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Infection and Immunity, August 2002, p. 4002-4008, Vol. 70, No. 8
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.8.4002-4008.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Up-Regulation of Fas (CD95) and Induction of Apoptosis in Intestinal Epithelial Cells by Nematode-Derived Molecules

Akio Kuroda,¹ Ryuichi Uchikawa,¹ Shinji Matsuda,² Minoru Yamada,¹ Tatsuya Tegoshi,¹ and Naoki Arizono^1*

Department of Medical Zoology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kyoto 602-8566,¹ Department of Parasitology, Akita University School of Medicine, Hondo Akita 010-8543, Japan²

Received 7 January 2002/ Returned for modification 25 February 2002/ Accepted 26 April 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
Infection by the intestinal nematode Nippostrongylus brasiliensis induces acceleration of apoptosis in the small intestinal villus epithelial cells in vivo. In the present study, we examined whether worm extract or excretory-secretory product induces apoptosis in the rat intestinal epithelial cell line IEC-6 in vitro. In the presence of worm extract or excretory-secretory product (6 µg/ml), IEC-6 cell growth was significantly suppressed, and there was a concomitant increase in the number of detached cells in culture dishes. Detached cells showed nuclear fragmentation, activation of caspase-3, and specific cleavage of poly(ADP-ribose) polymerase, suggesting that apoptosis was induced in these cells. Semiquantitative reverse transcription-PCR showed that expression of Fas (CD95) mRNA was up-regulated as early as 6 h after addition of excretory-secretory product, while Fas ligand expression and p53 expression were not up-regulated. Fluorescence-activated cell sorter analyses revealed a significant increase in Fas expression and a slight increase in FasL expression in IEC-6 cells cultured in the presence of excretory-secretory product, while control IEC-6 cells expressed neither Fas or FasL. These results indicated that N. brasiliensis worms produce and secrete biologically active molecules that trigger apoptosis in intestinal epithelial cells together with up-regulation of Fas expression, although the mechanism of induction of apoptosis remains to be elucidated.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Adult worms of the nematode Nippostrongylus brasiliensis are rejected from the small intestine of immunocompetent rodent hosts at around day 14 after infection by a T-cell-dependent mechanism (23). Before or around the time of worm rejection, a variety of pathological changes occur in the intestinal mucosa; these changes include edema, eosinophil infiltration, mastocytosis, and partial villus atrophy with crypt hyperplasia (9, 13, 25). However, the alterations resolve quickly after worm rejection, along with the occurrence of goblet cell hyperplasia with mucins that have altered sugar residues (15, 19, 21). Nawa et al. (21) demonstrated that mucins are highly selective and specific effectors for N. brasiliensis worm expulsion, and McKenzie et al. (18) reported a possible link between the Th2 cytokine interleukin-13 and the production of intestinal mucus. We reported previously that apoptosis of villus epithelial cells, which occurs at villus tips during the process of normal epithelial replacement, was significantly enhanced during N. brasiliensis infection (13). Thus, enhanced apoptosis in intestinal epithelial cells might be relevant to rapid removal of damaged cells and might contribute to remodeling of intestinal epithelial cells to cells that produce specific mucins and facilitate worm expulsion.

The mechanisms of the enhancement of apoptosis after nematode infection are not yet fully understood. Villus cell apoptosis may be triggered by NK or cytotoxic T cells through Fas-Fas ligand or tumor necrosis factor-tumor necrosis factor receptor interactions (11). Alternatively, it may be triggered directly by nematode-derived molecules. N. brasiliensis excretory-secretory product (ES) contains various biologically active molecules, such as acetylcholinesterase, proteases, and a factor that suppresses gamma interferon production (2, 4, 12, 14, 20, 29). In this context, it is of interest to clarify whether N. brasiliensis ES and/or worm extract (WE) contains factors that induce epithelial cell death. In the present study, we examined the effects of N. brasiliensis WE and ES on the intestinal epithelial cell line IEC-6, which was established from rat small intestinal crypt cells (27).

