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Infection and Immunity, September 2003, p. 5394-5397, Vol. 71, No. 9
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.9.5394-5397.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Differential Interleukin-8 and Nitric Oxide Production in Epithelial Cells Induced by Mucosally Invasive and Noninvasive Trypanosoma cruzi Trypomastigotes

C. S. Eickhoff,1 L. Eckmann,2 and D. F. Hoft1*

Department of Internal Medicine, Saint Louis University Health Sciences Center, St. Louis, Missouri,1 Department of Medicine, University of California at San Diego, La Jolla, California2

Received 20 March 2003/ Returned for modification 20 May 2003/ Accepted 20 June 2003


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ABSTRACT
 
Trypanosoma cruzi metacyclic trypomastigotes (MT), but not blood form trypomastigotes (BFT), are highly mucosally infective. We investigated the abilities of MT and BFT to induce inflammation and/or intracellular killing activity within mucosal epithelia. BFT, but not MT, induced marked increases in interleukin-8, GRO-{alpha}, MCP-1, and nitric oxide production in HeLa and AGS cells, despite similar infectivities. MT may avoid induction of inflammation as an important biological mechanism facilitating mucosal invasion.


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TEXT
 
An estimated 16 to 18 million people in South and Central America are infected with Trypanosoma cruzi, the protozoan parasite causing Chagas' disease (7). Fifty thousand deaths per year result from the cardiac and gastrointestinal pathology due to chronic T. cruzi infection. T. cruzi is most commonly transmitted to humans through contact with the infected feces of the reduviid bug. T. cruzi metacyclic trypomastigotes (MT) in reduviid bug feces can initiate infection through cuts in the skin or through contact with mucosal surfaces, including the eyes and mouth. T. cruzi MT infect epithelial cells, develop, and replicate intracellularly as amastigotes and are released from lysed host cells as blood form trypomastigotes (BFT). BFT are then able to infect new epithelial cells and most other types of mammalian host cells and repeat the intracellular replication cycle. T. cruzi MT are able to initiate infection through the conjunctival and gastrointestinal mucosa. On the other hand, we have shown that BFT are unable to infect through mucosal surfaces (14). The mechanisms responsible for the mucosal invasiveness of MT, but not BFT, are not known.

Tissue infection with T. cruzi elicits significant inflammation responsible for the pathology associated with Chagas' disease. BFT, but not MT, infection of macrophages induces proinflammatory cytokine and nitric oxide (NO) production (8). The effects of T. cruzi infection on proinflammatory responses in epithelial cells, however, have not been reported. We found that mice challenged orally with MT do not develop detectable mucosal inflammation until 4 to 7 days after infection, sufficient time for three to four cycles of replication and 100- to 1,000-fold amplification of BFT (16). We hypothesize that the inability of MT to induce inflammatory mediators could be important for T. cruzi mucosal infectivity. On the other hand, it may be advantageous for later stages of infection with BFT to induce inflammation, prolonging the life of the host by preventing uncontrolled parasite replication.

We studied the ability of T. cruzi strain Tulahuén MT and BFT to induce chemokine responses in mucosal epithelial cells. Culture-derived MT were prepared by subculture of axenically cultivated epimastigotes into modified Grace's medium (Sigma, St. Louis, Mo.) for 7 to 10 days. These culture-derived MT are morphologically similar to insect-derived MT and are mucosally infective (20). Insect-derived MT were prepared by allowing T. cruzi-infected reduviid bugs (Dipetalogaster maximus) to feed on anesthetized mice. Engorged insects were then incubated in glass vials for 3 to 5 h, excreta were pooled, and parasite concentrations were determined by direct counting with a hemocytometer. BFT were collected by sacrificing highly parasitemic mice and copurifying parasites with mononuclear cells in Ficoll-Paque density gradients (Amersham Pharmacia Biotech Inc., Piscataway, N.J.). Human cervical (HeLa) and gastric (AGS) epithelial cells obtained from the American Type Culture Collection (Manassas, Va.) were infected with MT and BFT in Dulbecco modified Eagle medium or McCoy's 5A medium (Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum, 2 mM L-glutamine, penicillin (50 U/ml), and streptomycin (50 µg/ml) in either 24-well culture plates or 4-well tissue culture slide chambers (Nalge Nunc International, Naperville, Ill.).

