TEXT

Top Abstract Text References

Cholera is traditionally considered to be a noninflammatory diarrheal disease caused by Vibrio cholerae serogroups O1 and O139 (10). Several groups of investigators have constructed a wide range of live attenuated V. cholerae O1 and O139 strains by deleting the CTX genes in order to immunize against cholera (2, 5, 9, 11, 16, 19). However, many of these strains have shown residual adverse properties (reactogenicity) in volunteer studies (1, 12, 22, 23). The cellular basis of reactogenicity is not clear, but there is some evidence of an intestinal inflammatory response. It was postulated that close proximity or contact of bacterial cells to the apical surface of the intestinal epithelium causes reactogenicity, due to the induction of a local inflammatory response; this theory was based on the lower reactogenicity observed for nonmotile mutants of V. cholerae compared with parental motile vaccine strains in volunteer studies (15). Furthermore, oral vaccination with CVD110, a reactogenic strain, produced copious amounts of lactoferrin and increased interleukin-8 (IL-8) levels in the stool of volunteers, while a nonreactogenic strain (CVD103HgR) did not (21). The proinflammatory cytokine IL-8 is a potent chemoattractant for polymorphonuclear leukocytes and T lymphocytes (17). Polymorphonuclear leukocytes, which are found in large numbers in inflammatory diarrheas (6, 20), can induce chloride secretion similar to that seen in toxigenic secretory diarrhea illnesses (14) and could play an important role in the diarrhea seen with live attenuated cholera vaccine strains. On the other hand, epithelial cells infected with several invasive and some noninvasive enteric pathogens produced an IL-8 response (8). However, the induction of proinflammatory signals by the attachment of V. cholerae strains to intestinal epithelial cell lines has not been investigated. Here, we examined the impact of attachment of reactogenic and nonreactogenic live cholera vaccine strains to human intestinal epithelial cells on the IL-8 response by using the undifferentiated HT29-18N2 cell line (7), a clone derived from the HT29 human adenocarcinoma cell line. This clone, when maintained in high-glucose Dulbecco's modified medium supplemented with fetal serum, does not produce mucus and mainly remains in the form of columnar enterocytes (18).

Strains, cell cultures, and infection protocol. The V. cholerae strains used in this work are summarized in Table 1 and were grown as semiconfluent streaks on Luria-Bertani agar. The HT29-18N2 cell line was provided by Tom E. Phillips (Department of Molecular Microbiology and Immunology, University of Missouri, Columbia) and was maintained in a high-glucose Dulbecco's modified medium supplemented with 10% fetal calf serum, penicillin-streptomycin (10 U and 10 µg per ml), and amphotericin B (0.5 µg/ml), hereafter referred to as complete medium. The cells were incubated at 37°C in complete medium in an atmosphere of 5% CO₂. For all experiments, undifferentiated HT29-18N2 cells were used to avoid mucus interference with adherence for all strains assayed. Attachment of V. cholerae vaccine strains to the intestinal epithelial cell line was essentially determined as described previously (3). For IL-8 mRNA and gene product detection, HT29-18N2 cells were seeded at 2 × 10⁴ to 3 × 10⁴ cells/cm² onto 25-mm-diameter coverglasses in six-well tissue culture plates in complete medium and incubated at 37°C in 5% CO₂ until confluent. Twenty-four hours before stimulation, the cell cultures were washed and maintained in fresh complete medium without fetal calf serum. Duplicate coverglasses were exposed to 10⁶ to 10⁷ CFU of each cholera vaccine strain for 30 min, washed three times, transferred to fresh Dulbecco's medium, and incubated for up to 8 h. In dose-dependency experiments, the V. cholerae inoculum size ranged from 10⁴ to 10⁷ CFU/well and RNA extractions were performed 4 h postinfection.

RNA extraction, RT-PCR analysis, and cytokine assay. Total cellular RNA was extracted 0.5, 2, 4, and 8 h after challenge of the epithelial cells with the different V. cholerae vaccine strains by using the acid guanidinium thiocyanate-phenol-chloroform method (4). First-strand cDNA synthesis was performed by using 1.5 µg of each purified RNA primed with 2 pmol of oligo(dT) primer in a 20-µl reaction volume. Reverse transcriptions (RTs) were made by using the Reverse Transcription system (Promega, Madison, Wis.). Qualitative PCR analysis was performed with 1 µl of each cDNA in a 50-µl reaction volume containing 0.2 mM deoxynucleoside triphosphate, 2 mM Mg²⁺, 0.5 µM concentrations of each sense and antisense primer, and 1.25 U of Taq DNA polymerase. All reagents for PCR amplification were purchased from Boehringer (Mannheim, Germany). The PCR profile included a 94°C denaturalization for 5 min, followed by 30 cycles of denaturalization at 94°C for 1 min, annealing at 60°C for 1 min, extension at 72°C for 2 min, and a final extension at 72°C for 10 min. The primers used and the molecular sizes of the corresponding products were as follows: -actin sense (5' GTG GGG CGC CCC AGG CAC CA 3') and antisense (5' CTC CTT AAT GTC ACG CAC GAT TTC 3') (548 bp); IL-8 sense (5' ATG ACT TCC AAG CTG GCC GTG 3') and antisense (5' TTA TGA ATT CTC AGC CCT CTT CAA AAA CTT CTC 3') (302 bp). The IL-8 concentration was determined at 0.5, 2, 4, and 8 h postinfection in culture supernatants of undifferentiated HT29-18N2 cells stimulated with reactogenic and nonreactogenic cholera vaccine strains by the Quantikine enzyme-linked immunosorbent assay (R & D Systems, Minneapolis, Minn.), with a detection limit of less than 10 pg/ml. Both the RT-PCR analysis and cytokine assay used unstimulated epithelial cell cultures as controls. Tumor necrosis factor alpha (TNF-)-treated epithelial cells were used as positive controls for IL-8 stimulation.

View larger version (45K): [in this window] [in a new window]   FIG. 1.   Time course of IL-8 mRNA induction in undifferentiated HT29-18N2 colonic epithelial cells by reactogenic and nonreactogenic cholera vaccine strains. Data shown are from a representative gel electrophoresis of three independent RT-PCR amplification products of -actin and IL-8 mRNAs from undifferentiated HT29-18N2 epithelial cells, after stimulation with four V. cholerae vaccine strains at various time intervals from 0 to 8 h. Numbers under each panel represent densitometry values and are expressed in arbitrary units. Sizes are indicated in base pairs (bp).

View larger version (23K): [in this window] [in a new window]   FIG. 2.   Dose-dependency experiments for IL-8 mRNA induction in undifferentiated HT29-18N2 colonic epithelial cells by reactogenic and nonreactogenic cholera vaccine strains. Data shown are from a representative gel electrophoresis of three independent RT-PCR amplification products of -actin and IL-8 mRNAs from undifferentiated HT29-18N2 epithelial cells, after 4 h of stimulation with two V. cholerae vaccine strains; inoculum size ranged from 104 to 107 CFU/well. Numbers under each panel represent densitometry values and are expressed as arbitrary units. Sizes are indicated in base pairs (bp).