Top Abstract Introduction Materials and Methods Results Discussion References

 
N. brasiliensis WE and ES. N. brasiliensis was maintained in our laboratory by serial passage in SD rats. WE and ES were prepared as described previously (30). Briefly, WE was prepared by homogenizing adult worms with phosphate-buffered saline (PBS), followed by centrifugation at 14,000 x g for 30 min, and the supernatant was stored at -80°C until it was used. For preparation of ES, adult worms were incubated in PBS (10,000 worms/15 ml) with 100 U of penicillin per ml and 100 µg of streptomycin per ml at 37°C for 24 h. The culture supernatant was collected, centrifuged at 14,000 x g for 30 min and stored at -80°C until it was used. For heat inactivation, WE or ES (700 µg/ml) was incubated at 55°C for 60 min and then at 95°C for 10 min. For protease digestion, WE or ES (700 µg/ml) was incubated with 100 µg of proteinase K (Sigma Chemical Co., St. Louis, Mo.) per ml at 55°C for 60 min and then at 95°C for 10 min.

Cell culture, cell counting, and nuclear staining. IEC-6 cells were obtained from Riken Cell Bank (Tsukuba, Japan) and were maintained in Dulbecco's modified Eagle's medium (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) supplemented with 5% (vol/vol) fetal calf serum, 5 µg of insulin per ml, 100 U of penicillin per ml, and 100 µg of streptomycin per ml at 37°C in a 5% CO₂ atmosphere. For cell growth experiments, IEC-6 cells were cultured with or without WE or ES in 96-well culture dishes. In some experiments, the protease inhibitor aprotinin, TLCK (N-p-tosyl-L-lysine chloromethyl ketone), or E-64 (Sigma) or antibodies against the amino terminus or the carboxy terminus of FasL (sc-834 and sc-6237, respectively; Santa Cruz Biotechnology Inc., Santa Cruz, Calif.) were added to the culture medium. To determine differential counts of detached and adherent cells, detached cells were first recovered from culture wells after gentle pipetting of the culture medium. Then adherent cells were harvested after treatment with trypsin and EDTA. Cell numbers were determined with a hemocytometer. For nuclear staining with Hoechst 33258 (Sigma), the dye was added to culture wells at a concentration of 20 µM and incubated for 30 min at 37°C. After recovery of detached and adherent cells, the cells were washed once with PBS and observed with a fluorescence microscope.

BrdU incorporation assay. IEC-6 cells were cultured with or without WE or ES in 96-well culture dishes for 2 or 3 days. Ten micromolar 5-bromo-2'-deoxyuridine (BrdU) (Sigma) was added to culture wells 2 h before termination of the culture. After the preparations were washed with PBS, adherent cells were harvested with trypsin and EDTA, and cells were counted. Aliquots consisting of 1 x 10⁴ cells resuspended in culture medium were plated in 96-well culture dishes and incubated at 37°C for 1 h in a 5% CO₂ atmosphere, during which time cells were fully attached to culture wells. The culture medium was discarded, and the wells were air dried. The wells were incubated with 70% ethanol for 30 min and then with 0.07 N NaOH for 15 min. The BrdU level in each well was measured with an enzyme-linked immunosorbent assay kit (Amersham Life Science Ltd., Amersham Place, United Kingdom) used according to the manufacturer's instructions. To generate a standard curve, BrdU-pulsed cells incubated without WE or ES were serially diluted and plated in 96-well culture dishes, and BrdU levels were determined as described above.

Immunoblotting. IEC-6 cells were cultured with or without WE in 25-cm² culture flasks for 24 h, and detached and adherent cells were recovered separately. Detached and adherent cells were heated at 95°C for 5 min in sodium dodecyl sulfate sample buffer containing 5% 2-mercaptoethanol. Samples were separated on a sodium dodecyl sulfate-4 to 20% polyacrylamide gradient gel and electrotransferred onto Immobilon P membranes (Millipore Corp., Bedford, Mass.). After the membranes were blocked with 5% nonfat dried milk, they were incubated with antibodies against caspase-3, caspase-8, poly(ADP-ribose) polymerase (PARP), or p53 (Santa Cruz) and then with Envision (DAKO, Carpinteria, Calif.).