We first measured the production of interleukin-8 (IL-8) in supernatants of cultures of human epithelial cells infected with BFT and culture-derived MT by enzyme-linked immunosorbent assay (ELISA) as previously described (11). We chose HeLa cells, which are commonly used for in vitro studies of T. cruzi infection, as an initial model for studies of chemokine responses produced by epithelial cells. Because we have shown that T. cruzi MT invade through the gastric epithelia after oral challenges (16), we chose AGS cells (derived from gastric adenocarcinoma tissue) as a more relevant model of T. cruzi mucosal infection. HeLa and AGS cells infected with BFT for 48 h produced marked increases in IL-8 in a dose-dependent manner (Fig. 1a and b, respectively). After the same period of infection, culture-derived MT induced only 20% as much IL-8 as did BFT in HeLa cells and failed to induce IL-8 in AGS cells, even at the highest infection level tested (>5:1 parasite-to-cell ratio). Insect-derived MT did not induce IL-8 in any of the cell lines tested (data not shown). In all cases, IL-8 induced by BFT was maximal in cultures by 48 h (data not shown). Increased IL-8 protein production after BFT infection was paralleled by increased IL-8 mRNA expression detected by reverse transcription-(RT) PCR at 24 h after T. cruzi infection (Table 1). BFT infection of HeLa cells induced >20-fold more IL-8 mRNA than did infection with culture-derived MT.



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  		FIG. 1. BFT induce a potent IL-8 response in a dose-dependent manner. HeLa (a) and AGS (b) cells were incubated overnight in 24-well plates at 2 x 105 and 3 x 105 cells per well, respectively, and then infected with BFT or culture-derived MT (CMT). After 24 h, infected cell monolayers were washed to remove extracellular parasites. Supernatants were collected 48 h after infection and tested by ELISA for IL-8. Results shown are representative of at least three experiments.


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  		TABLE 1. IL-8 RT-PCRa

The lack of IL-8 induction after MT infection could be related to a "silent" invasion of epithelial cells or active inhibition of the IL-8 responses in these cells. To test whether or not MT could actively inhibit IL-8 production, HeLa cells were infected with culture-derived MT and stimulated with IL-1ß (10 ng/ml), a potent activator of IL-8 secretion. Infection with culture-derived MT induced minimal IL-8 levels (<100 pg/ml), whereas IL-1ß stimulated >3,000 pg of IL-8 per ml, both in the presence and in the absence of culture-derived MT infection. Experiments were performed with insect-derived MT with similar results. Therefore, MT did not actively inhibit the induction of IL-8 by IL-1ß stimulation.

To determine whether the differential effects of BFT and MT infections of human epithelial cells are unique to IL-8 induction or more global differential effects on chemokine production are stimulated, we measured the levels of GRO-{alpha} (another CXC chemokine with neutrophil chemotactic properties) and MCP-1 (a representative CC chemokine) in supernatants from MT- and BFT-infected cultures by ELISA as previously described (11). GRO-{alpha} and MCP-1 levels in epithelial cell supernatants were both markedly increased in wells infected with BFT (Table 2). Maximal BFT infections induced >200 pg of GRO-{alpha} per ml in the HeLa and AGS cell lines, while culture-derived MT failed to induce detectable levels of this chemokine. In addition, BFT infection of HeLa cells induced MCP-1 production that was not seen in culture-derived MT-infected HeLa cell supernatants. Thus, these experiments demonstrate that infection with BFT and MT induced global differential effects on chemokine production in epithelial cells.


