Skip to main content
NCBI home page
The American Journal of Pathology logoLink to The American Journal of Pathology
. 1998 Apr;152(4):925–934.

Temporal expression of pro-inflammatory cytokines and inducible nitric oxide synthase in experimental acute Chagasic cardiomyopathy.

B Chandrasekar 1, P C Melby 1, D A Troyer 1, J T Colston 1, G L Freeman 1
PMCID: PMC1858258  PMID: 9546353

Abstract

To characterize the kinetics of myocardial cytokine and inducible nitric oxide synthase (iNOS) expression in acute Chagasic cardiomyopathy, we studied a rat model of acute Trypanosoma cruzi infection. Rats were euthanized 36 hours and 5, 10, and 15 days after infection, and hearts were collected for histology, mRNA, and protein analyses. Histological analysis of myocardium showed a progressive increase in the number of amastigotes and mononuclear inflammatory cells. Organisms were first detected 5 days after intraperitoneal inoculation as isolated nests and became numerous by day 15. Northern blot analysis of total RNA revealed no signal for interleukin (IL)-1beta or tumor necrosis factor (TNF)-alpha and a weak signal for IL-6 in control hearts. High levels of expression for the three genes were detected in the infected animals at 36 hours after infection. Although IL-1beta and IL-6 levels increased steadily up to 10 days, TNF-alpha levels were the highest at 5 days, remained high at 10 days, and declined thereafter. Western blot analysis showed similar results to that of mRNA expression. No signal was detected for iNOS in the controls, but both its mRNA and protein were found in the infected animals, with levels being highest at 15 days after infection. Immunohistochemistry revealed no iNOS immunoreactivity in uninfected animals, but intense iNOS staining was detected in blood vessels of infected animals, which decreased progressively with period of infection. Positive staining for iNOS in cardiomyocytes was first detected at 36 hours after infection (at a time when there was no histological inflammatory reaction), which steadily increased, being the highest at 15 days after infection. These results indicate that, in addition to mechanical damage by T. cruzi, substantial pro-inflammatory cytokine production within the myocardium is likely to participate in the pathophysiology of acute Chagasic cardiomyopathy.

Full text

PDF
925

Images in this article

928Figure 1
on p.928
929Figure 3
on p.929
929Figure 5
on p.929
930Figure 6
on p.930
931Figure 7
on p.931
932Figure 8
on p.932
932Figure 9
on p.932
932Figure 10
on p.932
933Figure 11
on p.933