Extraction of total RNA, cDNA synthesis, and PCR. IEC-6 cells were cultured with or without WE or ES in 24-well culture dishes. Total RNA was extracted with TRIZOL reagent (Life Technologies, Rockville, Md.). Five-microgram aliquots of RNA were reverse transcribed in 20 µl of reverse transcription (RT) buffer containing 5 mM MgCl₂, each deoxynucleoside triphosphate at a concentration of 1 mM, 1 U of RNase inhibitor per µl, 0.25 U of avian myeloblastosis virus reverse transcriptase per µl, and 0.125 µM oligo(dT) primer (Takara RNA LA PCR kit; Takara Biomedicals, Osaka, Japan) at 42°C for 50 min. Two-microliter aliquots of synthesized cDNA were added to PCR buffer containing 2.5 mM MgCl₂, each deoxynucleoside triphosphate at a concentration of 0.2 mM, 0.025 U of LA Taq DNA polymerase (Takara RNA LA PCR kit) per µl, 0.2 µM sense primer, and 0.2 µM antisense primer in a final volume of 25 µl. Each PCR was carried out by using cycles of 1 min at 94°C, 1 min at 62°C, and 1 min at 72°C. The following sense and antisense primers were used: 5'-AGAAGAGCTATGAGCTGCCTGACG-3' and 5'-CTTCTGCATCCTGTCAGCGATGC-3' for ß-actin (236-bp product); 5'-ATGGCCGACAAGGTCCTGAGGGCA-3' and 5'-ACTAATGTCCTGGGAAGAGGTAGA-3' for p53 (387-bp product); 5'-GCAATGCTTCTCTCTGTGACCACTG-3' and 5'-GCTGTTGTGCTCGATCTCATCG-3' for Fas (374-bp product); 5'-ATAGAGCTGTGGCTACCGGTG-3' and 5'-CTCCAGAGATCAAAGCAGTTCC-3' for Fas ligand (286-bp product); 5'-AAGTATGAGAAGCCTGGATC-3' and 5'-TCCAAGAGATGGTTGTCTGA-3' for fibronectin (570-bp product); 5'-GCCAGAAATCAAGAAAGGAA-3' and 5'-GCATTTGCATCCACATCTAG-3' for 2 laminin (354-bp product); 5'-CTCGAGGAAGCTGCTATCCA-3' and 5'-CGGGACTCACACACTACATC-3' for ß2 laminin (485-bp product); 5'-CTCGGAAGAGACAGACCTGG-3' and 5'-AAAGCAGCCTGTTGGTAGTG-3' for 1 laminin (451-bp product); 5'-CCAGTGTTTCTGCTATGGGC-3' and 5'-GTCCACGCGGTAGTCAAAAG-3' for 2 laminin (274-bp product); 5'-GAGAACGGTGGTCAAAGAGC-3' and 5'-TAATCGTAGTCCTGGTCCTG-3' for E-cadherin (384-bp product); and 5'-GACCCCAAGCCTTAGTAAAC-3' and 5'-ATGGTGGGTGCAGGAGTTTA-3' for ß-catenin (546-bp product).

Density analyses of PCR products. Aliquots (8 µl) of the amplified products were electrophoresed on agar gels and stained with ethidium bromide. The fluorescence images were saved with a charge-coupled device camera-image saver (ATTO Incorporated, Tokyo, Japan), and the density of each band was analyzed by using NIH Image. The band densities for p53, Fas, and Fas ligand were normalized relative to those for ß-actin.

Fluorescence-activated cell sorter (FACS) analysis. IEC-6 cells were harvested by incubation with 1.2 mM EDTA in PBS. Cells were washed in cold PBS containing 1% bovine serum albumin and incubated with anti-Fas or anti-FasL antibody (diluted to a concentration of 2 µg/ml; Santa Cruz) or with normal rabbit immunoglobulin G (IgG) (2 µg/ml), followed by fluorescein isothiocyanate (FITC)-conjugated antibody against rabbit IgG (diluted 1:400; Serotec Ltd., Oxford, United Kingdom). After the cells were washed, they were analyzed by FACScan (Becton Dickinson, San Jose, Calif.). Dead cells were gated out by forward and side light scattering.

