3456 K. Yamada et al. Eur. J. Immunol. 1997.27: 3456-3460

Bacterial invasion induces interleu kin-7 receptor expression in colonic epithelial cell line, T84

Keiko Yamada', Motomu Sh ima~ka '~ '~~ , Kenichi Nagayamal, Takachika Hiroi', Hiroshi Kiyono' and Takeshi Honda'

Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan

University, Osaka, Japan Intensive Care Unit, Osaka University Hospital, Osaka, Japan

* Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka

The intestinal epithelial layer forms the interface between the external and the internal envi- ronments of a host. Since the interleukin-7 (IL-7) and IL-7 receptor (IL-7R) signaling pathway has been shown to play an important role in the mucosal immune system, we studied the expression of IL-7R in T84, a colonic epithelial cell line, after cells were infected with several types of enteropathogenic bacteria, including Salmonella typhimurium, enteropathogenic Escherichia coli and enteroinvasive Escherichia coli. Bacterial invasion induced IL-7R expression in T84 assessed by a semi-quantitative reverse transcription-polymerase chain reaction technique and flow cytometry. The inhibition of bacterial invasion by cytochalasin D, a specific inhibitor of actin polymerization, led to a reduction in the expression of IL-7R. These data indicate that bacterial invasion into intestinal epithelial cells is likely to be an essential process in the induction of IL-7R. The communication between the epithelium and

revised Sept. 9, 1997:

mucosal lymphocytes which is mediated via IL-7 and IL-7R may be involved in the modula- tion of the mucosal inflammation which occurs in bacterial infection.

Key words: Interleukin-7 I Interleukin-7 receptor I Bacterial invasion I Epithelial cell

1 Introduction

The intestinal mucosa is continuously exposed to a variety of foreign and environmental antigens. The intes- tinal epithelial cells function not only as a site for diges- tion and absorption but also as a physiological and immunological barrier against ingested pathogens, including bacteria and viruses. Accumulated evidence suggests that the intestinal epithelial cells can induce and regulate the immune response, as well as initiate and modulate inflammatory processes in the mucosal immune system. When human intestinal epithelial cell lines are infected with bacteria in vitro, these cells express proinflammatory cytokines [l-31, which sug- gests that the intestinal epithelial cells play a pivotal role in chemotaxis and the activation of inflammatory re- sponses to bacterial infection in the gastrointestinal tract.

[I 169281

Abbreviations: IEL lntraepithelial lymphocyte RT: Reverse transcription EIEC: Enteroinvasive Escherichia coli EPEC: Enteropathogenic Escherichia coli

I accepted &pt. 19, 1997. I

Although IL-7 was originally discovered as a pre-B cell growth factor [4, 51, it is increasingly known that IL-7 is also a mediator for the development of T cells [6, 71. IL-7 that is produced by human and murine intestinal epithelial cells regulates the proliferation of mucosal lym- phocytes, including y 6 T cells, which constitutively express IL-7R [6, 81. IL-7 also enhances cytolytic T lym- phocyte activity and induces lymphokine-activated killer cells [9]. Considering the important role of y6 T cells in the regulation of mucosal immunity [lo], interaction between IL-7 and IL-7R must have a significant influence on the mucosal immune response. In the present study, to assess the possible effects of invasive bacterial infec- tion on the IL-7AL-7R system, intestinal epithelial cells were infected with different enteropathogenic bacteria. The changes in expression of this cytokine and its recep- tor were examined through the use of the semi- quantitative reverse transcription-polymerase chain reaction (RT-PCR) technique, enzyme-linked immuno- sorbent assays (ELISA) and flow cytometry (FCM) analy- ses.

001 4-298019711 21 2-3456$17.50 + ,5010 0 WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1997

Eur. J. Immunol. 1997.27: 3456-3460 Bacterial invasion induced IL-7R in T84 3457

2 Results

2.1 Bacterial invasion of epithelial cells

Of the bacteria tested, S. typhimurium were recovered in the highest numbers from the infected T84 cells (3.9 f 0.81 %)followed by ElEC (1.2 * 0.38 %) and EPEC (0.31 f 0.037 %). In contrast, non-pathogenic E. coli (HB101) was not recovered from the lysed T84. Cytocha- lasin D inhibited bacterial invasions by S. typhimurium (0.25 * 0.031 %), ElEC (0.061 f 0.032 %) and EPEC (0.020 f 0.008 %).

