Search tips
Search criteria 


Logo of molmedLink to Publisher's site
Mol Med. 1999 November; 5(11): 721–730.
PMCID: PMC2230482

Zinc finger transcription factors as molecular targets for nitric oxide-mediated immunosuppression: inhibition of IL-2 gene expression in murine lymphocytes.


BACKGROUND: Nitric oxide (NO) has frequently been shown to display immunosuppressive activities. We describe here a molecular mechanism contributing to this effect. MATERIALS AND METHODS: Murine T cell lymphoma EL4-6.1 cells were activated with the physiological stimulus interleukin (IL)-1beta to express IL-2 mRNA in the presence or absence of subtoxic concentrations of the physiological spontaneous NO donor S-nitrosocysteine (SNOC). Subsequently, semiquantitative RT-PCR and gel shift assays with nuclear extracts were performed to analyze the effects of NO on IL-2 mRNA expression and on the activity of the dominant regulating transcription factors Sp1, EGR-1, and NFATc. RESULTS: NO inhibits IL-1beta-induced IL-2 mRNA expression in EL4-6.1 cells. The suppressive activity of NO was concentration dependent and found to be completely reversible. Importantly, NO at the concentrations used induced neither apoptosis nor necrosis. Dominant regulation of IL-2 gene expression is known to reside in the zinc finger transcription factors Sp1 or EGR-1 and in the non-zinc finger protein NFAT. NO abrogates the DNA binding activities of recombinant Sp1 and EGR-1. More importantly, gel shift assays also showed a lack of DNA binding of native Sp1 derived from NO-treated nuclear extracts and that from NO-treated viable lymphocytes. This effect is selective, as the DNA binding activity of recombinant NFATc was not affected by NO. CONCLUSION: Inactivation of zinc finger transcription factors by NO appears to be a molecular mechanism in the immunosuppressive activity of NO in mammals, thus contributing to NO-mediated inhibition of IL-2 gene expression after physiological stimuli. The exact understanding of the molecular mechanism leading to NO-mediated, fully reversible suppression of immune reactions may lead to use of this naturally occurring tool as an aid in inflammatory diseases.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (2.1M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J. 1992 Sep;6(12):3051–3064. [PubMed]
  • Kröncke KD, Fehsel K, Kolb-Bachofen V. Inducible nitric oxide synthase and its product nitric oxide, a small molecule with complex biological activities. Biol Chem Hoppe Seyler. 1995 Jun;376(6):327–343. [PubMed]
  • Kolb H, Kolb-Bachofen V. Nitric oxide: a pathogenetic factor in autoimmunity. Immunol Today. 1992 May;13(5):157–160. [PubMed]
  • Bogdan C. The multiplex function of nitric oxide in (auto)immunity. J Exp Med. 1998 May 4;187(9):1361–1365. [PMC free article] [PubMed]
  • Kröncke KD, Fehsel K, Kolb-Bachofen V. Inducible nitric oxide synthase in human diseases. Clin Exp Immunol. 1998 Aug;113(2):147–156. [PubMed]
  • Wink DA, Mitchell JB. Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic Biol Med. 1998 Sep;25(4-5):434–456. [PubMed]
  • Hoffman RA, Langrehr JM, Billiar TR, Curran RD, Simmons RL. Alloantigen-induced activation of rat splenocytes is regulated by the oxidative metabolism of L-arginine. J Immunol. 1990 Oct 1;145(7):2220–2226. [PubMed]
  • Mills CD. Molecular basis of "suppressor" macrophages. Arginine metabolism via the nitric oxide synthetase pathway. J Immunol. 1991 Apr 15;146(8):2719–2723. [PubMed]
  • al-Ramadi BK, Meissler JJ, Jr, Huang D, Eisenstein TK. Immunosuppression induced by nitric oxide and its inhibition by interleukin-4. Eur J Immunol. 1992 Sep;22(9):2249–2254. [PubMed]
  • Sternberg J, McGuigan F. Nitric oxide mediates suppression of T cell responses in murine Trypanosoma brucei infection. Eur J Immunol. 1992 Oct;22(10):2741–2744. [PubMed]
  • Kolb H, Kolb-Bachofen V. Nitric oxide in autoimmune disease: cytotoxic or regulatory mediator? Immunol Today. 1998 Dec;19(12):556–561. [PubMed]
  • Marcinkiewicz J, Grabowska A, Chain BM. Is there a role for nitric oxide in regulation of T cell secretion of IL-2? J Immunol. 1996 Jun 15;156(12):4617–4621. [PubMed]