Statistics. Student's t test was employed to determine statistical significance.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Suppression of IEC-6 cell growth by N. brasiliensis-derived molecules. The effects of N. brasiliensis WE or ES on growth of rat intestinal epithelial cell line IEC-6 were examined. IEC-6 cells are rapidly growing cells that have a doubling time of approximately 12 h in the log proliferation phase. Addition of WE or ES to the culture medium induced dose-dependent suppression of the increase in cell number (Fig. 1a and b). The cell-growth-suppressive effect was significantly reduced by heating WE or ES at 55°C for 60 min and then at 95°C for 10 min and was eliminated completely by similar heat treatment in the presence of proteinase K (Fig. 1c). Morphological observation of IEC-6 cells incubated with WE or ES for more than 24 h showed that not only were the cell numbers reduced, but some of the cells also had degenerative features with star-shaped shrinkage and long filamentous cytoplasmic projections, which were not found in normally growing cells (Fig. 2). To determine whether the IEC-6 cell cycle was affected in the presence of WE or ES, BrdU uptake studies were carried out. IEC-6 cells cultured for 2 or 3 days with 50 µg of WE per ml or 50 µg of ES per ml exhibited a level of BrdU uptake as high as that in cultures without WE or ES (Fig. 3), suggesting that cell cycle arrest was not induced in these cells. There were also no significant differences in the percentage of mitotic cells between cultures with WE or ES and cultures without WE or ES (data not shown). These results raised the possibility that the suppression of the increase in cell number in the presence of WE or ES was due to sporadic detachment of degenerate IEC-6 cells, while unaffected cells continued to grow.

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FIG. 1. Effects of WE and ES on the growth of IEC-6 cells. (a and b) IEC-6 cells (0.5 x 103 cells/well) were cultured in the presence of ES (a) or WE (b) at different doses in 96-well culture dishes, and adherent cell numbers were determined after 1 to 4 days. (c) IEC-6 cells (0.5 x 103 cells/well) were cultured with 50 µg of untreated WE or ES per ml, 50 µg of heated WE or ES per ml, or 50 µg of proteinase K (Pr-K)-treated WE or ES per ml, and cell numbers were determined after 72 h. The data are means ± standard deviations based on quadruplicate cultures. The results are the results of one of three independent experiments. An asterisk indicates that the P value is <0.001. CNT, control.

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FIG. 2. Morphological alterations of IEC-6 cells cultured without ES (A) or with ES (50 µg/ml) (B) for 72 h. In the presence of ES, some cells exhibited shrinkage and long filamentous cytoplasmic projections. Giemsa staining. Magnification, x100.

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FIG. 3. Effects of ES and WE on BrdU incorporation in IEC-6 cells. IEC-6 cells were cultured in the presence or absence of 50 µg of ES per ml or 50 µg of WE per ml for 2 or 3 days and pulsed with BrdU (10 µM) for 2 h before cell harvest. Aliquots (1 x 104 cells) were replated in 96-well plates and fixed, and BrdU levels were determined by an enzyme-linked immunosorbent assay. The data are means + standard deviations based on four wells. The results are the results of one of three independent experiments. C, control; OD450 nm, optical density at 450 nm.

