Skip to main content
Log in

Quantitative PCR Analysis of TNF-α and IL-1β mRNA Levels in Pediatric IBD Mucosal Biopsies

  • Published:
Digestive Diseases and Sciences Aims and scope Submit manuscript

Abstract

Inflammatory bowel disease (IBD) is associatedwith increased activation of intestinal immune cells,whose overproduction of proinflammatory cytokines suchas tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) is implicatedin mediating the sustained inflammatory response.Studies to date have largely reported qualitativedifferences in cytokine gene expression between IBD andcontrols. Our aim was to perform quantitative analysis ofintestinal mucosal mRNA expression in colonic biopsiesfrom pediatric IBD patients using a competitivepolymerase chain reaction. IL-1β and TNF-αwere expressed in all IBD and control biopsies.Compared to controls, IL-1β mRNA levels wereincreased in involved tissue from Crohn's disease (CD)patients, but not in histologically uninvolved CD or in ulcerative colitis (UC) mucosa. IL-1βexpression in the latter groups were equivalent to thosefound in tissue from patients with eosinophilic colitis(EOC). Significantly higher levels of IL-1β mRNA were found in uninvolved mucosa from CDpatients who presented with a relapse of diseaseactivity, as compared to newly diagnosed cases withhistological features of CD at an early stage.TNF-α mRNA transcripts were also significantly elevated ininvolved CD mucosa, but not in the other groups.TNF-α gene expression in CD-involved tissuedecreased with disease duration. Follow-up of thepatients revealed that high cytokine expression inuninvolved CD tissue correlated with an early clinicalrelapse. In conclusion, quantitative determination ofproinflammatory cytokine gene expression revealsdifferences between the type, severity, and clinical coursein patients with IBD.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

REFERENCES

  1. Strober W, Ehrhardt RO: Chronic intestinal inflammation: An unexpected outcome in cytokine or T cell receptor mutant mice. Cell 75:203–205, 1993

    Google Scholar 

  2. Podolsky DK: Inflammatory bowel disease. N Engl J Med 325:928–937, 1991

    Google Scholar 

  3. Seidman E: Epidemiology and current etiological concepts. In Pediatric Inflammatory Bowel Disease. J Levine (ed). Philadelphia, Lippincott, 1994, pp 1–14

    Google Scholar 

  4. Braegger CP: Immunopathogenesis of chronic inflammatory bowel disease. Acta Paediatr 395:18–21, 1994

    Google Scholar 

  5. Schreiber S, Raedler A, Stenson WF, MacDermott RP: The role of mucosal immune system in inflammatory bowel disease. Gastroenterol Clin North Am 21:451–502, 1992

    Google Scholar 

  6. Sartor RB: Current concepts of the etiology and pathogenesis of ulcerative colitis and Crohn's disease. Gastroenterol Clin North Am 24:475–507, 1995

    Google Scholar 

  7. Sadlack B, Merz H, Schorle H, Schimpl A, Feller AC, Horak I: Ulcerative colitis-like disease in mice with disrupted interleukin-2 gene. Cell 75:253–261, 1993

    Google Scholar 

  8. Kühn R, Löhler J, Rennick D, Rajewsky K, Müller W: Interleukin-10 deficient mice develop chronic enterocolitis. Cell 75:263–274, 1993

    Google Scholar 

  9. Sartor B: Cytokines in intestinal inflammation: Pathophysiological and clinical considerations. Gastroenterology 106:533–539, 1994

    Google Scholar 

  10. McCabe RP, Secrist H, Botney M, Egan M, Peters MG: Cytokine mRNA expression in intestine from normal and inflammatory bowel disease patients. Clin Immunol Immunopathol 66:52–58, 1993

    Google Scholar 

  11. Brynskov J, Nielsen OH, Ahnfelt-Rønne I, Bendtzen K: Cytokines in inflammatory bowel disease. Scand J Gastroenterol 27:897–906, 1992

    Google Scholar 

  12. Beagley KW, Elson CO: Cells and cytokines in mucosal immunity and inflammation. Gastroenterol Clin North Am 21:347–366, 1992

