Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hu, L. Articles by Hickey, T. E. Search for Related Content PubMed PubMed Citation Articles by Hu, L. Articles by Hickey, T. E.

 Previous Article  |  Next Article 

Infection and Immunity, July 2005, p. 4437-4440, Vol. 73, No. 7
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.7.4437-4440.2005

Campylobacter jejuni Induces Secretion of Proinflammatory Chemokines from Human Intestinal Epithelial Cells

Lan Hu and Thomas E. Hickey^*

Naval Medical Research Center, Silver Spring, Maryland

Received 17 November 2004/ Returned for modification 6 December 2004/ Accepted 2 February 2005

Top Abstract Text References

 
Campylobacter jejuni is a common cause of diarrhea in humans. While the pathogenic mechanisms of C. jejuni are not completely understood, host inflammatory responses are thought to be contributing factors. In this report, C. jejuni 81-176 is shown to up-regulate chemokines essential to inflammatory responses. Growth-related oncogene (GRO), GRO, macrophage inflammatory protein 1, monocyte chemoattractant protein 1 (MCP-1), and gamma interferon-inducible protein 10 (IP-10) mRNA transcription in INT-407 cells was enhanced within 4 h of bacterial exposure. Infection with viable campylobacters was necessary for sustained chemokine transcription and was NF-B dependent. GRO, IP-10, and MCP-1 chemokine secretions were confirmed by immunological assays.

Top Abstract Text References

 
Campylobacters are a leading cause of diarrheal disease worldwide (2), yet very little is known about bacterial pathogenesis or bacterium-host interactions. Inflammation of the colon is a hallmark of campylobacter infections, and leukocytes and erythrocytes are almost always found in stool during active illness (13). Inflammation is thought to mediate, at least in part, host injury (1).

Intestinal epithelial cells constitute one of the first physical barriers to enteric pathogens and likely initiate the host response. In response to injury, epithelial cells secrete cellular factors that are capable of recruiting macrophages and other cellular components of the immune and inflammatory responses (6). During campylobacter infections, mononuclear phagocytes infiltrate the submucosal lining as a consequence of tissue injury (12). Additionally, human epithelial and monocytic cell lines liberate potent proinflammatory cytokines (interleukin-6 and interleukin-8) in response to Campylobacter jejuni exposure in vitro (4, 5). We demonstrate here that epithelial cells transcribe and secrete other key chemokines essential to the activation of the host's inflammatory response when exposed to C. jejuni 81-176 (4).

GRO gene transcription. The growth-related oncogene (GRO), GROß, and GRO chemokines are potent neutrophil chemoattractants produced by epithelial cells and a variety of other cell types (9, 10, 14). Expression of mRNA for these factors was assessed via reverse transcriptase (RT) PCR at 2, 4, and 24 h following infection of INT-407 cells with 81-176 (Fig. 1, lanes 2, 5, and 8). GRO message was slightly upregulated compared to uninfected controls at 2 and 4 h (Fig. 1, lanes 2 and 5). By 24 h, however, GRO mRNA transcription by cells cocultured with 81-176 was markedly enhanced compared to control cultures (lane 8). GRO message was readily detectable in 81-176-inoculated culture wells at 2 and 4 h but was most prominent 24 h after infection (Fig. 1, lanes 2, 5, and 8). Epithelial cells cultured with tumor necrosis factor alpha (TNF-) (20 ng/ml) served as positive controls for this assay and subsequent assays. GROß message was not up-regulated by either 81-176 or TNF- exposure but was detected in both uninoculated cultures and those cultured with campylobacters (Fig. 1, row 2).

View larger version (75K): [in this window] [in a new window]

FIG. 1. Transcription of proinflammatory chemokine message by human intestinal epithelial cells to campylobacter exposure. INT-407 monolayers were inoculated with 81-176 at a 100:1 ratio of bacteria to epithelial cells. After 2, 4, and 24 h, total RNA was extracted by Trizol reagent (Invitrogen). Extracted RNA was transcribed into first-strand cDNA by using the ProSTAR first-strand RT-PCR kit (Stratagene). Negative controls were constructed by omitting RT or RNA (not shown). PCR primer pairs specific for human ß-actin and chemokines were synthesized on an ABI 3400 DNA synthesizer as described previously (15). cDNA was amplified with Taq polymerase (Warrington, United Kingdom). ß-Actin transcription is included as a housekeeping control in this experiment and subsequent experiments. Lanes 1, 4, and 7 show uninfected controls. Lanes 2, 5, and 8 show cells inoculated with 81-176. Lanes 3, 6, and 9 show the positive controls of the TNF- assay.

