Vibrio cholerae-induced cellular responses of polarized T84 intestinal epithelial cells are dependent on production of cholera toxin and the RTX toxin.

To study the utility of in vitro-polarized intestinal cell monolayers for modeling Vibrio cholerae-host cell interactions, we added live V. cholerae bacteria to the apical surfaces of polarized T84 cell monolayers and monitored changes in electrical properties. We found that both classical and El Tor strains produce cholera toxin after addition to the monolayer, but induction is most likely due to medium components rather than bacterium-cell interactions. We also found that the RTX toxin is produced by El Tor strains. This toxin caused a loss of the barrier function of the paracellular tight junction that was measured as a decrease in transepithelial resistance. This decrease occurred when bacteria were added to either the apical or basolateral surfaces, indicating that the RTX toxin receptor is expressed on both surfaces. These results are discussed with regard to the applicability of the polarized T84 cell monolayers as an in vitro model of host-pathogen interactions.     

Translocation of Shiga toxin across polarized intestinal cells in tissue culture.

Escherichia coli strains producing Shiga toxins (Stx) 1 and 2 colonize the lower gastrointestinal tract in humans and are associated with gastrointestinal and systemic diseases. Stx are detectable in the feces of infected patients, and it is likely that toxin passes from the intestinal tract lumen to underlying tissues. The objective of this study was to develop an in vitro model to study the passage of Stx across intact, polarized cell monolayers. Translocation of biologically active Stx was examined in four cell lines grown on polycarbonate filters. Stx1 translocated across intestinal cell monolayers (CaCo2A and T84 cells) in an energy-requiring and saturable manner, while the monolayers maintained a high level of electrical resistance. Stx1 had no effect on electrical resistance or inulin movement across these cell lines for at least 24 h. Induction of specific Stx receptors with sodium butyrate reduced the proportion of toxin translocated across CaCo2A monolayers but had no major effect on the movement of horseradish peroxidase or [3H]inulin. We have shown that biologically active Stx1 is capable of moving across intact polarized intestinal epithelial cells without apparent cellular disruption, probably via a transcellular pathway. The data also suggest that the presence of Stx receptors on the apical surface of intestinal epithelial cells may offer some protection against the absorption of luminal Stx1.     

Entry of genital Chlamydia trachomatis into polarized human epithelial cells.

To study the initial invasion process(es) of genital chlamydiae, a model system consisting of hormonally maintained primary cultures of human endometrial gland epithelial cells (HEGEC), grown in a polarized orientation on collagen-coated filters, was utilized. After Chlamydia trachomatis inoculation of the apical surface of polarized HEGEC, chlamydiae were readily visualized, by transmission electron microscopy, in coated pits and coated vesicles. This was true for HEGEC maintained in physiologic concentrations of estrogen (proliferative phase) and of estrogen plus progesterone (secretory phase), despite the finding that association of chlamydiae with secretory-phase HEGEC is significantly reduced (P = 0.025; A.S. Maslow, C.H. Davis, J. Choong, and P.B. Wyrick, Am. J. Obstet. Gynecol. 159:1006-1014, 1988). In contrast, chlamydiae were rarely observed in the clathrin-associated structures if the HEGEC were cultured on plastic surfaces. The same pattern of coated pit versus noncoated pit entry was reproducible in HeLa cells. The quantity of coated pits associated with isolated membrane sheets derived from HeLa cells, grown on poly-L-lysine-coated cover slips in medium containing the female hormones, was not significantly different as monitored by radiolabeling studies and by laser scanning microscopy. These data suggest that culture conditions which mimic in vivo cellular organization may enhance entry into coated pits for some obligate intracellular pathogens.     