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  		TABLE 2. GRO-{alpha} and MCP-1 induction by BFTa

To test whether these differences in IL-8, GRO-{alpha}, and MCP-1 production were the result of differential infectivity of BFT and culture-derived MT in vitro, infected HeLa cells were Giemsa stained and examined microscopically. HeLa cells infected with BFT and culture-derived MT were found to have similar percentages of infected cells; 48 h after infection, 7 to 8% of cells were infected by both BFT and culture-derived MT. Infected cells also contained similar numbers of intracellular parasites (three to four amastigotes per infected cell). In fact, by day 4 of infection, the percentage of cells infected by culture-derived MT was almost twice that of cells infected by BFT, suggesting that the lack of chemokine induction by MT was accompanied by enhanced intracellular replication of T. cruzi. The infectivities of culture-derived MT and BFT in HeLa cells were also tested with a T. cruzi-specific semiquantitative PCR assay as previously described (15). At 48 h after infection, similar molecular equivalents of T. cruzi DNA were seen in MT- and BFT-infected HeLa cells (data not shown). These combined results demonstrate that the failure of MT to induce chemokine production was not due to decreased infectivity or intracellular replication.

The above results suggested that MT infection was associated with higher levels of T. cruzi replication by 4 days postinfection compared with BFT-infected cells. CC chemokines have been shown to promote NO-dependent trypanocidal activity in murine and human macrophages (2, 24). Because BFT, and not MT, infection induced chemokine production in human epithelial cells, we therefore next investigated the possibility that the induction of chemokines by BFT could induce NO production in human epithelial cells and thereby reduce parasite numbers detectable at the later time points. Culture supernatants from infected epithelial monolayers were tested for stable NO end products with a modified Griess reaction as previously described (12, 17, 22). NO production (20 to 30 µM) was detected in BFT-infected HeLa cell supernatants. NO was not detectable in cultures infected with culture-derived MT (Fig. 2). Similar results were obtained with AGS cells. Therefore, these results indicate that induction of chemokines and NO by BFT could have trypanocidal effects in human epithelial cells and reduce intracellular T. cruzi replication.



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  		FIG. 2. BFT infection induces a potent NO response. HeLa cells were incubated overnight in 24-well plates and then infected with BFT or culture-derived MT at a 1:1 parasite-to-target cell ratio. After 24 h, infected cell monolayers were washed to remove extracellular parasites. Supernatants were collected 48 h after infection and tested for NO with a modified Griess reaction and for IL-8 by ELISA. Results shown are representative of at least two experiments.

T. cruzi undergoes a complex life cycle, with initial human infections usually caused by invasion of insect-derived MT through cutaneous and/or mucosal epithelial surfaces. We have shown previously that T. cruzi MT are uniquely capable of initiating mucosal infection (14). BFT, which are infectious for mammalian cells in vitro and in vivo, are nevertheless unable to infect mice after oral inoculation. We have shown in this study that infections with BFT, but not MT, induce chemokine responses in different epithelial cells, including the production of IL-8, GRO-{alpha}, and MCP-1. In addition, epithelial cells produced NO after infection by BFT but not after infection by MT. Multiple studies have demonstrated that T. cruzi parasites express stage-specific proteins and other molecules, including some that are generated by stage-specific posttranscriptional and posttranslational modifications (1, 5, 6, 10, 13, 18, 21). It is possible that the differential chemokine and NO responses induced in epithelial cells by BFT and MT are due to the distinct glycoconjugates present on their surface membranes shown previously to differentially induce cytokines and NO production in macrophages (3, 4, 8, 9). BFT and MT were equally infective in epithelial cells, indicating that the lack of chemokine and NO responses in epithelial cells infected with MT could not be explained by decreases in infection. In fact, higher levels of parasite replication were detected 4 days after infection with MT, suggesting that lower levels of microbicidal activity were induced in these cultures. These findings are consistent with our hypothesis that the inability of BFT to establish infection in mice after mucosal challenge may be due to potent trypanocidal activity induced in epithelial cells upon mucosal invasion.

It may benefit the parasite for BFT to induce inflammation and microbicidal mechanisms so that overwhelming infection does not kill the host, allowing the host-parasite relationship to continue. Studies have shown that NO levels in mice correlate with protection but not eradication of parasite infection (23). In addition, other groups have shown that removal of the inducible NO synthase gene in knockout mice results in extreme susceptibility to acute T. cruzi infection (19). Therefore, NO production induced by inflammatory signals may be important for the maintenance of a stable host-parasite relationship. However, inflammatory diseases of the gut, as well as the heart, are the major cause of morbidity and mortality in Chagas' disease patients. Treatments decreasing the production of chemokines and other inflammatory mediators could be useful in limiting or slowing the progression of pathology associated with Chagas' disease.