Secretion of GRO by intestinal epithelial cells. The concentrations of GRO in supernatants of INT-407 cells were evaluated through enzyme-linked immunosorbent assay (ELISA) at 4 and 24 h after infection (Fig. 2A). Supernatants from 81-176-inoculated cultures demonstrated a slight increase in GRO levels (means ± standard deviations) compared to uninoculated culture wells as early as 4 h postinoculation (49 ± 76 pg/ml). However by the 24-hour time point, epithelial cells cocultured with 81-176 secreted 670 ± 81 pg/ml GRO (P 0.001). TNF-supplemented cultures secreted 1,134 ± 163 pg/ml GRO at 4 h and 1,261 ± 284 at 24 h (P < 0.001). Chemokine levels detected in uninoculated controls were negligible at this time point (17 ± 30 pg/ml).

View larger version (16K): [in this window] [in a new window]

FIG. 2. Chemokine secretion by INT-407 cells following exposure to 81-176. INT-407 cells were cocultured with 81-176 at a 100:1 ratio of bacteria to epithelial cells as described previously (4). Cell culture supernatants were harvested at 4 and 24 h. Chemokine levels were assayed by ELISA per the manufacturer's instructions (R&D Systems). (A) Secretion of GRO. (B) Secretion of MCP-1. (C) IP-10 secretion. Solid black bars, media control; solid grey bars, 81-176 coculture; white bars, TNF--supplemented cultures. Values (pg/ml) were calculated based upon standard curves generated with recombinant proteins. Cultures incubated with TNF- served as a positive experimental control (n = 3). P values, in comparison to uninoculated control wells, are given in the text.

Transcription of MCP-1 and MIP-1 message. Monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein 1 (MIP-1) are essential components of the immune response to enteric pathogens (3, 15). MCP-1 is a monocyte/basophil chemoattractant and activating factor produced by epithelial cells in response to physical assaults (7, 14). MIP-1, a chemoattractant for B cells, eosinophils, and killer T cells, is produced by a variety of cell types (15). Within 2 hours of inoculation, MCP-1 message in tissue culture exposed to 81-176 was up-regulated. Substantial levels of MCP-1 message were detected in these cells by 24 h (Fig. 1, lanes 2, 5, and 8). MIP-1 message detected in 81-176-inoculated cultures remained at levels similar to those found in uninoculated control cultures, with only a slight up-regulation detected at 24 h (Fig. 1, lane 8). The TNF--treated cultures also demonstrated moderate induction of MIP-1 message compared to uninoculated cultures (Fig. 1, lane 9).

Secretion of MCP-1. The production of MCP-1 by epithelial cells was evaluated by ELISA at 4 and 24 h (Fig. 2B). INT-407 cells inoculated with 81-176 secreted moderate amounts of MCP-1 at 4 h. Supernatants from cells cultured with 81-176 produced 128 ± 43 pg/ml MCP-1, compared to 6.2 ± 10 pg/ml detected in uninoculated cultures (P 0.01). By 24 h, 793 ± 174 pg/ml MCP-1 was detected in 81-176-inoculated cultures, compared to 141 ± 68 pg/ml found in controls (P 0.003).

Expression of interferon-inducible protein (IP-10). Gamma interferon-inducible protein IP-10 is a monocyte and T-lymphocyte chemoattractant and is frequently present in inflamed tissue (11, 15). IP-10 message in INT-407 cells cocultured with 81-176 was up-regulated. Within 4 h of exposure to campylobacters, message was strongly up-regulated compared to that in uninoculated control wells, and levels of IP-10 message remained elevated through 24 h (Fig. 1, lanes 5 and 8). INT-407 cultures supplemented with 20 ng/ml TNF- up-regulated IP-10 message within 4 h, and message remained elevated through 24 h, similar to results seen with 81-176-inoculated cultures (Fig. 1, lanes 6 and 9).

IP-10 protein was detectable by ELISA in 24-hour cultures (Fig. 2C). Supernatants from 24-hour INT-407 cells cultured with 81-176 contained 1,356 ± 204 pg/ml IP-10 (P 0.0004). Normal or media control cultures produced negligible amounts of the cytokine (35 ± 61 pg/ml). TNF--supplemented cultures produced 1,862 ± 276 pg/ml of the chemokine by 24 h (P 0.0004). At the earlier time point, there was no statistically significant difference in chemokine levels of 81-176 and TNF- cultures compared to normal control cultures; P values were 0.98 and 0.493, respectively.