    Google Scholar 

  13. Braegger CD, Nicholls S, Murch SH, Stephens S, MacDonald TT: Tumor necrosis factor alpha in stool as a marker of intestinal inflammation. Cancer 339:89–91, 1992

    Google Scholar 

  14. Murch SH, Lamkin VA, Savage MO, Walker-Smith JA, MacDonald TT: Serum concentrations of tumor necrosis factor alpha in childhood chronic inflammatory bowel disease. Gut 32:913–917, 1991

    Google Scholar 

  15. Hyams J, Treem W, Eddy E, Wyzgz N, Moore R: Tumor necrosis factor alpha is not elevated in children with inflammatory bowel disease. J Pediatr Gastroenterol Nutr 12:233–276, 1991

    Google Scholar 

  16. Capello M, Keshav S, Prince C, Jewell DP, Gordon S: Detection of mRNAs for macrophage products in inflammatory bowel disease by in situ hybridization. Gut 33:1214–1219, 1992

    Google Scholar 

  17. Stevens C, Walz G, Singaram C, Lipman ML, Zanker B, Muggia A, Antonioli D, Peppercorn MA, Strom TB: Tumor necrosis factor-α, interleukin-1β, and interleukin-6 expression in inflammatory bowel disease. Dig Dis Sci 37:818–826, 1992

    Google Scholar 

  18. Isaacs KL, Sartor RB, Haskill S: Cytokine messenger RNA profiles in inflammatory bowel disease mucosa detected by polymerase chain reaction amplification. Gastroenterology 103:1587–1595, 1992

    Google Scholar 

  19. Mahida YR, Wu K, Jewell DP: Enhanced production of interleukin-1β by mononuclear cells isolated from mucosa with ulcerative colitis or Crohn's disease. Gut 30:835–838, 1989

    Google Scholar 

  20. Ligumski M, Simon DL, Karmeli F, Rachmilewitz D: Role of interleukin 1 in inflammatory bowel disease-enhanced production during active disease. Gut 31:686–689, 1990

    Google Scholar 

  21. Mahida YR, Lamming CED, Gallagher A, Hawthorne A, Hawkey CJ: 5-Aminosalicylic acid is a potent inhibitor of interleukin 1β production in organ culture of colonic biopsy specimens from patients with inflammatory bowel disease. Gut 32:50–54, 1991

    Google Scholar 

  22. Brynskov J, Trede N, Vilien M, Andersen CB, Bentzen K: Increased concentrations of interleukin 1β, interleukin 2, and soluble interleukin-2 receptors in endoscopical mucosal biopsy specimens with active inflammatory bowel disease. Gut 33:55–58, 1992

    Google Scholar 

  23. Youngman KR, Simon PL, West GA, Cominelli F, Rachmilewitz D, Klein JS, Fiocchi C: Localization of intestinal interleukin 1 activity and protein and gene expression to lamina propria cells. Gastroenterology 104:749–758, 1993

    Google Scholar 

  24. Reinecker HC, Steffen M, Witthoef T, Pflueger I, Schreiber S, MacDermott RP, Raedler A: Enhanced secretion of tumor necrosis factor-alpha, IL-6, and IL-1β by isolated lamina propria mononuclear cells from patients with ulcerative colitis and Crohn's disease. Clin Exp Immunol 94:174–181, 1993

    Google Scholar 

  25. Mullin GE, Lazenby AJ, Harris MC, Bayless TM, James SP: Increased interleukin 2 messenger RNA in the intestinal mucosal lesions of Crohn's disease but not ulcerative colitis. Gastroenterology 102:1620–1627, 1992

    Google Scholar 

  26. Izutani R, Ohyanagi H, MacDermott RP: Quantitative PCR for detection of femtogram quantities of interleukin-8 mRNA expression. Microbiol Immunol 38:233–237, 1994

    Google Scholar 

  27. Andus T, Targan SR, Deem R, Toyoda H: Measurement of tumor necrosis factor α mRNA in small numbers of cells by quantitative polymerase chain reaction. Reg Immunol 5:11–17, 1993