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abel F. L. Myocardial function in sepsis and endotoxin shock. Am J Physiol. 1989 Dec;257(6 Pt 2):R1265–R1281. doi: 10.1152/ajpregu.1989.257.6.R1265. [DOI] [PubMed] [Google Scholar]
  2. Andrade Z. A. Mechanisms of myocardial damage in Trypanosoma cruzi infection. Ciba Found Symp. 1983;99:214–233. doi: 10.1002/9780470720806.ch12. [DOI] [PubMed] [Google Scholar]
  3. Andrade Z. A. Pathogenesis of Chagas' disease. Res Immunol. 1991 Feb;142(2):126–129. doi: 10.1016/0923-2494(91)90021-a. [DOI] [PubMed] [Google Scholar]
  4. Balligand J. L., Ungureanu D., Kelly R. A., Kobzik L., Pimental D., Michel T., Smith T. W. Abnormal contractile function due to induction of nitric oxide synthesis in rat cardiac myocytes follows exposure to activated macrophage-conditioned medium. J Clin Invest. 1993 May;91(5):2314–2319. doi: 10.1172/JCI116461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chandrasekar B., Colston J. T., Freeman G. L. Induction of proinflammatory cytokine and antioxidant enzyme gene expression following brief myocardial ischaemia. Clin Exp Immunol. 1997 May;108(2):346–351. doi: 10.1046/j.1365-2249.1997.d01-1017.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chandrasekar B., Freeman G. L. Induction of nuclear factor kappaB and activation protein 1 in postischemic myocardium. FEBS Lett. 1997 Jan 13;401(1):30–34. doi: 10.1016/s0014-5793(96)01426-3. [DOI] [PubMed] [Google Scholar]
  7. Chandrasekar B., Melby P. C., Troyer D. A., Freeman G. L. Induction of proinflammatory cytokine expression in experimental acute Chagasic cardiomyopathy. Biochem Biophys Res Commun. 1996 Jun 14;223(2):365–371. doi: 10.1006/bbrc.1996.0900. [DOI] [PubMed] [Google Scholar]
  8. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  9. Dinarello C. A., Wolff S. M. The role of interleukin-1 in disease. N Engl J Med. 1993 Jan 14;328(2):106–113. doi: 10.1056/NEJM199301143280207. [DOI] [PubMed] [Google Scholar]
  10. Eichenholz P. W., Eichacker P. Q., Hoffman W. D., Banks S. M., Parrillo J. E., Danner R. L., Natanson C. Tumor necrosis factor challenges in canines: patterns of cardiovascular dysfunction. Am J Physiol. 1992 Sep;263(3 Pt 2):H668–H675. doi: 10.1152/ajpheart.1992.263.3.H668. [DOI] [PubMed] [Google Scholar]
  11. Fahey T. J., 3rd, Yoshioka T., Shires G. T., Fantini G. A. The role of tumor necrosis factor and nitric oxide in the acute cardiovascular response to endotoxin. Ann Surg. 1996 Jan;223(1):63–69. doi: 10.1097/00000658-199601000-00009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Finkel M. S., Oddis C. V., Jacob T. D., Watkins S. C., Hattler B. G., Simmons R. L. Negative inotropic effects of cytokines on the heart mediated by nitric oxide. Science. 1992 Jul 17;257(5068):387–389. doi: 10.1126/science.1631560. [DOI] [PubMed] [Google Scholar]
  13. Freeman G. L., Colston J. T., Zabalgoitia M., Chandrasekar B. Contractile depression and expression of proinflammatory cytokines and iNOS in viral myocarditis. Am J Physiol. 1998 Jan;274(1 Pt 2):H249–H258. doi: 10.1152/ajpheart.1998.274.1.H249. [DOI] [PubMed] [Google Scholar]
  14. Geller D. A., Nussler A. K., Di Silvio M., Lowenstein C. J., Shapiro R. A., Wang S. C., Simmons R. L., Billiar T. R. Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):522–526. doi: 10.1073/pnas.90.2.522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Goldring C. E., Narayanan R., Lagadec P., Jeannin J. F. Transcriptional inhibition of the inducible nitric oxide synthase gene by competitive binding of NF-kappa B/Rel proteins. Biochem Biophys Res Commun. 1995 Apr 6;209(1):73–79. doi: 10.1006/bbrc.1995.1472. [DOI] [PubMed] [Google Scholar]