Viable campylobacters are required for sustained chemokine mRNA transcription. INT-407 monolayers were inoculated with 81-176, either viable or heat killed, at a multiplicity of infection of 100 bacteria to each epithelial cell. Heat-killed 81-176 was incubated at 70°C for 30 min and then plated to verify the absence of viability. To control for the effects of bacterial lipooligosaccharide (LOS)/lipopolysaccharide (LPS) on chemokine transcription, Escherichia coli LPS (Sigma Chemical Co., St. Louis, MO) was included in the assay at 50 µg/ml. Chemokine message was assayed at 4 and 24 h. Sustained GRO, MCP-1, MIP-1, and IP-10 transcription required viable campylobacters or heat-sensitive bacterial products. Heat-killed 81-176 mediated a moderate and short-lived transcription of GRO and MCP-1 genes that were detectable at 4 h but waned by the later time point compared to uninoculated control cultures (Fig. 3, lanes 1, 4, 5, and 8). A moderate MIP-1 response was also detected at 4 and 24 h (Fig. 3, lanes 4 and 8). With the exception of GRO transcription, similar responses were observed in cultures incubated with E. coli LPS (Fig. 3, lanes 3 and 7). E. coli LPS induced GRO transcription as late as 24 h, a response not detected when epithelial cells were inoculated with heat-killed campylobacters (Fig. 3, lane 7 and 8). Viable campylobacters induced marked transcription of each chemokine gene by 24 h (Fig. 3, lanes 2 and 6). These data indicate that viable 81-176 or possibly heat-sensitive or synthesized bacterial products other than LOS are required for inflammatory chemokine production by human epithelial cells.

View larger version (50K): [in this window] [in a new window]

FIG. 3. Viable campylobacters are required for sustained chemokine mRNA transcription. INT-407 cells were cocultured with 81-176, either viable or heat killed, at a 100:1 bacteria-to-epithelial-cell ratio. To control for the possible effects of bacterial LOS/LPS on chemokine transcription, E. coli LPS was included in the assay at 50 µg/ml. RT-PCR was conducted as described in the legend for Fig. 1. Lanes 1 through 4, mRNA transcription at 4 h; lanes 5 through 8, chemokine RNA levels detected at 24 hours. ß-Actin mRNA was assayed as a housekeeping control. Lanes 1 and 5, uninoculated controls; lanes 2 and 6, cultures incubated with viable 81-176; lanes 3 and 7, LPS control; lanes 4 and 8, cultures incubated with heat-killed 81-176. These data are representative of three independent experiments.

Requirement of NF-B activation for chemokine transcription. The requirement of transcription factor NF-B activation for chemokine mRNA synthesis was evaluated through inhibition studies. Intestinal epithelial cells were preincubated with caffeic acid phenethyl ester (CAPE) (Sigma Chemical Co., St. Louis, Mo.) at 50 µM (8) for 1 h prior to inoculation with viable 81-176 at a multiplicity of infection of 100:1. CAPE is a potent and specific inhibitor of NF-B activation and is known for its immunomodulatory and anti-inflammatory effects on a range of eukaryotic cell types (8). Preincubation of epithelial cells with CAPE prevented transcription of IP-10 message at 4 and 24 h (Fig. 4, lanes 3 and 6). Inhibition of NF-B activation also greatly reduced MCP-1 and MIP-1 transcription at both time points. These data suggest that transcription of the genes examined proceeds, at least in part, through the activation of transcription factor NF-B. The incomplete inhibition of MCP-1 and MIP-1 might be due to the activity of compensatory transcription factors not affected by CAPE.

View larger version (42K): [in this window] [in a new window]

FIG. 4. Requirement of NF-B activation for chemokine transcription. INT-407 monolayers were inoculated with viable 81-176 as described earlier. Transcription of chemokine genes was assayed as described earlier. Lanes 1 through 3, chemokine message detected at 4 h; lanes 4 through 6, message detected in 24-hour cultures. Lanes 1 and 4, uninoculated control cultures; lanes 2, 3, 5, and 6, mRNA transcription in cultures incubated with 81-176. Lanes 3 and 6, chemokine mRNA detected in cultures pretreated with the NF-B inhibitor CAPE at 50 µM prior to inoculation with 81-176 (8). These data are representative of three independent experiments.