Replication and budding of simian immunodeficiency virus in polarized epithelial cells

Simian immunodeficiency virus (SIV) infection of primates provides an important model for infection of humans by HIV. Since mucosal epithelium is likely to be an important portal of entry, we decided to study aspects of the interaction of SIV with epithelial cells. SIV was shown to produce virus efficiently in polarized epithelial cells (Vero C1008) transfected with SIVmac239 proviral DNA. The virus titer in the epithelial cell culture fluid reached 10(3) TCID50/ml at day 3 posttransfection. Initially after transfected epithelial cells were plated on a permeable membrane, virus budded at both the apical and the basolateral domains. However, after the cells formed a tight monolayer, 95-100% of the virus particles budded basolaterally, as assessed by release of p27 antigen into the fluid above and below the monolayer. This finding was confirmed by electron microscopy, which showed that the mature virus budded basolaterally in polarized cells. After introduction of the CD4 gene into Vero cells by a retrovirus vector, polarizable cells were able to be infected by cell-free SIVmac239 virus. The virus titer reached 10(4) TCID50/ml in culture fluid and virions also budded basolaterally, the same as the virus from transfected cells. Two viruses (SIVmac1A11 and SIVmac251) that contain truncated TMgp28 instead of TMgp41 also budded basolaterally. Furthermore, we found that HIV-1 with full-length or truncated TMgp41 also budded basolaterally.     

Coronavirus entry and release in polarized epithelial cells: a review

Most coronaviruses cause respiratory or intestinal infections in their animal or human host. Hence, their interaction with polarized epithelial cells plays a critical role in the onset and outcome of infection. In this paper, we review the knowledge regarding the entry and release of coronaviruses, with particular emphasis on the severe acute respiratory syndrome and Middle East respiratory syndrome coronaviruses. As these viruses approach the epithelial surfaces from the apical side, it is not surprising that coronavirus cell receptors are exposed primarily on the apical domain of polarized epithelial cells. With respect to release, all possibilities appear to occur. Thus, most coronaviruses exit through the apical surface, several through the basolateral one, although the Middle East respiratory syndrome coronavirus appears to use both sides. These observations help us understand the local or systematic spread of the infection within its host as well as the spread of the virus within the host population. Copyright © 2014 John Wiley & Sons, Ltd.     

Resistance of mouse cytolytic cells to pore-forming protein-mediated cytolysis

Pore-forming protein (perforin, PFP) was isolated from a mouse large granular lymphocyte (LGL) [natural killer (NK-like)] cell line. Purified PFP lysed a variety of mouse tumor cell lines and helper T lymphocyte cell lines. However, LGL and cytotoxic T lymphocyte cell lines were resistant to PFP-mediated cell lysis. The presence of hemolytic activity in the granule was examined in these resistant cell lines. Four out of five of these resistant cell lines had hemolytically active granules. We determined whether NK cells freshly isolated from BALB/c nude mouse spleens were resistant to PFP-mediated cytolysis. Nylon column-passed spleen cells with an enriched content of NK cells exhibited more resistance than whole spleen cells. Moreover, when spleen cells were treated with PFP the remaining live cells showed enriched NK activity suggesting that normal peripheral cells with NK activity are resistant to PFP. These results indicate that cytolytic cells containing PFP have developed defense mechanisms to inhibit PEP-mediated cell lysis.     

Interaction of Neisseria meningitidis with a polarized monolayer of epithelial cells.