Enhanced desquamation and apoptosis of IEC-6 cells by N. brasiliensis-derived molecules. To determine whether cellular detachment from culture dishes was enhanced in the presence of WE or ES, semiconfluent IEC-6 cells were incubated with WE or ES for 6 or 16 h, and detached and adherent cells were counted. After 6 h, there were no morphological alterations, and the number of detached cells was less than 0.1% of the total number of cells in either the absence or the presence of WE or ES. After 16 h, however, the numbers of detached cells were significantly increased in cultures with 50 µg of WE per ml or 50 µg of ES per ml (Fig. 4a). Hoechst 33258 nuclear staining showed that the majority of detached cells exhibited nuclear fragmentation, while adherent cells did not (Fig. 4b). It has been reported that most types of apoptotic cells show activation of caspase-3 with generation of active caspase-3 subunits p17 and p12. Activated caspase-3 then induces proteolytic cleavage of caspase-3-specific substrates, such as laminins, PARP, and ß-catenin (7, 11, 22). To examine whether caspase-3 activation occurred in cells cultured with WE, we first analyzed the total cells, including both adherent and detached cells. Caspase-3 immunoblotting, however, revealed only the 32-kDa proenzyme (data not shown). As detached cells comprised only 1 to 2% of the total cells even in the presence of WE, it might have been difficult to detect the cleaved form of caspase-3 by immunoblotting. Then we collected detached cells and adherent cells separately from 25-cm² culture flasks, and each cell lysate was subjected to immunoblotting analyses. Immunoblotting of adherent cell lysate revealed uncleaved 32-kDa procaspase-3, while detached cells from cultures incubated in the absence or presence of WE contained a 17-kDa molecule but no procaspase-3, suggesting that caspase-3 activation occurred in detached cells (Fig. 5). PARP immunoblotting analyses of adherent cell lysate revealed a 116-kDa molecule, while detached cell lysate contained an 85-kDa apoptosis-related cleavage fragment (Fig. 5). These results indicated that detached cells, but not adherent cells, exhibited apoptosis accompanied by activation of caspase-3 and cleavage of PARP. Detached cells that occurred spontaneously in control cultures and detached cells that appeared in the presence of WE showed the same caspase-3 and PARP cleavage patterns. This implied that detached cells underwent the same molecular apoptotic events regardless of whether detachment was induced naturally or triggered by a worm product.

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FIG. 4. Short-term effects of WE and ES on IEC-6 cells. (a) Semiconfluent IEC-6 cells were incubated with WE or ES at two doses (10 and 50 µg/ml) for 16 h. Numbers of adherent and detached cells were counted separately as described in Materials and Methods. The percentage of detached cells was calculated as follows: 100 x (number of detached cells)/(number of adherent cells + number of detached cells). The data are averages and standard errors for quadruplicate cultures. Asterisks indicate that values are significantly different from the control culture value (one asterisk, P < 0.05; two asterisks, P < 0.01). (b) Typical apoptotic nuclei in detached cells stained with Hoechst 33258. Magnification, x100.

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FIG. 5. Immunoblot analyses of PARP and caspase-3 in adherent and detached cells. IEC-6 cells were cultured without (-) or with (+) 50 µg of WE per ml for 24 h. Cell lysates of adherent and detached cells were prepared, and the same amounts of protein were loaded and electrophoresed on a 4 to 20% polyacrylamide gradient gel and then transferred onto a nitrocellulose membrane. Immunodetection was carried out with antibodies against PARP and caspase-3.

Up-regulation of Fas expression in IEC-6 cells by N. brasiliensis-derived molecules. As apoptosis was induced in IEC-6 cells in culture medium containing WE or ES, expression of apoptosis-related molecules (p53, Fas, and Fas ligand) in adherent IEC-6 cells was examined by RT-PCR. To determine the optimal numbers of PCR cycles, the densities of electrophoresed PCR products of control IEC-6 cells were analyzed after different numbers of PCR cycles (Fig. 6a). Fixed numbers of PCR cycles that allowed comparison of levels of gene expression were determined and employed in subsequent studies; 24, 30, 30, and 20 cycles were used for p53, Fas, Fas ligand, and ß-actin, respectively. For comparison of gene expression in cells cultured with WE or ES and gene expression in cells cultured without WE or ES, the densities of PCR products of p53, Fas, and Fas ligand were normalized to the density of the PCR product of ß-actin, and the levels of expression were expressed relative to the level for untreated cells, determined as follows: level of expression = (value normalized to value for ß-actin in cells cultured with WE or ES)/(value normalized to the value for ß-actin in untreated cells). As shown in Fig. 6b and c, IEC-6 cells cultured in the presence of 50 µg of ES per ml for 2 days showed significant up-regulation of Fas expression, whereas Fas ligand expression and p53 expression were unaltered compared to expression in control cultures. The effect of WE on Fas expression was significantly less than that of ES. The effect of ES on Fas expression was examined at different ES doses. Up-regulation of Fas was observed in cells cultured with ES at concentrations as low as 6 µg/ml (Fig. 6d). Time course studies showed that up-regulation of Fas occurred as early as 6 h after addition of ES to culture medium (Fig. 6e). The effect of ES on Fas expression was eliminated by preheating or proteinase K treatment of ES (Fig. 7).