2.2 Induction of IL-7R mRNA expression in epithelial cells by bacterial invasion

After infections with S. typhimurium, ElEC and EPEC, the levels of IL-7R-specific mRNA expression were

Figure 1 . Induction of IL-7R mRNA by T84 following bacte- rial invasion. RT-PCR amplified cDNA were examined for p- actin (548 bp), 11-7 (681 bp), IL-7R (362 bp), and IL-8 (288 bp) in T84 infected for 2 h with S. typhimurium, ElEC or EPEC (A). The effect of cytochalasin D on the expressions of IL-8 and IL-7R mRNA in infected T84 cells (B). Samples col- lected as a control were not exposed to bacteria. RT-PCR

Medium Sal ElEC EPEC TNFa ~ Medium Sal ElEC EPEC TNFa

Figure 2. Relative levels of IL-7R and IL-8 mRNA expression by T84 infected with S. fyphimurium, EIEC, EPEC or stimul- ated with TNF-a. The amounts of PCR products were mea- sured by capillary electrophoresis, and the concentrations of cytokine-specific molecules and p-actin were determined by the analysis and comparison of individual peak areas. Black and white columns show results in the absence or the pres- ence of cytochalasin D (10 ng/ml), respectively. Values represent the mean * SE of three different assays. *p < 0.05.

increased in T84 cells (Fig. IA). Furthermore, the extent of expression of IL-7R-specific mRNA correlated well with the invasion rate of each bacteria into T84. As a control, the expression of IL-8 was also examined, since the level of production of this cytokine has been shown to be influenced by bacterial invasion of epithelial cells [2]. The expression of IL-8 mRNA was also enhanced after bacterial infection (Fig. 1A). On the other hand, when T84 was co-cultured with non-pathogenic E. coli HB101, the level of IL-7R mRNA hardly changed, and IL-8 mRNA was not up-regulated (data not shown). When cytochalasin D was added, the increased expre- ssions of IL-7R mRNA were blocked in proportion to the rate of inhibition of bacterial invasion to T84 (Fig. 1 B). To evaluate the relative changes of mRNA expression fol- lowing bacterial invasion, the amounts of PCR products were measured by capillary electrophoresis. The levels of IL-7R mRNA were increased by bacterial infection (Fig. 2). Although TNF-a (200 ng/ml) also induced expression of IL-7R and IL-8 mRNA in T84 (17 h culture), this cytokine-induced IL-7R and IL-8 mRNA was not influenced by the addition of cytochalasin D (Fig. 2).

2.3 Bacterial infection induces cytokine production by T84 cells

IL-8 secretions were enhanced subsequent to bacterial infection and the amount of IL-8 secretions induced with S. typhimurium (1 287 * 26 pg/ml) and EPEC (797 f 51 pg/ml) was significantly larger than with non-

was carried out as described in Sect. 4.4. pathogenic E. coli HB101 (50 f 12 pg/ml). These find-

3458 K. Yamada et al. Eur. J. Immunol. 1997.27: 3456-3460

Figure 3. FCM analyses of IL-7R expression by T84 infected with S. typhimurium. In the experiments shown in (A) and (B), mouse mAb anti-human IL-7R was used, while biotiny- lated rhlL-7 was used for (C) and (D). In (B) and (D), the effect of cytochalasin D on the induction of IL-7R in the infected T84 was examined. The dotted and solid lines represent uninfected and S. typhimurium-infected T84, respectively. FCM was carried out as described in Sect. 4.6.

ings are consistent with previous results [2]. The IL-8 secretions induced by the infection with S. typhimurium were not inhibited by cytochalasin D treatment (1494 f 20 pg/ml). Detectable levels of IL-7 were not induced subsequent to bacterial invasion (data not shown). Although no remarkable change was detected, the expression of IL-7 mRNA had a tendency to be elevated during the first hour following the infection, and was decreased thereafter (data not shown).