  16. Goossens V., Grooten J., De Vos K., Fiers W. Direct evidence for tumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity. Proc Natl Acad Sci U S A. 1995 Aug 29;92(18):8115–8119. doi: 10.1073/pnas.92.18.8115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hagar J. M., Rahimtoola S. H. Chagas' heart disease. Curr Probl Cardiol. 1995 Dec;20(12):825–924. [PubMed] [Google Scholar]
  18. Herskowitz A., Choi S., Ansari A. A., Wesselingh S. Cytokine mRNA expression in postischemic/reperfused myocardium. Am J Pathol. 1995 Feb;146(2):419–428. [PMC free article] [PubMed] [Google Scholar]
  19. Hosenpud J. D. The effects of interleukin 1 on myocardial function and metabolism. Clin Immunol Immunopathol. 1993 Aug;68(2):175–180. doi: 10.1006/clin.1993.1115. [DOI] [PubMed] [Google Scholar]
  20. Hunter C. A., Slifer T., Araujo F. Interleukin-12-mediated resistance to Trypanosoma cruzi is dependent on tumor necrosis factor alpha and gamma interferon. Infect Immun. 1996 Jul;64(7):2381–2386. doi: 10.1128/iai.64.7.2381-2386.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Junquero D. C., Scott-Burden T., Schini V. B., Vanhoutte P. M. Inhibition of cytokine-induced nitric oxide production by transforming growth factor-beta 1 in human smooth muscle cells. J Physiol. 1992 Aug;454:451–465. doi: 10.1113/jphysiol.1992.sp019273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kelly R. A., Balligand J. L., Smith T. W. Nitric oxide and cardiac function. Circ Res. 1996 Sep;79(3):363–380. doi: 10.1161/01.res.79.3.363. [DOI] [PubMed] [Google Scholar]
  23. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  24. Lane J. R., Neumann D. A., Lafond-Walker A., Herskowitz A., Rose N. R. Interleukin 1 or tumor necrosis factor can promote Coxsackie B3-induced myocarditis in resistant B10.A mice. J Exp Med. 1992 Apr 1;175(4):1123–1129. doi: 10.1084/jem.175.4.1123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Le J., Vilcek J. Tumor necrosis factor and interleukin 1: cytokines with multiple overlapping biological activities. Lab Invest. 1987 Mar;56(3):234–248. [PubMed] [Google Scholar]
  26. Masters D. B., Griggs C. T., Berde C. B. High sensitivity quantification of RNA from gels and autoradiograms with affordable optical scanning. Biotechniques. 1992 Jun;12(6):902-6, 908-11. [PubMed] [Google Scholar]
  27. Matsumori A., Yamada T., Suzuki H., Matoba Y., Sasayama S. Increased circulating cytokines in patients with myocarditis and cardiomyopathy. Br Heart J. 1994 Dec;72(6):561–566. doi: 10.1136/hrt.72.6.561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Murray D. R., Freeman G. L. Tumor necrosis factor-alpha induces a biphasic effect on myocardial contractility in conscious dogs. Circ Res. 1996 Jan;78(1):154–160. doi: 10.1161/01.res.78.1.154. [DOI] [PubMed] [Google Scholar]
  29. Pagani F. D., Baker L. S., Hsi C., Knox M., Fink M. P., Visner M. S. Left ventricular systolic and diastolic dysfunction after infusion of tumor necrosis factor-alpha in conscious dogs. J Clin Invest. 1992 Aug;90(2):389–398. doi: 10.1172/JCI115873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Postan M., McDaniel J. P., Dvorak J. A. Comparative studies of the infection of Lewis rats with four Trypanosoma cruzi clones. Trans R Soc Trop Med Hyg. 1987;81(3):415–419. doi: 10.1016/0035-9203(87)90155-6. [DOI] [PubMed] [Google Scholar]
  31. Rivera M. T., Marques de Araujo S., Lucas R., Deman J., Truyens C., Defresne M. P., de Baetselier P., Carlier Y. High tumor necrosis factor alpha (TNF-alpha) production in Trypanosoma cruzi-infected pregnant mice and increased TNF-alpha gene transcription in their offspring. Infect Immun. 1995 Feb;63(2):591–595. doi: 10.1128/iai.63.2.591-595.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rossi M. A., Bestetti R. B. The challenge of chagasic cardiomyopathy. The pathologic roles of autonomic abnormalities, autoimmune mechanisms and microvascular changes, and therapeutic implications. Cardiology. 1995;86(1):1–7. doi: 10.1159/000176822. [DOI] [PubMed] [Google Scholar]