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FIG. 6. Effects of WE or ES on Fas, FasL, and p53 expression in IEC-6 cells. Total RNA isolated from IEC-6 cells was subjected to semiquantitative RT-PCR analyses. (a) To determine appropriate numbers of PCR cycles, the optical densities of the PCR product were measured after different numbers of cycles. Act, ß-actin. (b) IEC-6 cells were cultured with or without WE or ES (50 µg/ml) for 48 h, and Fas expression, FasL expression, and p53 expression were analyzed. ß-Actin was used as an internal control (CNT). (c) IEC-6 cells were cultured under the same conditions as described above for panel b. The densities of each PCR product were determined with an image analyzer and standardized by using the levels of ß-actin. The data are the means + standard errors based on four independent experiments and show expression levels relative to those in control cultures. An asterisk indicates that the level was significantly different from the control level (P < 0.01). (d) IEC-6 cells were cultured with different doses of ES for 48 h, and Fas expression was determined by RT-PCR. (e) IEC-6 cells were cultured with 50 µg of ES per ml, and Fas expression was determined by RT-PCR after different incubation times.

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FIG. 7. Effects of heat and proteinase K digestion on ES-induced up-regulation of Fas expression. IEC-6 cells were cultured for 48 h without ES (C1), with heat-inactivated proteinase K (C2), with 50 µg of nontreated ES per ml, with ES after heat treatment, or with ES after proteinase K treatment, and Fas expression was determined by RT-PCR. The data are means + standard errors based on four experiments and show expression levels relative to those of IEC-6 cells cultured for 48 h without ES. Asterisks indicate that the levels are significantly different from control levels (one asterisk, P < 0.05; two asterisks, P < 0.01).

To determine whether Fas protein was expressed on IEC-6 cells, FACS analyses were performed. IEC-6 cells cultured in the presence of 50 µg of ES per ml for 2 days exhibited a significant increase in Fas expression, while control IEC-6 cells showed no Fas expression (Fig. 8). ES was also shown to induce a slight increase in FasL expression on IEC-6 cells as determined by FACS analyses, although RT-PCR indicated no up-regulation of FasL (Fig. 8). Immunoblot analyses showed that p53 did not accumulate in IEC-6 cells in either the presence or the absence of ES (data not shown).

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FIG. 8. FACS analyses of Fas and FasL expression on IEC-6 cells. IEC-6 cells were cultured without ES (fine lines) or with 50 µg of ES per ml (thick lines) for 48 h, and harvested cells were incubated with anti-Fas or FasL antibody and then with FITC-conjugated goat anti-rabbit IgG. Shaded area, IEC-6 cells cultured with 50 µg of ES per ml, incubated with normal rabbit serum and then with FITC-conjugated goat anti-rabbit IgG, and subjected to FACS analyses.

To determine whether Fas and FasL molecules are involved in WE- or ES-induced apoptosis or cell growth suppression, IEC-6 cells were cultured in the presence of anti-FasL antibodies. However, WE- or ES-induced cell growth suppression was not inhibited by the presence of anti-FasL antibodies (Fig. 9). In addition, caspase-8 immunoblotting did not reveal the cleaved form of caspase-8 in either adherent or detached cells induced by WE or ES (data not shown). Next, to determine whether nematode-related molecules affect cellular adhesion of IEC-6 cells, adhesion molecule mRNA expression was examined by RT-PCR. There were no significant changes in expression of fibronectin, 2, ß2, and 1 laminins, E-cadherin, and ß-catenin in IEC-6 cells cultured in the presence or absence of 50 µg of ES per ml or 50 µg of WE per ml for 1 or 3 days (Fig. 10a and b). Expression of 2 laminin was negligible. As cellular detachment might be due to nematode-derived proteases, IEC-6 cells were cultured with ES or WE in the presence of proterase inhibitors. Addition of aprotinin, TLCK, or E-64 did not affect the suppressive effects of ES or WE on cell growth (Fig. 10c).