An important step in the pathogenesis of Neisseria meningitidis is the crossing of two cellular barriers, one in the nasopharynx and one in the brain. To approach the mechanisms by which this bacterium can achieve these goals, we studied the interactions between N. meningitidis and a monolayer of polarized tight junction-forming T84 cells grown on filter units. A capsulated, piliated, Opa-, and Opc- N. meningitidis strain is shown to be capable of adhering to and crossing this monolayer several orders of magnitude more efficiently than an isogenic nonpiliated derivative. This bacterial interaction does not affect the barrier function of tight junctions, as assessed by (i) the absence of modification of the transepithelial resistance, (ii) the lack of increase of [3H]inulin penetration across the monolayer, and (iii) the absence of delocalization of ZO-1, a tight junction protein. Electron microscopy studies and confocal examinations demonstrated that N. meningitidis (i) induces cytoskeletal rearrangements with actin polymerization beneath adherent bacteria, (ii) is intimately attached to the apical membrane of the cells, and (iii) can be internalized inside cells. Immunofluorescent staining with antipilus antibodies showed evidence that meningococcal piliation was dramatically reduced at later time points of bacterial cell interaction compared to the early phase of this interaction. In addition, adhesive bacteria recovered from an infected monolayer are piliated, capsulated, Opa-, and Opc-, a phenotype similar to that of the parental strain. Taken together, these data demonstrate that following pilus-mediated adhesion, N. meningitidis is involved in an intimate attachment which requires a bacterial component different from Opa and Opc and that meningococci cross a monolayer of tight-junction-forming epithelial cells by using a transcellular pathway rather than a paracellular route.     

Long-term cultures of polarized airway epithelial cells from patients with cystic fibrosis

The poor ability of respiratory epithelial cells to proliferate and differentiate in vitro into a pseudostratified mucociliated epithelium limits the general use of primary airway epithelial cell (AEC) cultures generated from patients with rare diseases, such as cystic fibrosis (CF). Here, we describe a procedure to amplify AEC isolated from nasal polyps and generate long-term cultures of the respiratory epithelium. AEC were seeded onto microporous permeable supports that carried on their undersurface a preformed feeder layer of primary human airway fibroblasts. The use of fibroblast feeder layers strongly stimulated the proliferation of epithelial cells, allowing the expansion of the cell pool with successive passages. AEC at increasing passage were seeded onto supports undercoated with airway fibroblasts and exposed to air. Either freshly isolated or amplified AEC could differentiate into a pseudostratified mucociliated epithelium for at least 10 mo. Thus, CF epithelia cultures showed elevated Na+ transport, drastic hyperabsorption of surface liquid, and absence of cAMP-induced Cl- secretion as compared with non-CF cultures. They were also characterized by thick apical secretion that hampered the movement of cell surface debris by cilia. However, CF respiratory epithelia did not show increased production of mucins or IL-8. The method described here is now routinely used in our laboratory to establish long-term cultures of well differentiated respiratory epithelia from human airway biopsies.     

Surface dynamics of aerolysin on the plasma membrane of living cells

Aerolysin secreted by the human pathogen Aeromonas hydrophila belongs to a group of bacterial toxins that are hemolytic and form channels in biological membranes. The toxin is secreted as an inactive precursor proaerolysin that must be proteolytically processed at its C-terminus to become active. The toxin then polymerizes into a heptameric ring that is amphipathic and can insert into a lipid bilayer and form a pore. We have examined these various steps at the surface of target cells. The toxin binds to specific receptors. Various receptors have been identified, all of which are anchored to the plasma membrane via a glycosylphosphatidyl inositol (GPI)-anchored moiety. The GPI anchor confers to the protein that is linked to it two usual properties: (i) the protein has a higher lateral mobility in a phospholipid bilayer than its transmembrane counterpart, (ii) the protein has the capacity to transiently associate with cholesterol-glycosphingolipid-rich microdomains. We have shown that both these properties of GPI-anchored proteins are exploited by proaerolysin bound to its receptor. The high lateral mobility within the phosphoglyceride region of the plasma membrane favors the encounter of the protoxin with its converting enzyme furin. The ability to associate with microdomains on the other hand favors the oligomerization process presumably by concentrating the toxin locally.     

Entamoeba histolytica interactions with polarized human intestinal Caco-2 epithelial cells.