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FOOTNOTES
 
* Corresponding author. Mailing address: Division of Infectious Diseases and Immunology, Saint Louis University Health Sciences Center, 3635 Vista Ave., St. Louis, MO 63110. Phone: (314) 577-8648. Fax: (314) 771-3816. E-mail: hoftdf{at}slu.edu. Back

Editor: J. M. Mansfield


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REFERENCES
 
    1
  1. Abuin, G., L. H. Freitas-Junior, W. Colli, M. J. Alves, and S. Schenkman. 1999. Expression of trans-sialidase and 85-kDa glycoprotein genes in Trypanosoma cruzi is differentially regulated at the post-transcriptional level by labile protein factors. J. Biol. Chem. 274:13041-13047.[Abstract/Free Full Text]
    2. 2
  2. Aliberti, J. C. S., F. S. Machado, J. T. Souto, A. P. Campanelli, M. M. Teixeira, R. T. Gazzinelli, and J. S. Silva. 1999. ß-Chemokines enhance parasite uptake and promote nitric oxide-dependent microbiostatic activity in murine inflammatory macrophages infected with Trypanosoma cruzi. Infect. Immun. 67:4819-4826.[Abstract/Free Full Text]
    3. 3
  3. Almeida, I. C., M. M. Camargo, D. O. Procopio, L. S. Silva, A. Mehlert, L. R. Travassos, R. T. Gazzinelli, and M. A. Ferguson. 2000. Highly purified glycosylphosphatidylinositols from Trypanosoma cruzi are potent proinflammatory agents. EMBO J. 19:1476-1485.[CrossRef][Medline]
    4. 4
  4. Almeida, I. C., and R. T. Gazzinelli. 2001. Proinflammatory activity of glycosylphosphatidylinositol anchors derived from Trypanosoma cruzi: structural and functional analyses. J. Leukoc. Biol. 70:467-477.[Abstract/Free Full Text]
    5. 5
  5. Astolfi, F. S., M. F. De Sa, and E. S. Gander. 1984. Protein synthesis in purified trypo- and epimastigote forms of Trypanosoma cruzi. Acta Trop. 41:109-115.[Medline]
    6. 6
  6. Avila, A. R., S. F. Yamada-Ogatta, V. da Silva Monteiro, M. A. Krieger, C. V. Nakamura, W. De Souza, and S. Goldenberg. 2001. Cloning and characterization of the metacyclogenin gene, which is specifically expressed during Trypanosoma cruzi metacyclogenesis. Mol. Biochem. Parasitol. 117:169-177.[CrossRef][Medline]
    7. 7
  7. Bresson, J. L., and I. Nuttall. 1991. Control of Chagas’ disease. WHO Tech. Rep. Ser. 811.
    8. 8
  8. Camargo, M. M., I. C. Almeida, M. E. A. Pereira, M. A. J. Ferguson, L. R. Travassos, and R. Gazzinelli. 1997. Glycosylphosphatidylinositol-anchored mucin-like glycoproteins isolated from Trypanosoma cruzi trypomastigotes initiate the synthesis of proinflammatory cytokines by macrophages. J. Immunol. 158:5890-5901.[Abstract]
    9. 9
  9. Campos, M. A., I. C. Almeida, O. Takeuchi, S. Akira, E. P. Valente, D. O. Procopio, L. R. Travassos, J. A. Smith, D. T. Golenbock, and R. T. Gazzinelli. 2001. Activation of Toll-like receptor-2 by glycosylphosphatidylinositol anchors from a protozoan parasite. J. Immunol. 167:416-423.[Abstract/Free Full Text]
    10. 10
  10. Dallagiovanna, B., C. Plazanet-Menut, S. F. Ogatta, A. R. Avila, M. A. Krieger, and S. Goldenberg. 2001. Trypanosoma cruzi: a gene family encoding chitin-binding-like proteins is posttranscriptionally regulated during metacyclogenesis. Exp. Parasitol. 99:7-16.[CrossRef][Medline]
    11. 11
  11. Denney, C. F., L. Eckmann, and S. L. Reed. 1999. Chemokine secretion of human cells in response to Toxoplasma gondii infection. Infect. Immun. 67:1547-1552.[Abstract/Free Full Text]