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FIG. 9. Effects of anti-FasL antibodies on WE-induced cell growth inhibition. IEC-6 cells were cultured with WE (50 µg/ml) in the presence of IgG antibodies against the amino terminus of FasL (N), the carboxy terminus of FasL (C), or normal rabbit IgG (CNT) at different doses, and adherent cell numbers were determined after 72 h. The percentage of inhibition was determined as follows: [1 - (cell numbers in cultures with WE/cell numbers in cultures without WE)] x 100. The data are means + standard deviations based on quadruplicate cultures.

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FIG. 10. Effects of WE or ES on adherent molecules in IEC-6 cells. (a) IEC-6 cells were cultured with or without WE (50 µg/ml) for 24 h, and ß-actin expression (lanes 1 and 2), fibronectin expression (lanes 3 and 4), 2 laminin expression (lanes 5 and 6), ß2 laminin expression (lanes 7 and 8), 1 laminin expression (lanes 9 and 10), and 2 laminin expression (lanes 11 and 12) were examined by RT-PCR (28 PCR cycles for all experiments except the ß-actin experiment, in which 20 cycles were used). C, control; W, WE. (b) IEC-6 cells were cultured with or without WE or ES (50 µg/ml) for 72 h, and ß-actin expression (lanes 1 to 3), E-cadherin expression (lanes 4 to 6), and ß-catenin expression (lanes 7 to 9) were examined by RT-PCR (28 PCR cycles for all experiments except the ß-actin experiment, in which 20 cycles were used). C, control; W, WE; E, ES. (c) IEC-6 cells were cultured with WE or ES (50 µg/ml) in the presence or absence of 100 µg aprotinin per ml, 10 µM TLCK, or 10 µM E-64, and adherent cell numbers were determined after 72 h. The percentage of inhibition was determined as follows: [1 - (cell numbers in cultures with WE or ES/cell numbers in cultures without WE or ES)] x 100. The data are means + standard deviations based on quadruplicate cultures. Non, no aprotinin, TLCK, or E-64 added.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study showed that IEC-6 cell growth was suppressed in a dose-dependent fashion in culture medium containing N. brasiliensis-derived molecules. There was no parallel suppression of BrdU uptake in IEC-6 cells, suggesting that IEC-6 cells continued to divide in medium containing even high concentrations of nematode-derived molecules. On the other hand, differential counts of adherent and detached cells indicated that the numbers of detached cells increased significantly in the presence of nematode-derived molecules. Thus, it seemed that the growth inhibition in the presence of nematode-derived molecules was a result of increased levels of cell loss due to cellular detachment.

The detached cells exhibited nuclear fragmentation, activation of caspase-3, and specific cleavage of PARP, indicating that apoptosis occurred in these cells. There are several possible mechanisms for initiation of apoptosis, including forced cellular detachment, accumulation of p53, and death ligand-receptor interactions. In vitro studies have shown that apoptosis occurs in epithelial or endothelial cells when these cells are experimentally displaced from the extracellular matrix (3, 10, 28). The mechanism responsible for this detachment-induced apoptosis remains unclear, but it has been ascribed to the loss of integrin-mediated survival signals derived from the extracellular matrix (3, 10, 28). Thus, it is possible that nematode-derived molecules specifically affected production of adherent molecules in IEC-6 cells and that loss of cellular anchorage resulted in detachment-induced caspase-3 activation and apoptosis. However, we could not detect significant alterations in levels of expression of genes encoding cellular adherence molecules, such as fibronectin, 2, ß2, 1, and 2 laminins, E-cadherin, and ß-catenin. Furthermore, protease inhibitors could not eliminate the cell growth inhibition induced by nematode-derived molecules, although it is possible that certain nematode-derived proteases which could not be efficiently inhibited by the protease inhibitors used in this study are responsible for the induction of cellular detachment. The nuclear phosphoprotein p53, which accumulates under a variety of cellular stress conditions, induces growth arrest or apoptosis (1, 26). The present results, however, showed neither up-regulation of p53 mRNA nor accumulation of p53 protein in IEC-6 cells. On the other hand, we found up-regulation of Fas mRNA expression in IEC-6 cells cultured with nematode-derived molecules. The levels of Fas mRNA expression in the presence of nematode-derived molecules were dose dependent and increased as early as 6 h after ES administration. FACS analyses also showed that there was a significant increase in Fas protein expression in IEC-6 cells. In contrast, FACS analyses of FasL on IEC-6 cells revealed that there was only a slight increase. Thus, up-regulation of Fas might not have been relevant to the induction of IEC-6 cell apoptosis. In fact, caspase-8 activation was not found in either adherent or detached cells in cultures with or without nematode-derived molecules, and anti-FasL antibodies did not eliminate the cell growth inhibition induced by nematode-derived molecules. Thus, up-regulation of Fas might be an epiphenomenon, occurring independent of the induction of apoptosis.

