesion, the cells of the stratum spinosum showed the greatest intensity of labelling for viral proteins. Importantly, these cells had the most intense labelling for
v
6 in uninfected tissues (Fig. 2
). However, in the middle of the lesion, the infected cells appeared to express little or no
v
6. At present we cannot be certain why these cells appear to express less surface
v
6. However, it is likely that these cells are at a more advanced stage of infection than the cells at the edge of the lesion and consequently normal protein expression may be reduced as both host-cell protein synthesis and protein secretory pathways are inhibited by infection of FMDV in vitro (Belsham et al., 2000
; Moffat et al., 2005
).
Although shown to be a receptor for FMDV on cultured cells, v
3 was not detected on any of the epithelial cells investigated but was detected on the vasculature (Fig. 1
). This observation is in agreement with studies of other mammalian species, which have concluded that
v
3 normally predominates in endothelial rather than epithelial cells (Breuss et al., 1993
, 1995
; Clark et al., 1996
; Damjanovich et al., 1992
; Mette et al., 1993
; Singh et al., 2000
, 2001
). However, although we did not detect expression of
v
3 on epithelial cells, it should be acknowledged that expression of
v
3 has been reported for epithelial cells lining the bile duct and small intestine of pig and cattle (Singh et al., 2001
). With this in mind, we cannot rule out the possibility that
v
3 is expressed on the epithelial cells investigated but at levels below that can be detected using our methods. Similarly, we cannot completely exclude a role for
v
3 as a receptor at later stages of infection.
Integrin expression is upregulated during processes such as wound healing and inflammation (Clark et al., 1996; Gailit et al., 1994
; Haapasalmi et al., 1995
, 1996
; Hakkinen et al., 2000
; Larjava et al., 1993
; Pilewski et al., 1997
; Wang et al., 1996
; Zambruno et al., 1995
). This phenomenon suggests that integrin expression on epithelial cells could increase on FMDV infection. Although our data are limited to the interdigital-skin, we did not detect noticeable changes in the level of
v
6 protein on the cells at the edge of the lesion, perhaps because within the interdigital-skin,
v
6 is expressed constitutively at a high level. Similarly, we did not observe upregulation of
v
3 on the cells at the edge of the lesion. These observations suggest that within the interdigital-skin the integrins
v
3 and
v
6 are not upregulated on epithelial cells at sites of infection.
In vivo, many different integrin species are likely to be activated as a result of infection (Burns et al., 2001; Gonzalez-Amaro et al., 1998
; Springer, 1990
) and for viruses, such as FMDV, this activation could effectively switch-on receptors for gaining entry into cells. Therefore, we investigated changes in
1 integrin activation of infected interdigital-skin using mAb 9EG7. mAb 9EG7 recognizes
1 integrins that are in their active state (Bazzoni et al., 1998
). Using this mAb we did not detect noticeable changes in activated
1 integrins on infection as, in both uninfected and infected cattle, activated
1 integrins were detected primarily on the basal membrane of the basal cells and on blood vessels.
Two other v integrins, namely
v
1 and
v
8 (Jackson et al., 2002
, 2004
) have been shown to serve as receptors for FMDV in vitro. The integrin
v
8 has been shown to be expressed on human airway epithelial cells (Cambier et al., 2000
; Fjellbirkeland et al., 2003
). However, we have not been able to study this integrin in cattle, as cross-reactive antibodies for bovine
v
8 that can be used with fixed tissues have not been identified. Similarly, we could not study the tissue distribution of
v
1, since antibodies specific for the
v
1 heterodimer are currently not available.
At present we cannot be certain what role the various integrin receptors play in FMDV tropism and pathogenesis. However, our data show that v
6 is expressed constitutively at high levels on the epithelial cells normally targeted by FMDV in cattle and suggest that
v
6, rather than
v
3, is the major receptor that determines the tropism of this virus.
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ACKNOWLEDGEMENTS |
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Received 10 May 2005;
accepted 22 July 2005.
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