    12. 12
  12. Green, L. C., D. A. Wagner, J. Glogowski, P. L. Skipper, J. S. Wishnok, and S. R. Tannenbaum. 1982. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal. Biochem. 126:131-138.[CrossRef][Medline]
    13. 13
  13. Haynes, P. A., and G. A. Cross. 1996. Differential glycosylation of epitope-tagged glycoprotein Gp72 during the Trypanosoma cruzi life cycle. Mol. Biochem. Parasitol. 83:253-256.[CrossRef][Medline]
    14. 14
  14. Hoft, D. F. 1996. Differential mucosal infectivity of different life stages of T. cruzi. Am. J. Trop. Med. Hyg. 55:360-364.
    15. 15
  15. Hoft, D. F., and C. S. Eickhoff. 2002. Type 1 immunity provides optimal protection against both mucosal and systemic Trypanosoma cruzi challenges. Infect. Immun. 70:6715-6725.[Abstract/Free Full Text]
    16. 16
  16. Hoft, D. F., P. L. Farrar, K. Kratz-Owens, and D. Shaffer. 1996. Gastric invasion by Trypanosoma cruzi and induction of protective mucosal immune responses. Infect. Immun. 64:3800-3810.[Abstract]
    17. 17
  17. Hoft, D. F., A. R. Schnapp, C. S. Eickhoff, and S. T. Roodman. 2000. Involvement of CD4+ Th1 cells in systemic immunity protective against primary and secondary challenges with Trypanosoma cruzi. Infect. Immun. 68:197-204.[Abstract/Free Full Text]
    18. 18
  18. Krieger, M. A., A. R. Avila, S. F. Ogatta, C. Plazanet-Menut, and S. Goldenberg. 1999. Differential gene expression during Trypanosoma cruzi metacyclogenesis. Mem. Inst. Oswaldo Cruz. 94(Suppl. 1):165-168.
    19. 19
  19. Michailowsky, V., N. M. Silva, C. D. Rocha, L. Q. Vieira, J. Lannes-Vieira, and R. T. Gazzinelli. 2001. Pivotal role of interleukin-12 and interferon-gamma axis in controlling tissue parasitism and inflammation in the heart and central nervous system during Trypanosoma cruzi infection. Am. J. Pathol. 159:1723-1733.[Abstract/Free Full Text]
    20. 20
  20. Neira, I., F. A. Silva, M. Cortez, and N. Yoshida. 2003. Involvement of Trypanosoma cruzi metacyclic trypomastigote surface molecule gp82 in adhesion to gastric mucin and invasion of epithelial cells. Infect. Immun. 71:557-561.[Abstract/Free Full Text]
    21. 21
  21. Rondinelli, E., R. Silva, J. F. Carvalho, S. de Almeida, E. F. de Carvalho, and F. T. de Castro. 1988. Trypanosoma cruzi: an in vitro cycle of cell differentiation in axenic culture. Exp. Parasitol. 66:197-204.[CrossRef][Medline]
    22. 22
  22. Schnapp, A. R., C. S. Eickhoff, D. Sizemore, R. Curtiss III, and D. F. Hoft. 2002. Cruzipain induces both mucosal and systemic protection against Trypanosoma cruzi in mice. Infect. Immun. 70:5065-5074.[Abstract/Free Full Text]
    23. 23
  23. Vespa, G. N., F. Q. Cunha, and J. S. Silva. 1994. Nitric oxide is involved in control of Trypanosoma cruzi-induced parasitemia and directly kills the parasite in vitro. Infect. Immun. 62:5177-5182.[Abstract/Free Full Text]
    24. 24
  24. Villalta, F., Y. Zhang, K. E. Bibb, J. C. Kappes, and M. F. Lima. 1998. The cysteine-cysteine family of chemokines RANTES, MIP-1{alpha}, and MIP-1ß induce trypanocidal activity in human macrophages via nitric oxide. Infect. Immun. 66:4690-4695.[Abstract/Free Full Text]

Infection and Immunity, September 2003, p. 5394-5397, Vol. 71, No. 9
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.9.5394-5397.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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