THE MOUSE INTERLEUKIN 1 RECEPT ANTAGONIST PROTEIN: GENE REGULATION IN VITRO Kamyar A. Zahedi,’ Clarissa M. Uhlar,2 Miriam Rits,3 Anne E. Prada,’ Alexander S. Whitehead The interleukin 1 receptor antagonist (IL-lra) protein is an inhibitor of the pro-inflamma- tory cytokine interleukin 1. We have sequenced the mouse gene encoding the monocyte form of IL-lra (IL-lrn) and compared it with the sequence of the human homologue. In addition to high levels of similarity between the coding regions of the two genes, portions of the introns show surprisingly high levels of identity. In order to develop an in vitro model system to investigate the regulation of IL-lra induction, three differently responding mouse macro- phage cell lines were stimulated with lipopolysaccharide. The kinetics and magnitude of IL- lra mRNA accumulation was cell-line specific indicating that IL-lra synthesis in response to inducing agents varies according to the phenotype of the cell. Analysis of the relative tran- scription rate and the half life of the mouse IL-lra mRNA indicate that IL-lra mRNA accumulation in macrophages following LPS treatment is due primarily to an increase in transcription rate rather than to increased stability. Inflammation is a complex physiological response to infection or trauma and is initiated, maintained and coordinated principally by a range of mediator mol- ecules (cytokines).’ The action of proinflammatory cytokines is modified, diminished and superceded by a variety of counterbalancing mechanisms including cytokine antagonists. The interleukin 1 receptor an- tagonist protein (IL-lra) is a 22-25 Kd glycoprotein with potent anti-inflammatory activities.233 It is a member of the interleukin 1 (IL-l) gene family4 which also includes the two isoforms of the proinflammatory cytokine IL-1 (IL-lcx and IL-1B). IL-lra exhibits a moderate degree of amino acid sequence similarity to both IL-la and IL-lp.2,5 In the mouse, the IL-lra is encoded by a single gene (IL-lm) on chromosome 2;6 From the 1 Department of Pediatrics, University of Cincinnati Medical School, and the Division of Nephrology, The Childrens Hospital Research Foundation, Cincinnati, OH, USA; ’ Depart- ment of Genetics and Biotechnology Institute, Trinity College, University of Dublin, Dublin, Ireland; ’ Division of Hematology/ Oncology, Children’s Hospital, Boston, MA, USA Correspondence to: Alexander S. Whitehead, Department of Gen- etics, Trinity College, University of Dublin, Lincoln Place Gate, Dublin 2, Ireland Received 7 April 1993; revised and accepted for publication 14 June 1993 0 1994 Academic Press Limited 1043-4666/94/010001+09 $08.00/O KEY WORDS: Mouse ILlra/gene structure/gene expressiomin- flammatory mediators CYTOKINE, Vol. 6, No. 1 (January), 1994: pp 1-9 the genes encoding IL-la and IL-1B are also located on mouse chromosome 2,7 though not in close link- age. All three human genes have remained in close linkage on chromosome 2q14-q21.’ Many in vivo functions of IL-l, including its ability to induce neutrophilia, and to induce an acute phase response can be blocked by IL-lra.539 Adminis- tration of recombinant IL-lra also reduces mortality from endotoxic shock in rabbits’“7” and mice.” In vitro, IL-lra inhibits the IL-l mediated induction of collagenase and PGEz production by synovial cells and fibroblasts’3,‘4 and abrogates the synthesis of gly- cosaminoglycans by human articular cartilage cul- tures. l5 IL-1 acts on target cells by binding to surface receptors through which a pro-inflammatory signal is transduced. Two types of IL-l receptor have been characterized: type I expressed on T-cells, fibroblasts and endothelial cells, and type II present on B-cells, neutrophils and macrophages.r6, i7 Each bind both IL- la and IL-1B. i8,r9 IL-lra abrogates the proinflamma- tory activities of both IL-la and IL-1B by competitive inhibition of their binding to IL-1 receptors.3320 Although IL-lra binds to both type I and type II IL-1 receptors, 21 its association with the type II receptor is of very low avidity. Consequently it fails to block the binding of IL-la or IL-lp and the resulting transduc- tion of signal; its interaction with the type II receptor may therefore be physiologically insignificant.3.53921 IL-lra, like IL-l, is primarily synthesized by cells 1