2.4 IL-7R on the cell surfce of infected T84 cells

FCM analysis using anti-human IL-7R mAb revealed induction of IL-7R on the surface of T84 infected with S. typhimurium compared with uninfected T84 cells (Fig. 3A). These results were confirmed by analysis using biotinylated rhlL-7 although the intensity of positive cells was lower in comparison to the data obtained by using IL-7R mAb (Fig. 3C). Induction of IL-7R was similarly

detected in T84 infected with EIEC and EPEC. Further- more, when cytochalasin D was added, the increased expression of IL-7R was inhibited (Fig. 3B and D).

3 Discussion

In the present study, we have demonstrated that the expression of IL-7R in both levels of mRNA and protein was enhanced in T84 cells infected with various types of invasive bacteria. IL-7R induced by the bacterial invasion of epithelial cells was the biologically active form since it could bind to rhlL-7. These findings suggest that bacte- rial invasion can exert some influence on the IL-7AL-7R signaling pathway in intestinal epithelial cells.

Recent investigations have suggested that the IL-71 IL-7R signaling pathway plays an important role in the induction and regulation of the mucosal immune system. IL--/-deficient mice are highly lymphopenic [ l 11 and IL-7R-deficient mice lack y6 T cells [12, 131. IL-7 trans- genic mice have been shown to develop progressive cutaneous disorder [14] and severe dermatitis [15] due to y6 T cell infiltration. Our present study provides addi- tional evidence that epithelial cells express IL-7R, which was up-regulated subsequent to bacterial invasion. It is possible that bacterial invasion may stimulate the signal- ing pathways for both IL-7 and IL-7R. However, the acti- vation of signal transduction for 11-7 may occur before the expression of the corresponding receptor. With regard to this, our results showed an elevation of IL-7- specific mRNA during the first hour after infection, while the level of IL-7R specific mRNA was increased after 2 h. The early expression of IL-7 may promote the activation of neighboring mucosal T cells, which in turn leads to the production of regulatory Thl and Th2 type cytokines (e.g. IFN-y, IL-2, IL-4 and IL-6) [16, 1 7 that affect epi- thelial cells and lamina propria lymphocytes. Alterna- tively, IL-7 produced by epithelial cells following bacterial infection may immediately bind to corresponding recep- tors which are expressed on the surface of the expre- ssing cell in an autocrine mechanism. This may explain why, although secreted IL-7 was not able to find in the culture supernatants of the infected T84, IL-7-specific mRNA was detected in the total RNA preparation obtained from an aliquot of the infected cells by RT-PCR. Thus, the IL-7AL-7R interactions may initiate and modul- ate the inflammatory response to bacterial infection.

As bacterial invasion of the epithelial cells is a crucial step in the destruction of the mucosal barrier, the means of bacterial entry into naturally nonphagocytic cells have recently been the focus of much attention [18]. Bacterial entry is thought to be triggered by complex interactions between microbial factors and cell surface receptors and

Eur. J. Immunol. 1997.27: 3456-3460 Bacterial invasion induced IL-7R in T84 3459

achieved by endocytosis [18]. Our results demonstrate that the extent of IL-7R mRNA expression correlated well with the rate of invasion of bacteria into T84. Also, cyto- chalasin D attenuated both the bacterial invasion and IL- 7R expression. Thus, bacterial invasion may be an essential process for the induction of IL-7R in T84. Recent studies have suggested that these complex inter- actions interfere with intracellular signal transduction pathways; this causes rearrangement of the actin cyto- skeleton and leads to bacterial internalization [19-211. However, the relationship between signal transduction involved in bacterial infection and induction of inflamma- tory response is still unclear. We postulate that the bac- terial invasion and subsequent activation of a signal transduction pathway may be involved in the induction of IL-7R expression. Further investigation will be needed to elucidate the issues that remain.

4 Materials and methods

4.1 Bacterial strains

ASalmonella typhimurium strain (RIMD 198501 5), an entero- invasive fscherichia coli (EIEC) strain (RIMD 0509935) and an enteropathogenic E. coli (EPEC) strain (RIMD 0509829) were all provided by the Laboratory for Culture Collections (Osaka University). A common laboratory strain of a non- pathogenic E. coli, HBlOl, was used as a negative control.