  • Taylor-Robinson AW. Inhibition of IL-2 production by nitric oxide: a novel self-regulatory mechanism for Th1 cell proliferation. Immunol Cell Biol. 1997 Apr;75(2):167–175. [PubMed]
  • Chang RH, Feng MH, Liu WH, Lai MZ. Nitric oxide increased interleukin-4 expression in T lymphocytes. Immunology. 1997 Mar;90(3):364–369. [PubMed]
  • Bauer H, Jung T, Tsikas D, Stichtenoth DO, Frölich JC, Neumann C. Nitric oxide inhibits the secretion of T-helper 1- and T-helper 2-associated cytokines in activated human T cells. Immunology. 1997 Feb;90(2):205–211. [PubMed]
  • van der Veen RC, Dietlin TA, Pen L, Gray JD. Nitric oxide inhibits the proliferation of T-helper 1 and 2 lymphocytes without reduction in cytokine secretion. Cell Immunol. 1999 May 1;193(2):194–201. [PubMed]
  • Serfling E, Avots A, Neumann M. The architecture of the interleukin-2 promoter: a reflection of T lymphocyte activation. Biochim Biophys Acta. 1995 Sep 19;1263(3):181–200. [PubMed]
  • Kadonaga JT, Carner KR, Masiarz FR, Tjian R. Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain. Cell. 1987 Dec 24;51(6):1079–1090. [PubMed]
  • Klug A, Schwabe JW. Protein motifs 5. Zinc fingers. FASEB J. 1995 May;9(8):597–604. [PubMed]
  • Skerka C, Decker EL, Zipfel PF. A regulatory element in the human interleukin 2 gene promoter is a binding site for the zinc finger proteins Sp1 and EGR-1. J Biol Chem. 1995 Sep 22;270(38):22500–22506. [PubMed]
  • Decker EL, Skerka C, Zipfel PF. The early growth response protein (EGR-1) regulates interleukin-2 transcription by synergistic interaction with the nuclear factor of activated T cells. J Biol Chem. 1998 Oct 9;273(41):26923–26930. [PubMed]
  • Kröncke KD, Fehsel K, Schmidt T, Zenke FT, Dasting I, Wesener JR, Bettermann H, Breunig KD, Kolb-Bachofen V. Nitric oxide destroys zinc-sulfur clusters inducing zinc release from metallothionein and inhibition of the zinc finger-type yeast transcription activator LAC9. Biochem Biophys Res Commun. 1994 Apr 29;200(2):1105–1110. [PubMed]
  • Berendji D, Kolb-Bachofen V, Meyer KL, Grapenthin O, Weber H, Wahn V, Kröncke KD. Nitric oxide mediates intracytoplasmic and intranuclear zinc release. FEBS Lett. 1997 Mar 17;405(1):37–41. [PubMed]
  • Kröncke KD, Kolb-Bachofen V. Detection of nitric oxide interaction with zinc finger proteins. Methods Enzymol. 1996;269:279–284. [PubMed]
  • 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. [PubMed]
  • Gubler U, Hoffman BJ. A simple and very efficient method for generating cDNA libraries. Gene. 1983 Nov;25(2-3):263–269. [PubMed]
  • Kashima N, Nishi-Takaoka C, Fujita T, Taki S, Yamada G, Hamuro J, Taniguchi T. Unique structure of murine interleukin-2 as deduced from cloned cDNAs. Nature. 313(6001):402–404. [PubMed]
  • LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed]
  • Lowenthal JW, Cerottini JC, MacDonald HR. Interleukin 1-dependent induction of both interleukin 2 secretion and interleukin 2 receptor expression by thymoma cells. J Immunol. 1986 Aug 15;137(4):1226–1231. [PubMed]
  • Knoepfel L, Steinkühler C, Carrì MT, Rotilio G. Role of zinc-coordination and of the glutathione redox couple in the redox susceptibility of human transcription factor Sp1. Biochem Biophys Res Commun. 1994 Jun 15;201(2):871–877. [PubMed]
  • Ammendola R, Mesuraca M, Russo T, Cimino F. The DNA-binding efficiency of Sp1 is affected by redox changes. Eur J Biochem. 1994 Oct 1;225(1):483–489. [PubMed]
  • Peng HB, Libby P, Liao JK. Induction and stabilization of I kappa B alpha by nitric oxide mediates inhibition of NF-kappa B. J Biol Chem. 1995 Jun 9;270(23):14214–14219. [PubMed]
  • Matthews JR, Botting CH, Panico M, Morris HR, Hay RT. Inhibition of NF-kappaB DNA binding by nitric oxide. Nucleic Acids Res. 1996 Jun 15;24(12):2236–2242. [PMC free article] [PubMed]
  • Tabuchi A, Sano K, Oh E, Tsuchiya T, Tsuda M. Modulation of AP-1 activity by nitric oxide (NO) in vitro: NO-mediated modulation of AP-1. FEBS Lett. 1994 Aug 29;351(1):123–127. [PubMed]
  • Chen L, Glover JN, Hogan PG, Rao A, Harrison SC. Structure of the DNA-binding domains from NFAT, Fos and Jun bound specifically to DNA. Nature. 1998 Mar 5;392(6671):42–48. [PubMed]

Articles from Molecular Medicine are provided here courtesy of The Feinstein Institute for Medical Research at North Shore LIJ