doi:10.1006/cyto.2002.1014, available online at http://www.idealibrary.com on THIOL ANTIOXIDANTS INHIBIT THE FORMATION OF THE INTERLEUKIN-12 HETERODIMER: A NOVEL MECHANISM FOR THE INHIBITION OF IL-12 PRODUCTION Daniela Mazzeo,† Silvano Sacco, 2 Pietro Di Lucia, 1 * Giuseppe Penna, 1 * Luciano Adorini, 1 * Paola Panina-Bordignon, 1 * Pietro Ghezzi 2 IL-12 is a 75 kDa heterodimeric cytokine composed of two disulfide-linked subunits, p35 and p40, which plays an important role in the regulation of the immune response. We tested the hypothesis that thiol antioxidants might interfere with dimerization of the two IL-12 subunits. We thus studied the effect of reduced glutathione (GSH) and N-acetyl-cysteine (NAC) on IL-12 p75 production by human THP-1 cell stimulated with IFN- and Staphylococcus aureus Cowan strain I (SAC), using ELISAs specific for IL-12 p75 or the p40 subunit. NAC and GSH, but not cystine, at concentrations of 5–10 mM inhibited production of IL-12 p75 but not of the p40 subunit. NAC did not inhibit p40 or p35 mRNA expression in dendritic cells or THP-1 cells, or NF-B activation in THP-1 cells. The effect of NAC was specific for IL-12 p75, as NAC did not affect induction of MHC class II expression by IFN--stimulated THP-1 cells. IL-12 dimer formation appears to be reduced by NAC also in vivo, because pretreatment with NAC (1 g/kg, orally), before LPS injection in mice, inhibited peak IL-12 p75 serum levels without affecting those of p40. We conclude that thiol levels regulate IL-12 p75 production and that assembly of the heterodimer is a step that might represent a target for pharmacological intervention.  2002 Elsevier Science Ltd. All rights reserved. IL-12 is a 75 kDa heterodimeric cytokine com- posed of two disulfide-linked glycosylated subunits, p35 (35 kDa) and p40 (40 kDa). 1 This cytokine, pro- duced by stimulated myelomonocytic cells 2 and den- dritic cells, 3 has an essential role in innate and adaptive immunity. IL-12 acts mainly on T and NK cells promoting their cytolitic activity and stimulating IFN- production. Furthermore, IL-12 plays a critical role in the development of T helper 1 (Th 1 ) cell- mediated immune responses. 4–6 IL-12-driven Th 1 responses can result in autoimmune disorders. 7 For this reason, a better understanding of the regulation of IL-12 production can have therapeutic relevance. The expression of p35 and p40 subunits is differ- entially regulated. While p35 subunit is secreted only as part of the heterodimer, p40 is also secreted, in large excess, as free subunit. 2 This suggests that p35 could be a limiting factor for the formation of the heterodimer. 8 Previous studies have shown that the expression of p40 and p35 subunits is regulated at the transcriptional level. 9 Two recent papers 10,11 indicate that p35 secretion is also regulated post-transcriptionally. The processing of p35 signal peptide requires two sequen- tial cleavages occurring in the ER first, and then in the Golgi compartment. Furthermore, glycosylation and sialylation are also required for the secretion of the p35 subunit. We investigated if the formation of the IL-12 p75 heterodimer might be influenced by the redox status of the cell. Our attention focused on the intra- cellular levels of the major water-soluble, non-protein sulphydryl compound, the tripeptide glutathione (GSH, -glutamyl-cysteinyl-glycine). Formation of From the 1 Roche Milano Ricerche, 20132 Milano, Italy; and 2 Laboratory Neuroimmunology, Istituto di Ricerche Farmacologiche ‘‘Mario Negri’’, 20157 Milano, Italy Correspondence to: Pietro Ghezzi, ‘‘Mario Negri’’ Institute for Pharmacological Research, via Eritrea 62, 20157 Milan, Italy. E-mail: ghezzi@marionegri.it, or Paola Panina-Bordignon, Ph.D., Roche Milano Ricerche, Via Olgettina 58, I-20132 Milan, Italy. E-mail: paola.panina@bioxell.com †Present address: Division of Cell Biology and Immunology, Wellcome Trust Biocentre, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK *Present address: BioXell SpA, Via Olgettina 58 I-20129 Milano, Italy Abbreviations: GSH, glutathione; GSSG, oxidized glutathione; NAC, N-acetyl-cysteine; PDTC, pirrolidine dithiocarbamate; SAC, Staphilococcus aureus Cowan strain I Received 15 November 2001; received in revised form 4 February 2002; accepted for publication 6 February 2002 1043–4666/02/$-see front matter  2002 Elsevier Science Ltd. All rights reserved. KEY WORDS: IL-12/N-acetylcysteine/glutathione CYTOKINE, Vol. 17, No. 6 (21 March), 2002: pp 285–293 285