  33. Rottenberg M. E., Castaños-Velez E., de Mesquita R., Laguardia O. G., Biberfeld P., Orn A. Intracellular co-localization of Trypanosoma cruzi and inducible nitric oxide synthase (iNOS): evidence for dual pathway of iNOS induction. Eur J Immunol. 1996 Dec;26(12):3203–3213. doi: 10.1002/eji.1830261254. [DOI] [PubMed] [Google Scholar]
  34. Schreck R., Baeuerle P. A. Assessing oxygen radicals as mediators in activation of inducible eukaryotic transcription factor NF-kappa B. Methods Enzymol. 1994;234:151–163. doi: 10.1016/0076-6879(94)34085-4. [DOI] [PubMed] [Google Scholar]
  35. Sehgal P. B. Regulation of IL6 gene expression. Res Immunol. 1992 Sep;143(7):724–734. doi: 10.1016/0923-2494(92)80011-9. [DOI] [PubMed] [Google Scholar]
  36. Starobinas N., Russo M., Minoprio P., Hontebeyrie-Joskowicz M. Is TNF alpha involved in early susceptibility of Trypanosoma cruzi-infected C3H/He mice? Res Immunol. 1991 Feb;142(2):117–122. doi: 10.1016/0923-2494(91)90019-f. [DOI] [PubMed] [Google Scholar]
  37. Stein B., Frank P., Schmitz W., Scholz H., Thoenes M. Endotoxin and cytokines induce direct cardiodepressive effects in mammalian cardiomyocytes via induction of nitric oxide synthase. J Mol Cell Cardiol. 1996 Aug;28(8):1631–1639. doi: 10.1006/jmcc.1996.0153. [DOI] [PubMed] [Google Scholar]
  38. Suffredini A. F., Fromm R. E., Parker M. M., Brenner M., Kovacs J. A., Wesley R. A., Parrillo J. E. The cardiovascular response of normal humans to the administration of endotoxin. N Engl J Med. 1989 Aug 3;321(5):280–287. doi: 10.1056/NEJM198908033210503. [DOI] [PubMed] [Google Scholar]
  39. Tanowitz H. B., Kirchhoff L. V., Simon D., Morris S. A., Weiss L. M., Wittner M. Chagas' disease. Clin Microbiol Rev. 1992 Oct;5(4):400–419. doi: 10.1128/cmr.5.4.400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tarleton R. L. Tumour necrosis factor (cachectin) production during experimental Chagas' disease. Clin Exp Immunol. 1988 Aug;73(2):186–190. [PMC free article] [PubMed] [Google Scholar]
  41. Titus R. G., Sherry B., Cerami A. The involvement of TNF, IL-1 and IL-6 in the immune response to protozoan parasites. Immunol Today. 1991 Mar;12(3):A13–A16. doi: 10.1016/S0167-5699(05)80005-2. [DOI] [PubMed] [Google Scholar]
  42. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Truyens C., Angelo-Barrios A., Torrico F., Van Damme J., Heremans H., Carlier Y. Interleukin-6 (IL-6) production in mice infected with Trypanosoma cruzi: effect of its paradoxical increase by anti-IL-6 monoclonal antibody treatment on infection and acute-phase and humoral immune responses. Infect Immun. 1994 Feb;62(2):692–696. doi: 10.1128/iai.62.2.692-696.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Xie Q. W., Kashiwabara Y., Nathan C. Role of transcription factor NF-kappa B/Rel in induction of nitric oxide synthase. J Biol Chem. 1994 Feb 18;269(7):4705–4708. [PubMed] [Google Scholar]
  45. Zentella A., Manogue K., Cerami A. The role of cachectin/TNF and other cytokines in sepsis. Prog Clin Biol Res. 1991;367:9–24. [PubMed] [Google Scholar]
  46. Zhang L., Tarleton R. L. Characterization of cytokine production in murine Trypanosoma cruzi infection by in situ immunocytochemistry: lack of association between susceptibility and type 2 cytokine production. Eur J Immunol. 1996 Jan;26(1):102–109. doi: 10.1002/eji.1830260116. [DOI] [PubMed] [Google Scholar]

Articles from The American Journal of Pathology are provided here courtesy of American Society for Investigative Pathology

RESOURCES