    Google Scholar 

  28. de Dombal FT, Burton IL, Clamp SE, Goligher JC: Short term course and prognosis of Crohn's disease. Gut 15:435–441, 1974

    Google Scholar 

  29. Chomczynski P: A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. Biotechniques 15:532–535, 1993

    Google Scholar 

  30. Gurbindo C, Russo P, Sabbah S, Lohoues MJ, Seidman E: Interleukin-2 activity of colonic lamina propria mononuclear cells in a rat model of experimental colitis. Gastroenterology 104:964–972, 1993

    Google Scholar 

  31. McCall RD, Haskill S, Zimmermann EM, Lund PK, Thompson RC, Sartor RB: Tissue interleukin 1 and interleukin-1 receptor antagonist expression in enterocolitis in resistant and susceptible rats. Gastroenterology 106:960–972, 1994

    Google Scholar 

  32. Siebert PD: Quantitative RT-PCR. Methods and Applications, book 3. Clontech Laboratories, Palo Alto, 1993

    Google Scholar 

  33. Innis MA, Gelfand DH: Optimization of PCRs. In PCR Protocols: A guide to methods and applications. MA Innis, DH Gelfand, JJ Sninsky, TJ White (eds). San Diego, Academic Press, 1990, pp 3–12

    Google Scholar 

  34. Landgraf A, Wolfes H: Taq polymerase (EC 2.7.7.7.). With particular emphasis on its use in PCR protocols. Methods Mol Biol 16:31–58, 1993

    Google Scholar 

  35. Wang AM, Doyle MV, Mark DF: Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci USA 86:9717–9721, 1989

    Google Scholar 

  36. Siebert PD, Larrick JW: Competitive PCR. Nature 359:557–558, 1992

    Google Scholar 

  37. He Q, Marjamäki M, Soini H, Mertsola J, Viljanen MK: Primers are decisive for sensitivity of PCR. Biotechniques 17:82–86, 1994

    Google Scholar 

  38. Wiesner RJ: Direct quantification of picomolar concentrations of mRNAs by mathematical analysis of a reverse transcription/exponential polymerase chain reaction assay. Nucleic Acids Res 20:5863–5864, 1992

    Google Scholar 

  39. Robinson MO, Simon MI: Determining transcript number using the polymerase chain reaction: PgK-2, mPz, and PGK-2 transgene mRNA levels during spermatogenesis. Nucleic Acids Res 19:1557–1562, 1991

    Google Scholar 

  40. Siebert PD, Larrick JW: PCR mimics: Competitive DNA fragment for use as internal standards in quantitative PCR. Biotechniques 14:244–249, 1993

    Google Scholar 

  41. Trapnell BC: Quantitative evaluation of gene expression in freshly isolated human respiratory epithelial cells. Am J Physiol 264:L199–L212, 1993

    Google Scholar 

  42. Grayson DR, Bovolin P, Santi MR: Absolute quantitation of γ-aminobutyric acid A receptor subunit messenger RNAs by competitive polymerase chain reaction. Methods Neurosci 12:191–207, 1993

    Google Scholar 

  43. Becker-André M: Absolute levels of mRNA by polymerase chain reaction-aided transcript titration assay. Methods Enzymol 218:420–445, 1993

    Google Scholar 

  44. Scheuermannn RH, Bauer SR: Polymerase chain reaction-based mRNA quantitation using an internal standard: analysis of oncogene expression. Methods Enzymol 218:446–473, 1993

    Google Scholar 

  45. D'Addario M, Roulston A, Wainberg MA, Hiscott J: Coordinate enhancement of cytokine gene expression in human immunodeficiency virus type 1 infected promonocytic cells. J Virol 64:6080–6089, 1990

    Google Scholar 

  46. Kotewicz ML, Sampson CM, D'Alessio JM, Gerard GF: Isolation of cloned moloney murine leukemia virus reverse transcriptase lacking ribonuclease H activity. Nucleic Acids Res 16:265–277, 1988

