Génétique Virale-NRSN, Département Neuroscience1, Interactions Lymphoépithéliales2 and Plate-forme de microscopie électronique3, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris cedex 15, France
Author for correspondence: Florence Colbère-Garapin. Fax +33 1 40 61 33 67. e-mail fcolbere{at}pasteur.fr
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() |
---|
![]() |
Main text |
---|
![]() ![]() ![]() ![]() |
---|
Since Bodians studies in the 1950s, it has been widely held that the primary sites of PV replication in the gut are Peyers patches (PP) (Bodian, 1955 ; Minor, 1997
). The epithelium covering lymphoid follicles of PP contains a variable number of scattered epithelial M (microfold) cells, which play a central role in the uptake of foreign antigens and microorganisms and their delivery to the underlying mucosal-associated lymphoid tissue (MALT) (Neutra et al., 1996a
, b
; Owen, 1977
). M cells can be distinguished from their neighbouring enterocytes by characteristic morphological features, including a diminished apical brush border and its membrane-anchored glycoprotein network, the glycocalyx, and a deep invagination of the basolateral plasma membrane, which contains lymphocytes and phagocytic leukocytes (Gebert, 1996
; Madara et al., 1984
; Madara & Trier, 1994
; Niedergang & Kraehenbuhl, 2000
; Owen & Jones, 1974
). In addition to their physiological role of antigen sampling (Kraehenbuhl & Neutra, 1992
), M cells can be exploited by a large number of pathogenic microorganisms (Siebers & Finlay, 1996
). Particular viruses, such as reoviruses, gain access to distant organs by passing through the epithelial barrier via M cells (Amerongen et al., 1994
; Bass et al., 1988
; Wolf et al., 1981
, 1987
).
There has only been one study of the interaction of PV with M cells: human tissue samples were infected at 20 °C with PV type 1 (Sicinski et al., 1990 ). Virions were found specifically adhering to the surface projections of M cells and in vesicles in M cells (Sicinski et al., 1990
). However, the experimental conditions prevented the further transport of the virus because the ultrastructure of the epithelial cells deteriorated during in vitro incubations lasting longer than 1 h. No virions were found under the basement membrane of the epithelium. Thus, to our knowledge, the transcytosis of infectious PV, or any other enterovirus, through M cells has never been demonstrated.
We used an in vitro model previously developed (Kerneis et al., 1997). This model reproduces the main characteristics of M cells and lymphoid follicle-associated epithelium by cultivation of murine PP lymphocytes with differentiated absorptive enterocyte monolayers of the Caco-2 cell line. When they are grown on permeable filters, Caco-2 cells express genes that are specifically activated in villus absorptive enterocytes of the small intestine. This indicates that, despite their colonic origin, Caco-2 cells differentiate into small intestine enterocyte-like cells (Costa de Beauregard et al., 1995 ; Pinto et al., 1983
). Caco-2 cells grown on porous filters polarize and differentiate after 1214 days of culture. Lymphocytes from freshly isolated PP of mice can then be dropped into the basolateral chamber of Transwell devices, where in a few hours they settle into the epithelial monolayer, inducing enterocyte/M-like cell conversion in 24 days (Kerneis et al., 2000
). Using this model, M cell-specific transcytosis of 200 nm diameter latex beads (El Bahi et al., 2002; Kerneis et al., 1997
), bacteria (Gullberg et al., 2000
; Kerneis et al., 1997
) and the pathogenic form of the prion protein (PrPsc) (Heppner et al., 2001
) have been demonstrated. Translocation through M cells is temperature-dependent. There is a latency period of 510 min, which corresponds to the half-time of trancytosis in enterocytes previously described for trafficking proteins and receptors, such as the Fc receptor and the poly-Ig receptor (Dickinson et al., 1999
; Mostov et al., 2000
). The transcytosis reaches a maximal level after 24 h for the particles or microorganisms so far tested.