4.2 The epithelial cell line T84

T84 (ATCC CCL248) colonic epithelial cell line was kindly provided by the Health Science Research Resources Bank (Osaka, Japan). T84 was grown in 50 % Dulbecco's modified Eagle's medium, 50 % Ham F-12 medium, supplemented with 5 % heat-inactivated FCS at 37 "C with 5 % COP. In the experiments involving ELISA, RT-PCR, invasion assay and FCM, T84 was seeded at 5 x 105/ml in 48-well plates (1 ml/ well) or 6-well plates (4 ml/well) (Corning, Cambridge, MA) and incubated for 48 h to form a confluent monolayer.

rial invasion on the induction of cytokines and their receptor, T84 was incubated with cytochalasin D (10 ng/ml) (Sigma Chemical Co., St. Louis, MO) for 30 min before infection. Cytochalasin D has been shown to specifically prevent poly- merization and microfilament formation, thus it interferes with the process of endocytosis and consequently inhibits bacterial entry into eukaryotic cells [23].

4.4 Cytokine-specific semi-quantitative RT-PCR

For the detection of mRNA specific for IL-7 and IL-7R in T 84, amplification was performed using the SuperscriptTM Preamplification System (GibcoBRL, Gaithersburg, MD). Details of this procedure have been previously described [16]. The reaction protocol consisted of a sequence of 35 cycles of 95 "C for 1 min, 55 "C for 1 min, 72 "C for 1 rnin, using a programmed thermal cycler (GeneAmpTM9600, Per- kin Elmer Corp., Emeryville, CA). Sequences for human IL-7 and IL-7R PCR primers were described previously [8]. Primers for human IL-8 were: S'primer, 5'-ATGAClTC- CAAGCTGGCCG-3'; 3'primer, 5'-CTCAGCCCTCTCAA- AAACTT-3'; amplified fragment, 288 bp. Primers for human p-actin were: 5' primer, 5'-GTGGGGCGCCCCAGGC- ACCA-3'; S'primer, 5'-CTCCTTAATGTCACGCACGAlT TC-3'; amplified fragment, 548 bp. PCR products were ana- lyzed on l .8 % agarose gels. The amounts of PCR products were measured by capillary electrophoresis with a laser- induced fluorescence detection system (LIF-P/ACETM, Beck- man Instruments, Fullerton, CA), and the concentration of cytokine-specific targets and p-actin were determined by the analysis and comparison of individual peak areas [24].

4.5 ELSA

T84 was prepared in the same way as for invasion assay and incubated for additional an 8 h in a medium containing 10 pg/ml gentamicin. To determine the concentration of se- creted cytokine, the cell culture supernatants were collected and assayed using an IL-8 ELISA kit (R&D Systems, Minnea- polis, MN; detection limit S 6 pg/ml.) and an IL-7 ELISA kit (Amersham, Arlington Heights, IL; detection limit 5 4 pg/ ml.).

4.3 Invasion assay 4.6 FCM analyses

Bacterial invasion, including S. typhimurium, EIEC and EPEC, was measured by the gentamicin survival assay as described previously [22]. Briefly, monolayers of T84 were infected with 1 O5 colony-forming units of each bacteria for 2 h and incubated further for 1 h with medium containing 100 pg/ml of gentamicin to eliminate extracellular bacteria. T84 was lysed with 0.1 % Triton X-100, then the released intracellular bacteria were collected on tryptic soy agar to determine their number. The invasion potential was expressed as percentages of intracellular bacteria which were recovered from T84. To assess the influence of bacte-

T84 was prepared in the same way as for ELISA. After additional incubation for 8 h, cells were harvested with PBS containing 5 mM EDTA. The expression of IL-7R on T84 was confirmed by two different methods. First, immunofluores- cent staining with mouse anti-human IL-7R mAb (Genzyrne, Cambridge, MA) and FITC-conjugated goat anti-mouse IgG Ab were used. Second, IL-7R was detected by FluorokineTM (R&D Systems) including biotinylated rhlL-7 and FITC- conjugated avidin. In both methods, cells were analyzed with a FACScan flow cytometer (Becton Dickinson, CA).

3460 K. Yamada et at. Eur. J. Immunol. 1997.27: 3456-3460

Acknowledgment: The authors thank Drs. K. Yamamoto, 1. Takahashi and T. lida for critical comments; Drs. H. lijima, M. Yanagida, Mr. Y. Akeda, T. Oguchi and K. lwatani for helpful discussion; and Mrs. J. Takeda and N. Kitagaki for technical assistance. This study was supported in part by a grant-in-aid from the Ministry of Education, Culture, Sports and Science, Japan.