Cryptosporidium parvum induces apoptosis in intestinal or biliary epithelial cells in vitro (5, 8, 17, 24). It has been reported that C. parvum stimulated FasL membrane surface translocation, increased both FasL and Fas protein expression in infected biliary epithelia, and induced a marked increase in the level of soluble FasL, suggesting that C. parvum induces apoptosis in biliary epithelia by a Fas-FasL-dependent mechanism involving both autocrine and paracrine pathways (5). On the other hand, C. parvum infection attenuated epithelial apoptosis induced by proapoptotic agents (17). However, our preliminary experiments showed that the presence of ES or WE did not accelerate or suppress actinomycin D- or cycloheximide-induced apoptosis in IEC-6 cells.

A factor(s) that is responsible for induction of growth inhibition and/or up-regulation of Fas was not identified in the present study. The effects of ES on the induction of apoptosis and up-regulation of Fas were eliminated by preheating or proteinase digestion of ES, suggesting that the factor(s) is not endotoxin-like. Relatively large doses of WE or ES (6 µg/ml) were required for growth inhibition or up-regulation of Fas. These results raise the question of whether nematode-derived molecules have an active role in the induction of apoptosis in vivo. However, ES is produced at levels of >200 µg per day by 1,000 adult N. brasiliensis worms in vitro (30). In addition, in an experimental infection with 2,000 N. brasiliensis L3 larvae in rats, more than 1,000 adult worms parasitized a narrow region of the jejunum, usually 15 to 35 cm from the pyloric ring, suggesting that the local ES concentration might increase to a level that can affect epithelial cells. In fact, immunoelectron microscopic studies have revealed high levels of ES immunoreactivity in the cytoplasmic vacuoles and the intercellular spaces of villus epithelial cells in rats parasitized with N. brasiliensis (unpublished data).

The effects of parasite-derived molecules on lymphocytes have been reported previously (6, 16). Hookworm secretions had proapoptotic effects on activated human T cells but not on resting peripheral blood lymphocytes (6), and Schistosoma soluble egg antigens induced splenic and granuloma CD4⁺ T-cell apoptosis and stimulated expression of FasL on splenic but not granuloma CD4⁺ T cells, CD8⁺ T cells, and CD19⁺ B cells (16). These results suggested that parasite-derived molecules affect not only local epithelial cells but also lymphocytes and modulate the evolution of pathology and immune responses.

Taken together, the results of the present study showed that the intestinal nematode N. brasiliensis produces molecules that affect intestinal epithelial cells and induce up-regulation of Fas, although up-regulation of Fas in epithelial cells might not be involved in induction of apoptosis in the in vitro system described here. The role of the nematode-derived molecules in vivo should be clarified in future studies.

 
This study was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and the Toray Research Institute.

 
* Corresponding author. Mailing address: Department of Medical Zoology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kyoto 602-8566, Japan. Phone: 81-75-251-5325. Fax: 81-75-251-5328. E-mail: arizonon{at}basic.kpu-m.ac.jp.

Editor: W. A. Petri, Jr.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Infection and Immunity, August 2002, p. 4002-4008, Vol. 70, No. 8
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.8.4002-4008.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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