    Google Scholar 

  47. Ma TS, Brink PA, Perryman B, Roberts R: Improved quantification with validation of multiple mRNA species by polymerase chain reaction: Application to human myocardial creatine kinase M and B. Cardiovasc Res 28:464–471, 1994

    Google Scholar 

  48. Surawicz CM, Haggitt RC, Husseman M, McFarland LV: Mucosal biopsy diagnosis of colitis: acute self-limited colitis and idiopathic inflammatory bowel disease. Gastroenterology 107:755–763, 1994

    Google Scholar 

  49. Shanahan F: Pathogenesis of ulcerative colitis. Lancet 342: 407–411, 1993

    Google Scholar 

  50. Breese E, Braegger CP, Corrigan CJ, Walker-Smith JA, MacDonald TT: Interleukin-2 and interferon-γ secreting T cells in normal and diseased human intestinal mucosa. Immunology 78:127–131, 1993

    Google Scholar 

  51. Lionetti P, Breese E, Braegger CP, Murch SH, Taylor J, MacDonald TT: T-cell activation can induce either mucosal destruction or adaptation in cultured human fetal small intestine. Gastroenterology 105:373–381, 1993

    Google Scholar 

  52. Mahida YR: The role of macrophages in the pathogenesis of inflammatory bowel disease. Can J Gastroenterol 7:83–86, 1993

    Google Scholar 

  53. Weldon MJ, Maxwell JD: Lymphocyte and macrophage interleukin receptors in inflammatory bowel disease: A more selective target for therapy? Gut 35:867–871, 1994

    Google Scholar 

  54. Thomas TK, Will PC, Srivastava A: Evaluation of an interleukin-1 receptor antagonist in the rat acetic acid-induced colitis model. Agents Actions 34:187–190, 1991

    Google Scholar 

  55. Cominelli F, Nast CC, Duchini A, Lee M: Recombinant interleukin-1 receptor antagonist blocks the proinflammatory activity of endogenous interleukin-1 in rabbit immune colitis. Gastroenterology 103:65–71, 1992

    Google Scholar 

  56. Mazlam MZ, Hodgson HSF: Interrelations between interleukin-6, interleukin-1β, plasma C-reactive protein values, and in vitro C-reactive protein generation in patients with inflammatory bowel disease. Gut 35:177–183, 1994

    Google Scholar 

  57. Olson AD, Ayass M, Chensue S: Tumor necrosis factor and IL-1β expression in pediatric patients with inflammatory bowel disease. J Pediatr Gastroenterol Nutr 16:241–246, 1993

    Google Scholar 

  58. Murch SH, Braegger CP, Walker-Smith J, MacDonald TT: Location of tumor necrosis factor α by immunohistochemistry in chronic inflammatory bowel disease. Gut 34:1705–1709, 1993

    Google Scholar 

  59. Breese EJ, Michie CA, Nicholls SW, Murch SH, Williams CB, Domizio P, Walker-Smith JA, MacDonald TT: Tumor necrosis factor α-producing cells in the intestinal mucosa of children with inflammatory bowel disease. Gastroenterology 106:1455–1466, 1994

    Google Scholar 

  60. Plevy SE, Targan SR, Deem RL, Toyoda H: Increased mucosal TNFα mRNA levels and numbers of TNFα producing cells are unique to Crohn's disease mucosal inflammation. Gastroenterology 106:A754, 1994

    Google Scholar 

  61. Schreiber S, Koop I, Bauditz J, Nikolaus S, Lochs H, Raedler A: Increased secretion of pro-inflammatory cytokines by LPMNC is a predictor for relapse of IBD. Gastroenterology 108:A913, 1995

    Google Scholar 

Download references

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Dionne, S., Hiscott, J., D'Agata, I. et al. Quantitative PCR Analysis of TNF-α and IL-1β mRNA Levels in Pediatric IBD Mucosal Biopsies. Dig Dis Sci 42, 1557–1566 (1997). https://doi.org/10.1023/A:1018895500721

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1018895500721

Navigation