Department of Virology, University of Bremen, Leobener Straße/UFT, D-28359 Bremen, Germany
Correspondence
Andreas Dotzauer
dotzauer{at}uni-bremen.de
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ABSTRACT |
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Only weak evidence has been provided for replication of HAV in intestinal cells of animal models (Karayiannis et al., 1986; Asher et al., 1995
; Hornei et al., 2001
), and Blank et al. (2000)
demonstrated, by the observation of vectorial apical (lumenal) entry and release of HAV from Caco-2 cells, that infection of polarized intestinal cells by HAV does not result in penetration of the intestinal epithelium.
An alternative mechanism is transcytosis of HAV across the epithelial barrier of the intestinal tract without infection of this area. However, transcytosis of HAV virions by intestinal epithelial cells could not be detected (Blank et al., 2000) and transcytosis by M cells (Silvey et al., 2001
; Ouzilou et al., 2002
) has never been demonstrated.
One additional possibility for HAV to overcome the intestinal barrier is carrier-mediated transport. In an earlier study, we demonstrated that HAVanti-HAV IgA complexes (HAVIgA) are infectious for human hepatocytes by IgA-mediated endocytosis via the IgA-specific hepatocellular asialoglycoprotein receptor (ASGPR) (Dotzauer et al., 2000). This IgA carrier mechanism may contribute to the efficiency of HAV infections and may even enable prolonged or relapsing courses of disease (Glikson et al., 1992
), which occur in the presence of otherwise neutralizing antibodies. We speculated that, seemingly contrary to the view of IgA function in mucosal defence (reviewed by Rojas & Apodaca, 2002
), intestinal HAVIgA may enter the bloodstream by existing transport activities of intestinal cells specific for IgA, allowing transepithelial passage of HAVIgA complexes.
IgA transport across the intestinal epithelial layer is carried out by the epithelial polymeric immunoglobulin receptor (pIgR) (Mostov, 1994) and, in the following, we will show that HAVIgA translocation from the intestinal lumen into blood is possible via this receptor.
In order to examine transepithelial passage of HAVIgA, we used MadinDarby canine kidney (MDCK) cells transfected stably with the IgA-specific pIgR. In contrast to the Caco-2 cell model (Blank et al., 2000), MDCK cells are not permissive for HAV infection (Dotzauer et al., 1994
) and, therefore, results possibly confounded by HAV replication are excluded. MDCKpIgR cells develop a strong barrier to diffusion of macromolecules and are a well-characterized model of pIgR-mediated IgA transcytosis in polarized epithelium (Mostov & Deitcher, 1986
). The customary route of IgA transepithelial transport by the pIgR is from the basolateral (lamina propria) to the apical (intestinal lumen) cell surface. Basal-to-apical transcytosis of antigenIgA complexes was also demonstrated, a mechanism contributing to the elimination of pathogens (Kaetzel et al., 1991
; Gan et al., 1997
). However, as pIgR missorting to the apical membrane occurs (Mostov & Deitcher, 1986
) and IgA not released from the pIgR at the apical site can be transcytosed back and released basolaterally (Breitfeld et al., 1989
), we supposed that reverse transcytosis of antigenIgA complexes from the apical to the basolateral cell surface is possible, a pathway accessible to intestinal HAVIgA.
In order to have separate access to basolateral and apical cell surfaces necessary for studying this possibility of HAVIgA transcellular transport, MDCKpIgR cells were cultivated on porous-membrane filter chambers (0·45 µm pore size; Millicell HA, Millipore) inserted into six-well plates with Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal calf serum (FCS). Integrity and polarization of the cell monolayer were tested by measuring the transmembrane electrical resistance with a VoltOhm meter (Millicell-ERS; Millipore). As confirmed by impermeability to IgG conjugated with peroxidase, readings of >240 cm2 were required for cell use. This was achieved after 56 days in culture and was retained for at least 26 h, a time course relevant to the experiments.
HAVIgA complexes were prepared by incubating HAV (105 TCID50 ml1), which was obtained by triple freezethaw cycles and removal of cellular debris from FRhK-4 (fetal rhesus monkey kidney) cells infected with a tissue culture-adapted variant of strain HM175 (Dotzauer et al., 2000), with 20 µg monoclonal mouse anti-HAV IgA antibody 1.193 ml1 (Ping & Lemon, 1992
) for 2 h at room temperature in DMEM. As this antibody, which contains monomeric and pIgR-affine polymeric IgA molecules in nearly equal amounts (Dotzauer et al., 2000
), neutralizes HAV upon infection of FRhK-4 cells, complex formation was assayed by infection inhibition on FRhK-4 cells (Dotzauer et al., 2000
). Neutralization to a TCID50 titre of <102 ml1 indicated that >99 % of the virus was associated with IgA.