To model the initial pathogenic effects of Entamoeba histolytica trophozoites on intestinal epithelial cells, the interactions of E. histolytica HM1-IMSS trophozoites with polarized human intestinal Caco-2 cell monolayers grown on permeabilized filters were examined. Trophozoites, when incubated with the apical surface of the monolayers at 37 degrees C, induced a rapid decrease in transepithelial resistance over 15 to 60 min. The transmonolayer resistance response was not associated with changes in short-circuit current but was associated with an increase in mannitol flux, suggesting that the drop in resistance reflected a nonselective increase in epithelial permeability rather than stimulation of electrogenic ion transport. This response preceded the earliest detection of morphologic disruption of monolayer integrity by light or electron microscopy. Apical injury to the monolayer was detected by ultrastructural studies which revealed a loss of brush border in regions of contact between epithelial cells and amebas and by chromium release assays where a small increase in the apical release of 51Cr from the monolayer (6% over background) was observed. The transmonolayer resistance response was inhibited when the temperature was reduced to 4 degrees C and by addition of cytochalasin D (1 microgram/ml) to the medium at concentrations that did not directly affect transmonolayer resistance. Application of amebic lysates or medium conditioned by coincubation of amebas with Caco-2 monolayers failed to lower transmonolayer resistance, suggesting that this effect was not mediated by soluble amebic cytotoxins. Polarized Caco-2 monolayers grown on permeable filters provide a useful model for studying the initial interactions of E. histolytica trophozoites with intestinal epithelial cells.     

Plasmid-encoded toxin of enteroaggregative Escherichia coli is internalized by epithelial cells

We have previously described a 104-kDa protein termed Pet (for plasmid-encoded toxin) secreted by some strains of enteroaggregative Escherichia coli (EAEC). Through an unknown mechanism, this toxin (i) raises transepithelial short-circuit current (Isc) and decreases the electrical resistance of rat jejunum mounted in the Ussing chamber, (ii) causes cytoskeletal alterations in HEp-2 cells and HT29/C1 cells, and (iii) is required for histopathologic effects of EAEC on human intestinal mucosa. Pet is a member of the autotransporter class of secreted proteins and together with Tsh, EspP, EspC, ShMu, and SepA proteins comprises the SPATE subfamily. Here, we show that Pet is internalized by HEp-2 cells and that internalization appears to be required for the induction of cytopathic effects. Evidence supporting Pet internalization includes the facts that (i) the effects of Pet on epithelial cells were inhibited by brefeldin A, which interferes with various steps of intracellular vesicular transport; (ii) immunoblots using anti-Pet antibodies detected Pet in the cytoplasmic fraction of intoxicated HEp-2 cells; (iii) Pet was detected inside HEp-2 cells by confocal microscopy; and (iv) a mutant in the passenger domain cleavage site, which prevents Pet release from the bacterial outer membrane, did not produce cytopathic effects on epithelial cells, whereas the release of mutant Pet from the outer membrane with trypsin yielded active toxin. We have also shown that the Pet serine protease motif is required to produce cytopathic effects but not for Pet secretion. Our results suggest an intracellular mode of action for the Pet protease and are consistent with we our recent report suggesting an intracellular mode of action for Pet.     

The Bacteroides fragilis toxin fragilysin disrupts the paracellular barrier of epithelial cells.

Bacteroides fragilis is a member of the normal colonic microflora of most mammals and is the most commonly isolated anaerobe from human clinical specimens. Some strains produce a toxin (fragilysin, a zinc-metalloproteinase) implicated as a cause of diarrheal disease in farm animals and humans. Studies in our laboratory confirm that the proteolytic activity of this toxin is responsible for the fluid secretion and tissue damage observed in vivo. In this study, we investigated the effects of fragilysin on the paracellular barrier of epithelial cells. Researchers suggest that, since the toxin rapidly intoxicates HT-29 cells, it may be internalized. However, we could not prevent cell rounding by using inhibitors of receptor-mediated endocytosis, which indicates that the toxin may act outside the cell. Based on these observations, we studied the effects of the highly purified B. fragilis fragilysin on the barrier function of cultured epithelial cells. Fragilysin rapidly increased the permeability of the paracellular barrier of epithelial cells to ions (decrease in electrical resistance across monolayers) and to larger molecules (increase in mannitol flux across monolayers). We tested a human colon cell line and cell lines from the lung and the kidney; the human colon cell line was most sensitive, but all three were affected in the same manner. Our studies show that B. fragilis fragilysin alters the barrier function of the epithelial lining, possibly by degrading the tight junction proteins, such as ZO-1. The proteolytic activity is required to cause this effect. The toxin's action has been assumed to be limited to the intestine; however, our studies show that fragilysin could also contribute to the pathogenesis of B. fragilis in extraintestinal infections.     