The method for establishing cocultures containing M-like cells has previously been reported in detail (Kerneis et al., 2000 ). Briefly, 3x105 Caco-2 cells (clone 1) were seeded and cultivated for 2 weeks on 0·33 cm2, 3 µm pore Transwell device filters (Costar), in Dulbeccos modified essential medium (DMEM) supplemented with 10% foetal calf serum (DMEM-SVF). PP lymphocytes (1·5x106) were isolated from a BALB/c mouse and added to the basolateral chambers of Transwell devices containing tight monolayers of fully differentiated Caco-2 cells. Such cocultures can be maintained in DMEM-SVF for at least 34 days. In our study, the full differentiation of Caco-2 cell monolayers was checked by measuring transepithelial electrical resistance (TER), by fluorescence microscopy and by transmission electron microscopy. The lectin Ulex Europaeus Agglutinin 1 (UEA1), conjugated with fluorescein isothiocyanate (FITC), which binds to terminal fucose residues of the brush border glycocalyx on enterocyte-like Caco-2 cells, was used to monitor the integrity of the brush border of polarized enterocytes, whether or not cocultured with PP lymphocytes. The UEA1 labelling of cells decreased and appeared much more heterogeneous in Caco-2 cells in cocultures with PP lymphocytes than in those cultured alone (Fig. 1A
, B
), indicating lymphocyte-driven brush border disorganization at the surface of a large percentage of epithelial cells. This is as expected for enterocyte to M-like cell conversion. A reorganization of the F-actin apical network in cocultures was confirmed by labelling with rhodamine-conjugated phalloidin (Fig. 1C
, D
). In addition, cells were examined by transmission electron microscopy. Cell monolayers were fixed in 1% paraformaldehyde, 2·5% glutaraldehyde, post-fixed in 1% osmium tetroxide supplemented with 1% potassium ferricyanide and included in epoxy resin. Ultrathin sections (70 nm) were cut with a diamond knife in a Leica Ultracut UCT microtome, placed on to 200-mesh copper grids, stained with uranyl acetate and lead citrate, and examined with a JEOL 1200 EX transmission electron microscope operating at 80 kV. The apical side of the entire monolayer of Caco-2 cells cultured alone displayed a regular and homogeneous brush border (Fig. 1E
). In contrast, numerous epithelial cells with disorganized flat apical surfaces were observed in cocultures, consistent with M-like cell induction (Fig. 1F
). Tight junctions, responsible for the tightness of epithelial monolayers, were observed between epithelial cells in both types of cultures (Fig. 1E
, F
). This tightness was confirmed by measurements of TER using a Millicell-ERS apparatus at the beginning of each transcytosis experiment. The average values measured in Caco-2 cell monolayers cultured alone and in cocultures were 365±12 and 279±10 ohms.cm2, respectively, as expected for tight Caco-2 clone 1 monolayers. Immunofluorescence (as previously described by Pavio et al., 1996
) and confocal microscopy detected the PV receptor, CD155, on the apical and basolateral membranes of cells in both types of cell culture (Fig. 1GJ
). However, an M cell-specific marker will be required to determine whether or not M-like cells express CD155.
|
|
The kinetics of translocation of PV1/Mahoney were then studied in the two culture conditions (Fig. 3). It was first verified that the monolayers were tight by measuring the TER (means of 323±14 and 312±22 ohms.cm2 for Caco-2 cells alone and cocultures, respectively). PV1/Mahoney (0·8x108 TCID50) was then added to the apical chamber of the cultures, as described above. A pre-incubation period at 4 °C confirmed that transcytosis did not occur at 4 °C in both culture conditions (Fig. 3
). After a rapid shift to 37 °C, very small amounts of PV1/Mahoney were transported through Caco-2 monolayers cultured alone, confirming that enterocytes were almost unable to transcytose PV (Fig. 3
). In sharp contrast, high titres of PV1/Mahoney appeared in the basolateral medium of Caco-2 monolayers cultured with PP lymphocytes after 15 min at 37 °C, and the titres increased thereafter (Fig. 3
). The appearance of PV1/Mahoney in the basolateral compartment was consistent with the characteristics of transcytosis: it was inhibited at 4 °C and occurred within 3 h of the shift to 37 °C (Fig. 3
). The kinetics is compatible with the previously reported half-time of receptor-mediated transcytosis through epithelial cells (15 min) (Dickinson et al., 1999
; Mostov et al., 2000
). In contrast, it is not compatible with the time required for the synthesis of progeny virions after infection. Thus, these results constitute the first indication of PV translocation through M cells. Some membrane functions may still be active at 4 °C, but their role could not be investigated by incubating cells for several hours at 4 °C because this treatment affected the integrity of the cell monolayer.
|
After the initial replication cycle that occurs during a natural PV infection, the viral load on the apical side of M cells may be sufficient to cause a significant amount of virus translocation, thus contributing to viral pathogenesis. In vivo, potential target cells of the immune system are in contact with the basal membrane of M cells. This may facilitate PV spread to mesenteric lymph nodes and viral amplification in permissive cells, even if the translocation rate through M cells is low. This may also facilitate the spread to secondary organs, such as the central nervous system. The intestine is among the most innervated organs (Emmanuel et al., 2001 ) and it remains possible that some virions reach the CNS by using the local neural route. In this case, they may use CD155 expressed at the surface of neurons (Arita et al., 1999
; Racaniello & Ren, 1996
).