After rinsing the cells with PBS, 500 µl HAVIgA was added to the apical surface of MDCKpIgR cells. After 2 h at 37 °C for adsorbtion, DMEM/1 % FCS was added to both sides of the filter and cells were incubated at 37 °C for 24 h. Samples of the basolateral and apical media were analysed for intact HAVIgA. For this purpose, the complexes were separated (from 500 µl samples) by using biotin-labelled goat anti-mouse IgA (Kirkegaard and Perry Laboratories) bound onto streptavidin-coated magnetic beads (Dynabeads M-280; Dynal) (60 µl, incubation at room temperature for 1 h), which were prepared by incubation of 50 µg beads with 0·5 µg antibody for 1 h at room temperature, and tested for HAV by RT-PCR amplification of the viral 2C region. PCR products were visualized by dot-blot hybridization (Dotzauer et al., 1994) (upper half of Fig. 1
, dot-blot analysis). Quantification of HAV in the samples was performed by using a RealArt HAV LC RT-PCR kit (Artus) and a LightCycler instrument (Roche Diagnostics) (lower half of Fig. 1
, real-time RT-PCR analysis).
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To confirm that the apical-to-basolateral translocalization of HAVIgA observed was dependent on the pIgR pathway and, therefore, that reverse transcytosis occurred, several controls were included. After incubation at 4 °C instead of at 37 °C, only a small amount (1 %) of the complexes could be detected basolaterally (Fig. 1
, lane 2). This is indicative of the involvement of an active-transport mechanism and additionally shows that diffusion did not take place. Preincubation of the apical surface for 2 h with 100 µg non-specific IgA ml1 (polyclonal mouse anti-trinitrophenyl IgA antibody MOPC 315; Sigma) competitively inhibited translocation of the HAVIgA complexes by approximately 90 % (
2 % could be detected basolaterally) (Fig. 1
, lane 3). This demonstrates that the transcellular transport of the complexes was IgA-specific.
Virus alone in amounts equivalent to those in the immunocomplex inoculum added to the apical surface of the cells did not translocalize into the basolateral medium (<0·005 % was detected basolaterally) (Fig. 1, lane 4). These medium samples were tested for HAV by RT-PCR after RNA extraction with a QIAamp viral RNA kit (Qiagen). Also, HAVanti-HAV IgG, which was prepared as described for IgA-complex formation by using the monoclonal anti-HAV IgG antibody 7E7 (Mediagnost), did not traverse the MDCKpIgR epithelial cell sheets (<0·005 % was detected basolaterally) (Fig. 1
, lane 5). The samples were analysed as described for IgA complexes, using anti-mouse IgG for magnetic separation. These controls, again, showed that HAVIgA translocation from the apical to the basolateral compartment was dependent on IgA, that no diffusion occurred and that MDCK cells translocate neither HAV nor HAVIgG complexes from the apical to the basolateral site. Similarly, MDCK cells without the gene encoding the pIgR did not translocate HAVIgA from the apical into the basolateral compartment. In three independent experiments, apical inoculation with HAVIgA containing 9x106 IU viral RNA (1 IU is equivalent to 10 HAV genomes), as determined by real-time RT-PCR (Heitmann et al., 2005
), resulted in a mean basolateral HAV RNA amount of 396 IU (435, 407 and 346 IU), which corresponds to 0·004 % transcytosis. This indicates that the reverse transport of HAVIgA across MDCKpIgR cells was dependent on the pIgR.
These experiments show that free HAV is not translocated by transcytosis from the apical cell surface to the basolateral compartment of MDCK and MDCKpIgR cells, which are model epithelial cell lines. This is in accordance with findings using Caco-2 cells (Blank et al., 2000) that transcytosis of HAV across the epithelial barrier of the intestinal tract by epithelial cells does not occur. However, HAVanti-HAV IgA complexes can be translocated by reverse transcytosis from apical to basolateral compartments by epithelial cells via the pIgR. This seems contrary to the view of IgA function in mucosal defence. Polymeric IgA synthesized in the lamina propria binds to the pIgR, which is localized on the basolateral cell surface. After transcytosis to the apical surface, the extracellular, IgA-binding portion of the pIgR [secretory component (SC)] is cleaved off and released into the secretions of the small intestines, where the SC remains associated with IgA (Mostov, 1994
). These IgASC molecules (secretory IgA) cannot rebind to intact pIgR molecules present on the apical surface of the cells, which ensures that the IgApIgR pathway is directed vectorially towards the intestinal lumen.