Shiga toxins 1 and 2 translocate differently across polarized intestinal epithelial cells

Shiga toxin-producing Escherichia coli (STEC) is an important food-borne pathogen that causes hemolytic-uremic syndrome. Following ingestion, STEC cells colonize the intestine and produce Shiga toxins (Stx), which appear to translocate across the intestinal epithelium and subsequently reach sensitive endothelial cell beds. STEC cells produce one or both of two major toxins, Stx1 and Stx2. Stx2-producing STEC is more often associated with disease for reasons as yet undetermined. In this study, we used polarized intestinal epithelial cells grown on permeable filters as a model to compare Stx1 and Stx2 movement across the intestinal epithelium. We have previously shown that biologically active Stx1 is able to translocate across cell monolayers in an energy-dependent, saturable manner. This study demonstrates that biologically active Stx2 is also capable of movement across the epithelium without affecting barrier function, but significantly less Stx2 crossed monolayers than Stx1. Chilling the monolayers to 4 degrees C reduced the amount of Stx1 and Stx2 movement by 200-fold and 20-fold respectively. Stx1 movement was clearly directional, favoring an apical-to-basolateral translocation, whereas Stx2 movement was not. Colchicine reduced Stx1, but not Stx2, translocation. Monensin reduced the translocation of both toxins, but the effect was more pronounced with Stx1. Brefeldin A had no effect on either toxin. Excess unlabeled Stx1 blocks the movement of (125)I-Stx1. Excess Stx2 failed to have any effect on Stx1 movement. Our data suggests that, despite the many common physical and biochemical properties of the two toxins, they appear to be crossing the epithelial cell barrier by different pathways.     

Expression of SV40 receptors on apical surfaces of polarized epithelial cells.

We have investigated the interaction of SV40 virions with polarized monkey kidney epithelial cells. Virions were tagged with biotin to facilitate their detection and were found to retain full infectivity. When polarized Vero C1008 cells were incubated with biotinylated virions followed by a strepavidin-rhodamine conjugate, distinct cell populations were identified which expressed very different levels of SV40 receptors. The parental Vero C1008 cells yielded three types of cell clones which exhibited low, moderate, or predominantly high levels of SV40 binding. Virus-binding assays to each of these clones as well as to parental Vero C1008 cells indicated that the level of SV40 receptor expression is cell-cycle-dependent. The cellular receptors for influenza A virus (WSN strain) were also found to be distributed heterogeneously on polarized epithelial cells. In contrast, in several types of nonpolarized cells, SV40 receptors were found to be uniformly distributed over the monolayer. SV40 binding was not found to correlate with HLA expression on Vero C1008 cells or other cell types. Also in contrast to SV40 receptor expression, which is restricted to the apical domain, HLA was found to be distributed on both apical and basolateral domains of Vero C1008 cells.     

EBV BMRF-2 facilitates cell-to-cell spread of virus within polarized oral epithelial cells

We previously reported that the Epstein-Barr virus (EBV) BMRF-2 protein plays an important role in EBV infection of polarized oral epithelial cells by interacting with β1 and αv family integrins. Here we show that infection of polarized oral epithelial cells with B27-BMRF-2^low recombinant virus, expressing a low level of BMRF-2, resulted in significantly smaller plaques compared with infection by parental B95-8 virus. BMRF-2 localized in the trans-Golgi network (TGN) and basolateral sorting vesicles and was transported to the basolateral membranes of polarized epithelial cells. Mutation of the tyrosine- and dileucine-containing basolateral sorting signal, YLLV, in the cytoplasmic domain of BMRF-2 led to the failure of its accumulation in the TGN and its basolateral transport. These data show that BMRF-2 may play an important role in promoting the spread of EBV progeny virions through lateral membranes of oral epithelial cells.     