5 References

1 Hedges, S., Svensson, M. and Svanborg, C., Infect. Immun. 1992.60: 1295.

2 Eckmann, L., Kagnoff, M. F. and Fierer, J., Infect.

3 Jung, H. C., Eckmann, L., Yang, S.-K., Panja, A., Fie- rer, J., Morzycka-Wroblewska, E. and Kagnoff, M. F., J. Clin. Invest. 1995. 9 5 55.

4 Namen, A. E., Lupton, S., Hjerrild, K., Wignall, J., Mochizuki, D. Y., Schmierer, A., Mosley, B., March, C. J., Urdal, D., Gillis, S., Cosman, D. and Goodwin, R. G., Nature 1988.333 571.

5 Goodwin, R. G., Lupton, S., Schmierer, A., Hjerrild, K. J., Jerzy, R., Clevenger, W., Gillis, S., Cosman, D. and Namen, A. E., Proc. Natl. Acad. Sci. USA 1989.86 302.

6 Fujihashi, K., Kawabata, S., Hiroi, T., Yamamoto, M., McGhee, J. R., Nishikawa, S. and Kiyono, H., Proc. Natl. Acad. Sci. USA 1996. 9 3 361 3.

7 Watanabe, Y., Sudo, T., Minato, N., Ohnishi, A. and Katsura, Y., Int. Irnrnunol. 1991. 3 1067.

8 Watanabe, M., Ueno, Y., Yajima, T., Iwao, Y., Tsuchiya, M., Ishikawa, H., Aiso, S., Hibi, T. and Ishii, H., J. Clin. Invest. 1995. 9 5 2945.

9 Alderson, M. R., Sassenfeld, H. M. and Widmer, M. B., J. Exp. Med. 1990. 172 577.

Immun. 1993.61: 4569.

10 Janeway Jr, C. A., Nature 1988.333: 804.

11 von Freeden, J. U., Vieira, P., Lucian, L. A., McNeil, T., Burdach, S. E. and Murray, R., J. Exp. Med. 1995. 181: 1519.

12 Maki, K., Sunaga, S., Komagata, Y., Kodaira, Y., Mabuchi, A., Karasuyama, H., Yokomuro, K., Miya- zaki, J. and Ikuta, K., Proc. Natl. Acad. Sci. USA 1996. 9 3 7172.

13 He, Y. W. and Malek, T. R., J. Exp. Med. 1996.184: 289.

14 Rich, 6. E., Campos-Torres, J., Tepper, R. I., Morea- dith, R. W. and Leder, P., J. Exp. Med. 1993. 177: 305.

15 Uehira, M., Matsuda, H., Hirata, I., Sakata, T., Fuji- wara, H. and Nishimoto, H., Int. Immunol. 1993. 5 1619.

16 Yamamoto, M., Fujihashi, K., Beagley, K. W., McGhee,

17 Armitage, R. J., Macduff, B. M., Ziegler, S. F. and

18 Finlay, B. B. and Falkow, S., Microbiol. MoI. Biol. Rev.

19 Clerc, P. and Sansonetti, P. J., Infect. Immun. 1987.55:

J. R. and Kiyono, H., J. Irnrnunol. 1993. 150: 106.

Grabstein, K. H., Cyfokine 1992.4 461.

1997.61: 136.

2681.

20 Mounier, J., Ryter, A., Coquis-Rondon, M. and Sanso- netti, P. J., Infect. Immun. 1990. 58. 1048.

21 Pace, J., Hayman, M. J. and Galan, J. E., Cell 1993.72 505.

22 Elsinghorst, E. A., Methods Enzymol. 1994.236: 405.

23 Brenner, S. L. and Korn, E. D., J. Biol. Chem. 1979.254: 9982.

24 Yamamoto, M., Kawabata, K., Fujihashi, K., McGhee, J. R. van Dyke, T. E., Bamberg, T. V., Hiroi, T. and Kiy- ono, H., Am. J. Pathol. 1996. 148: 331.

Correspondence: Takeshi Honda, Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka University Yamadaoka 3-1, Suita, Osaka 565, Japan Fax: +81-6-879-8277 e-mail: honda@biken.osaka-u.ac.jp