In this context, it is an important finding that, after infection with HAV, the IgA response is extraordinarily fast, strong and, after reaching its peak titre 50 days post-infection (p.i.), it is long-lasting for up to 5 years (Lofgren et al., 1980; Sikuler et al., 1983
; Naudet, 1988
). The majority of the IgA is not secreted by the IgApIgR pathway (Stapleton et al., 1991
), but a significant fraction of this serum IgA is released into the intestinal tract via bile by liver functions (Mestecky et al., 1991
; Shimada et al., 1999
). Progeny HAV, also released from the liver to the gastrointestinal tract via bile, can bind to these IgA molecules, which are not associated with the pIgR SC. The presence of significant amounts of intestinal HAVIgA is demonstrated by the finding that HAV is partly transmitted as HAVIgA complexes (Locarnini et al., 1980
; Karayiannis et al., 1988
).
Previously, we demonstrated that HAVIgA is astonishingly stable in acidic environments (Dotzauer et al., 2000). We found that 100 % of the complexes remained stable at pH 3·5 for 3 h; 60 % remained associated after treatment with pH 2·5 for 3 h or treatment with pH 1·5 for 1 h. Therefore, after transmission via faeces, especially after transmission from person to person through close contacts, these complexes, which can pass through the harsh conditions of the stomach undamaged (Dotzauer et al., 2000
), may bind to missorted pIgR of intestinal cells (Breitfeld et al., 1989
) and IgA-mediated reverse transcytosis may allow HAV to enter the bloodstream and reach the liver, resulting in infection of the hepatocytes through uptake of the complexes via the ASGPR (Dotzauer et al., 2000
), which mediates uptake of both IgA (serum IgA) and IgASC (secretory IgA) (Tomana et al., 1985
, 1988
) (Fig. 2a
). As HAV shows only weak replication in the intestinal tract, this mechanism, which prevents transport of the IgASC immune complexes back to the intestinal lumen by the pIgR, would increase the amount of HAV available for infection of the liver and therefore support the infectious outcome.
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Also, enterohepatic cycling of HAV as HAVIgA may play a role in relapsing courses (Fig. 2b). As the clinical picture, including the IgM immune response, is similar to the primary phase (Schiff, 1992
), reinfections of the liver seem to occur in this case. These recurrent infections appear during reconvalescence between 30 and 90 days after the primary infection in up to 20 % of patients (Glikson et al., 1992
). As it was shown for acute infections that HAV is detectable in faeces for at least 90120 days after onset of icterus (Yotsuyanagi et al., 1996
) and faecal anti-HAV IgA persists at least for 120 days after the onset of symptoms (Yoshizawa et al., 1980
), HAVIgA may be the cause of recurrent infections, especially in the presence of significant amounts of neutralizing anti-HAV IgG. At last, eradication of HAV from the organism may result by displacement of the IgA in the HAVIgA complexes by neutralizing high-avidity IgG of the matured IgG response.
Although the significance of these IgA carrier mechanisms, which were found in cell culture, for HAV infections in vivo remains to be shown, they deserve some consideration. Different observations in vivo can be related to an association of HAV with IgA beside prolonged and relapsing courses. Although HAV is able to infect a number of non-liver cells in cell culture (Dotzauer et al., 1994), no extrahepatic sites of HAV replication have been identified clearly in the whole organism. In association with IgA, HAV would be neutralized for infection of IgA receptor-negative cells, so that no extrahepatic site of replication could be identified. Although viral RNA is detectable in blood for several weeks in significant amounts (Normann et al., 2004
), viral antigen is found only from week 3 until week 5 p.i. (viraemic phase) and in low amounts. Association of HAV with IgA would interfere with the detection of HAV antigen. We are presently planning to investigate the IgA carrier hypothesis for HAV infections by using a mouse model.
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ACKNOWLEDGEMENTS |
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Received 29 April 2005;
accepted 14 July 2005.
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