Secretion of cytokines and chemokines by polarized human epithelial cells from the female reproductive tract

BACKGROUND: Pro-inflammatory chemokines that attract and cytokines that activate immune cells contribute to normal physiological homeostasis in the female reproductive tract, and are needed to deal effectively with potential pathogenic microbes. Mucosal epithelial cells are capable of producing these factors that communicate with cells of the innate and adaptive immune systems. METHODS: Epithelial cells from Fallopian tube, endometrium and endocervix were isolated and grown to high transepithelial resistance in cell inserts from seven patients who had hysterectomies. Interleukin (IL)-8, IL-6, granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein-1 (MCP-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor-alpha (TNF-alpha) and macrophage inflammatory peptide-1beta (MIP-1beta) were assessed by Luminex bead analysis or enzyme-linked immunosorbent assay (ELISA) in epithelial cell conditioned media from the apical and basolateral compartments. RESULTS: With the exception of MCP-1, the seven chemokines/cytokines constitutively produced by the polarized epithelial cells were preferentially secreted apically. A concentration pattern was found in all cases, with IL-8 and IL-6 produced in the greatest quantity. CONCLUSIONS: The concentrations of IL-8, IL-6, G-CSF and MCP-1 are similar to the levels found in reproductive tract fluids of patients with infection. The constitutive secretion and compartmentalization of large quantities of bioactive chemokines and cytokines provide additional evidence for the role of epithelial cells as gatekeepers of innate immune protection in the female reproductive tract.     

Detergent-resistant membrane microdomains and apical sorting of GPI-anchored proteins in polarized epithelial cells

Detergent-insoluble microdomains or rafts play a crucial role in many cellular functions: membrane traffic, cell signalling and human diseases. In this work we investigate the role of rafts in the sorting of GPI-anchored proteins in polarized epithelial cells. In contrast to MDCK cells, the majority of endogenous GPI-anchored proteins are sorted to the basolateral surface of Fischer rat thyroid cells (Zurzolo et al., J. Cell Biol. 121, 1031-1039, 1993). We analyzed a set of transfected GPI proteins in order to understand the role of the GPI anchor and of association with rafts for apical sorting. We found that the GPI moiety is necessary but not sufficient for apical sorting of GPI proteins and that the ectodomain has a major role. We propose a new model in which the stabilization of proteins into rafts, probably mediated by interactions between protein ectodomains and a putative receptor, plays a crucial role in apical sorting.     

The cytolytic toxin aerolysin must aggregate to disrupt erythrocytes, and aggregation is stimulated by human glycophorin.

The hole-forming toxin aerolysin was shown to aggregate after binding to erythrocytes at 37 degrees C. Although the protein also bound and aggregated at 4 degrees C, hole formation was not observed, indicating that aggregation preceded penetration of the lipid bilayer. Aggregation, but not binding, could be blocked by pretreatment of the toxin with diethyl pyrocarbonate, a histidine-reactive reagent. This resulted in inactivation of the toxin. Incubation of aerolysin with glycophorin purified from human erythrocytes caused aggregation and complete inactivation. Erythrocytes which lacked glycophorin were less sensitive to the toxin. Proaerolysin, the inactive precursor of aerolysin, also bound to erythrocytes; however, it did not aggregate, nor did it aggregate when preincubated with glycophorin. The protoxin could be activated by treatment with trypsin even after it had bound to erythrocytes. Activation could also be achieved by reaction of proaerolysin with a variety of other proteases, each of which brought about a similar reduction in protein molecular weight. The activated protein was resistant to further proteolysis. These results indicate that aggregation is a necessary step in hole formation and that the sites on aerolysin required for binding and for aggregation and hole formation are separate.