In summary, secretion of select chemokines by human intestinal epithelial cells exposed to campylobacters may provide the initial signals for acute inflammatory responses. We demonstrate that transcription of GRO, GRO, MCP-1, MIP-1, and IP-10 chemokine genes are up-regulated by human intestinal epithelial cells following exposure to viable 81-176 or bacterial products other than LOS. These transcriptional responses were apparently mediated by NF-B activation. We also demonstrate the secretion of GRO, MCP-1, and IP-10 proteins by ELISA. These chemokines are essential components of the inflammatory immune response to enteric pathogens, and additionally, these data provide insight into the mechanisms of tissue injury by campylobacters. These data further support the premise that inflammation plays a significant role in C. jejuni pathogenesis and that the intestinal epithelial tissue likely play significant roles in initiating the inflammatory response.

 
This work was supported by Military Infectious Diseases Research Program Work Unit Number A30001-04-NM.

The opinions and assertions herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large.

 
* Corresponding author. Mailing address: Enterics Department, Naval Medical Research Center, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500. Phone: (301) 319-7579. Fax: (301) 319-7679. E-mail: hickeyt{at}nmrc.navy.mil.

Editor: J. T. Barbieri

Present address: Enterics and STD Laboratory, CBER, FDA, 29 Lincoln Drive, Room 424, Bethesda, MD 20892-4555.

Top Abstract Text References

 

1
1. Blaser, M. J., R. B. Parsons, and W. L. Wang. 1980. Acute colitis caused by Campylobacter fetus ss. jejuni. Gastroenterology 78:448-453.[Medline]
2
2. Coker, A. O., R. D. Isokpehi, B. N. Thomas, K. O. Amisu, and L. Obi. 2002. Human campylobacteriosis in developing countries. Emerg. Infect. Dis. 8:237-243.[Medline]
3
3. Futagami, S., T. Hiratsuka, A. Tatsuguchi, K. Suzuki, M. Kusunoki, Y. Shinji, K. Shinoki, T. Iizumi, T. Akamatsu, H. Nishigaki, K. Wada, K. Miyake, K. Gudis, T. Tsukui, and C. Sakamoto. 2003. Monocyte chemoattractant protein 1 (MCP-1) released from Helicobacter pylori stimulated gastric epithelial cells induces cyclooxygenase 2 expression and activation in T cells. Gut 52:1257-1264.[Abstract/Free Full Text]
4
4. Hickey, T. E., S. Baqar, A. L. Bourgeois, C. P. Ewing, and P. Guerry. 1999. Campylobacter jejuni stimulated secretion of interleukin-8 by INT407 cells. Infect. Immun. 67:88-93.[Abstract/Free Full Text]
5
5. Joens, M. A., S. Totemeyer, D. J. Maskell, C. E. Bryant, and P. A. Barrow. 2003. Induction of proinflammatory responses in the human monocytic cell line THP-1 by Campylobacter jejuni. Infect. Immun. 71:2626-2633.[Abstract/Free Full Text]
6
6. Jung, H. C., L. Eckmann, S. K. Yang, A. Panja, J. Fierer, E. Morzycka-Wroblewska, M. F. Kagnoff. 1995. A distinct array of proinflammatory cytokines is expressed in human colon epithelial cells in response to bacterial invasion. J. Clin. Investig. 95:55-65.
7
7. MacDermott, R. P. 1999. Chemokines in the inflammatory bowel diseases. J. Clin. Immunol. 19:266-272.[CrossRef][Medline]
8
8. Natarajan, K., S. Singh, T. R. Burke, Jr., D. Grunberger, and B. B. Aggarwal. 1996. Caffeic acid phenethyl ester is a potent and specific inhibitor of nuclear transcription factor NF-kappa B. Proc. Natl. Acad. Sci. USA 93:9090-9095.[Abstract/Free Full Text]
9
9. Sieveking, D., H. M. Mitchell, and A. S. Day. 2004. Gastric epithelial cell CXC chemokine secretion following Helicobacter pylori infection in vitro. J. Gastroenterol. Hepatol. 19:982-987.[Medline]
10
10. Thorpe, C. M., W. E. Smith, B. P. Hurley, and D. W. K. Acheson. 2001. Shiga toxins induce, superinduce, and stabilize a variety of C-X-C chemokine mRNAs in intestinal epithelial cells, resulting in increased chemokine expression. Infect. Immun. 69:6140-6147.[Abstract/Free Full Text]
11
11. Uguccioni, M., P. Gionchetti, D. F. Robbiani, F. Rizzello, S. Peruzzo, M. Campieri, and M. Baggiolini. 1999. Increased expression of IP-10, IL-8, MCP-1, and MCP-3 in ulcerative colitis. Am. J. Pathol. 155:331-336.[Abstract/Free Full Text]
12
12. van Spreeuwel, J. P., G. C. Duursma, C. J. L. M. Meijer, R. Bax, P. C. M. Rosekrans, and J. Lindeman. 1985. Campylobacter colitis: histological, immunohistochemical and ultrastructural findings. Gut 26:945-951.[Abstract/Free Full Text]
13
13. Wassenaar, T. M., and M. J. Blaser. 1999. Pathophysiology of Campylobacter jejuni infections of humans. Microbes Infect. 1:1023-1033.[CrossRef][Medline]
14
14. Yamaoka, Y., M. Kita, T. Kodama, N. Sawai, T. Tanahashi, K. Kashima, and J. Imanishi. 1998. Chemokines in the gastric mucosa in Helicobacter pylori infection. Gut 42:609-617.[Abstract/Free Full Text]
15
15. Yang, S. K., L. Eckmann, A. Panja, and M. F. Kagnoff. 1997. Differential and regulated expression of C-X-C, C-C, and C-chemokines by human colon epithelial cells. Gastroenterology 113:1214-1223.[CrossRef][Medline]