In conclusion, using an in vitro model of polarized epithelial cells reproducing some of the main structural and functional characteristics of M cells scattered in a monolayer of polarized enterocytes, we have shown that PV transcytosis toward the basolateral compartment of cells occurred. Transcytosis was much more efficient in cocultures containing M-like cells than in polarized Caco-2 cells cultured alone. Our results are consistent with PV using M cells as gateways for entry into the organism, and constitute a first step to understanding the molecular mechanisms by which PV and other enteroviruses breach the intestinal barrier. These mechanisms, in particular the role of CD155, remain to be elucidated. Direct delivery of virions by M cells to underlying MALT implicate immune cells as good candidates for PV transport to mesenteric lymph nodes. Our results also suggest that translocated PV may infect enterocytes through their basolateral, CD155-covered face. Infected cells would then liberate virions from their apical face in the intestinal lumen, as previously proposed (Tucker et al., 1993b ), allowing further amplification of PV infection in the gut.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() |
---|
Arita, M., Ohka, S., Sasaki, Y. & Nomoto, A. (1999). Multiple pathways for establishment of poliovirus infection. Virus Research 62, 97-105.[Medline]
Bass, D. M., Trier, J. S., Dambrauskas, R. & Wolf, J. L. (1988). Reovirus type I infection of small intestinal epithelium in suckling mice and its effect on M cells. Laboratory Investigation 58, 226-235.[Medline]
Blondel, B., Duncan, G., Couderc, T., Delpeyroux, F., Pavio, N. & Colbère-Garapin, F. (1998). Molecular aspects of poliovirus biology with a special focus on the interactions with nerve cells. Journal of Neurovirology 4, 1-26.[Medline]
Bodian, D. (1955). Emerging concept of poliomyelitis infection. Science 122, 105-108.[Medline]
Costa de Beauregard, M. A., Pringault, E., Robine, S. & Louvard, D. (1995). Suppression of villin expression by antisense RNA impairs brush border assembly in polarized epithelial intestinal cells. EMBO Journal 14, 409-421.[Abstract]
Dickinson, B. L., Badizadegan, K., Wu, Z., Ahouse, J. C., Zhu, X., Simister, N. E., Blumberg, R. S. & Lencer, W. I. (1999). Bidirectional FcRn-dependent IgG transport in a polarized human intestinal epithelial cell line. Journal of Clinical Investigation 104, 903-911.
El Bahi, S., Caliot, E., Bens, M., Bogdanova, A., Kerneis, S., Kahn, A., Vandewalle, A. & Pringault, E. (2002). Lympho-epithelial interactions trigger specific regulation of gene expression in the M-cell containing follicle-associated epithelium of Peyers patches. Journal of Immunology 168, 3713-3720.
Emmanuel, A. V., Mason, H. J. & Kamm, M. A. (2001). Relationship between psychological state and level of activity of extrinsic gut innervation in patients with a functional gut disorder. Gut 49, 209-213.
Gebert, A. (1996). M-cells in the rabbit tonsil exhibit distinctive glycoconjugates in their apical membranes. Journal of Histochemistry and Cytochemistry 44, 1033-1042.
Gullberg, E. M., Leonard, M., Karlsson, J., Hopkins, A. M., Brayden, D., Baird, A. W. & Artursson, P. (2000). Expression of specific markers and particle transport in a new human intestinal M-cell model. Biochemical and Biophysical Research Communications 279, 803-813.[Medline]
Heppner, F. L., Christ, A. D., Klein, M. A., Prinz, M., Fried, M., Kraehenbuhl, J. P. & Aguzzi, A. (2001). Transepithelial prion transport by M cells. Nature Medicine 7, 976-977.[Medline]
Kerneis, S., Bogdanova, A., Kraehenbuhl, J. P. & Pringault, E. (1997). Conversion by Peyers patch lymphocytes of human enterocytes into M cells that transport bacteria. Science 277, 949-952.
Kerneis, S., Caliot, E., Stubbe, H., Bogdanova, A., Kraehenbuhl, J. & Pringault, E. (2000). Molecular studies of the intestinal mucosal barrier physiopathology using cocultures of epithelial and immune cells: a technical update. Microbes and Infection 2, 1119-1124.[Medline]
Kraehenbuhl, J. P. & Neutra, M. R. (1992). Molecular and cellular basis of immune protection of mucosal surfaces. Physiological Reviews 72, 853-879.
Lopez, M., Jordier, F., Bardin, F., Coulombel, L., Chabannon, C. & Dubreuil, P. (1999). Identification of a new class of Ig superfamily antigens expressed in hemopoiesis. In Leucocyte Typing VI. White Cell Differentiation Antigens , pp. 1081. Edited by T. K. Kishimoto, H. Kikutani, A. E. G. von dem Borne, S. M. Goyert, D. Y. Mason, M. Miyasaka, M. Moretta, K. Okumura, S. Shaw, T. A. Springer, K. Sugamura & H. Zola. New York: Garland Publishing.
Madara, J. L. & Trier, J. S. (1994). The functional morphology of the mucosa of the small intestine. In Physiology of the Gastrointestinal Tract , pp. 1577. Edited by L. R. Johnson. New York: Raven Press.
Madara, J. L., Bye, W. A. & Trier, J. S. (1984). Structural features of and cholesterol distribution in M-cell membranes in guinea pig, rat and mouse Peyers patches. Gastroenterology 87, 1091-1103.[Medline]
Minor, P. D. (1997). Poliovirus. In Viral Pathogenesis , pp. 555-574. Edited by N. Nathanson. Philadelphia: LippincottRaven.
Mostov, K. E., Verges, M. & Altschuler, Y. (2000). Membrane traffic in polarized epithelial cells. Current Opinion in Cell Biology 12, 483-490.[Medline]
Neutra, M. R., Frey, A. & Kraehenbuhl, J. P. (1996a). Epithelial M cells: gateways for mucosal infection and immunization. Cell 86, 345-348.[Medline]
Neutra, M. R., Pringault, E. & Kraehenbuhl, J. P. (1996b). Antigen sampling across epithelial barriers and induction of mucosal immune responses. Annual Review of Immunology 14, 275-300.[Medline]
Niedergang, F. & Kraehenbuhl, J. P. (2000). Much ado about M cells. Trends in Cell Biology 10, 137-141.[Medline]
Owen, R. L. (1977). Sequential uptake of horseradish peroxidase by lymphoid follicle epithelium of Peyers patches in the normal unobstructed mouse intestine: an ultrastructural study. Gastroenterology 72, 440-451.[Medline]
Owen, R. L. & Jones, A. L. (1974). Epithelial cell specialization within human Peyers patches: an ultrastructural study of intestinal lymphoid follicles. Gastroenterology 66, 189-203.[Medline]
Pavio, N., Buc-Caron, M. H. & Colbère-Garapin, F. (1996). Persistent poliovirus infection of human fetal brain cells. Journal of Virology 70, 6395-6401.[Abstract]
Pinto, M., Robine-Leon, S., Appay, M., Kedinger, M., Triadou, N., Dussaulx, E., Lacroix, B., Simon-Assmann, P., Haffen, K., Fogh, J. & Zweibaum, A. (1983). Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture. Biology of the Cell 47, 323-330.
Racaniello, V. & Ren, R. (1996). Poliovirus biology and pathogenesis. Current Topics in Microbiology and Immunology 206, 305-325.[Medline]
Sicinski, P., Rowinski, J., Warchol, J. B., Jarzabek, Z., Gut, W., Szczygiel, B., Bielecki, K. & Koch, G. (1990). Poliovirus type 1 enters the human host through intestinal M cells. Gastroenterology 98, 56-58.[Medline]
Siebers, A. & Finlay, B. B. (1996). M cells and the pathogenesis of mucosal and systemic infections. Trends in Microbiology 4, 22-29.[Medline]
Tucker, S. P., Thornton, C. L., Wimmer, E. & Compans, R. W. (1993a). Bidirectional entry of poliovirus into polarized epithelial cells. Journal of Virology 67, 29-38.[Abstract]
Tucker, S. P., Thornton, C. L., Wimmer, E. & Compans, R. W. (1993b). Vectorial release of poliovirus from polarized human intestinal epithelial cells. Journal of Virology 67, 4274-4282.[Abstract]
Wimmer, E., Hellen, C. U. & Cao, X. M. (1993). Genetics of poliovirus. Annual Review of Genetics 27, 353-436.[Medline]
Wolf, J. L., Rubin, D. H., Finberg, R., Kauffman, R. S., Sharpe, A. H., Trier, J. S. & Fields, B. N. (1981). Intestinal M cells: a pathway for entry of reovirus into the host. Science 212, 471-472.[Medline]
Wolf, J. L., Dambrauskas, R., Sharpe, A. H. & Trier, J. S. (1987). Adherence to and penetration of the intestinal epithelium by reovirus type 1 in neonatal mice. Gastroenterology 92, 82-91.[Medline]
Received 11 March 2002;
accepted 29 April 2002.