---

Infection and Immunity, July 2005, p. 4437-4440, Vol. 73, No. 7
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.7.4437-4440.2005

This article has been cited by other articles:

• Stewart-Tull, D. E. S., Coote, J. G., Thompson, D. H, Candlish, D., Wardlaw, A. C., Candlish, A. (2009). Virulence spectra of typed strains of Campylobacter jejuni from different sources: a blinded in vivo study. J Med Microbiol 58: 546-553 [Abstract] [Full Text]  
• Friis, L. M., Keelan, M., Taylor, D. E. (2009). Campylobacter jejuni Drives MyD88-Independent Interleukin-6 Secretion via Toll-Like Receptor 2. Infect. Immun. 77: 1553-1560 [Abstract] [Full Text]  
• Zheng, J., Meng, J., Zhao, S., Singh, R., Song, W. (2008). Campylobacter-Induced Interleukin-8 Secretion in Polarized Human Intestinal Epithelial Cells Requires Campylobacter-Secreted Cytolethal Distending Toxin- and Toll-Like Receptor-Mediated Activation of NF-{kappa}B. Infect. Immun. 76: 4498-4508 [Abstract] [Full Text]  
• Janssen, R., Krogfelt, K. A., Cawthraw, S. A., van Pelt, W., Wagenaar, J. A., Owen, R. J. (2008). Host-Pathogen Interactions in Campylobacter Infections: the Host Perspective. Clin. Microbiol. Rev. 21: 505-518 [Abstract] [Full Text]  
• Yoder, J. M., Aslam, R. U., Mantis, N. J. (2007). Evidence for Widespread Epithelial Damage and Coincident Production of Monocyte Chemotactic Protein 1 in a Murine Model of Intestinal Ricin Intoxication. Infect. Immun. 75: 1745-1750 [Abstract] [Full Text]  
• MacCallum, A. J., Harris, D., Haddock, G., Everest, P. H. (2006). Campylobacter jejuni-infected human epithelial cell lines vary in their ability to secrete interleukin-8 compared to in vitro-infected primary human intestinal tissue. Microbiology 152: 3661-3665 [Abstract] [Full Text]  
• Chen, M. L., Ge, Z., Fox, J. G., Schauer, D. B. (2006). Disruption of Tight Junctions and Induction of Proinflammatory Cytokine Responses in Colonic Epithelial Cells by Campylobacter jejuni. Infect. Immun. 74: 6581-6589 [Abstract] [Full Text]  
• Long, K. Z., Santos, J. I., Estrada Garcia, T., Haas, M., Firestone, M., Bhagwat, J., DuPont, H. L., Hertzmark, E., Rosado, J. L., Nanthakumar, N. N. (2006). Vitamin A Supplementation Reduces the Monocyte Chemoattractant Protein-1 Intestinal Immune Response of Mexican Children. J. Nutr. 136: 2600-2605 [Abstract] [Full Text]  
• Johanesen, P. A., Dwinell, M. B. (2006). Flagellin-Independent Regulation of Chemokine Host Defense in Campylobacter jejuni-Infected Intestinal Epithelium.. Infect. Immun. 74: 3437-3447 [Abstract] [Full Text]  
• Hu, L., Bray, M. D., Osorio, M., Kopecko, D. J. (2006). Campylobacter jejuni Induces Maturation and Cytokine Production in Human Dendritic Cells.. Infect. Immun. 74: 2697-2705 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hu, L. Articles by Hickey, T. E. Search for Related Content PubMed PubMed Citation Articles by Hu, L. Articles by Hickey, T. E.