In these studies, we have identified DNA sequences and specific protein interactions necessary for transcriptional regulation of the human prointerleukin 113 (proIL-i1i) gene. A cell-type-independent lipopolysaccharide (LPS)-responsive enhancer element located between -3757 and -2729 bp upstream from the transcription start site (cap site) consisted of at least six discrete subregions which were essential to the maximal induction by LPS in transfected monocytes. The enhancer also appeared to mediate phorbol myristate acetate induction in monocytes and IL-1 responsiveness in fibroblasts. Deletion and base substitution mutations along with DNA binding studies demonstrated that the enhancer contained a minimum of three functional protein binding sequences, two of which appeared to be important for gene induction. One of the essential proteins which bound to the enhancer was similar or identical to members of the C/EBP family of transcription factors required for both IL-1-and LPS-specific induction of the IL-6 gene (i.e., the NF-IL6 proteins). When ligated to the proIL-1l3 cap site-proximal region (located between -131 to +12), both the proIL-1,3 and the simian virus 40 enhancer elements functioned more efficiently in monocytes than in HeLa cells, which are not normally competent for IL-11 expression. When ligated to the murine c-fos promoter, however, the proIL-1j3 enhancer was inducible in phorbol myristate acetate-stimulated HeLa cells, suggesting the existence of a proIL-1,3 promoter-proximal requirement for tissue specificity.Interleukin 11 (IL-113), a 17-kDa cytokine involved in inflammatory and immunological processes, is produced by activated monocytes/macrophages, fibroblasts, endothelial cells, and other cell types. The IL-1 proteins are translated as larger 31-kDa precursors (proIL-1) which are extracellularly processed into smaller 17-kDa forms (mature IL-1) (4, 20). There are two proIL-1 genes (proIL-lco and proIL-113) coding for distinct proteins which are observed in many different species (7, 18). The two proIL-1 genes are located on human chromosome 2 (11, 30, 48) and show a high conservation of exon/intron structure (7). IL-1 has a variety of biological activities on a wide range of tissues (see reference 13 for a review). For example, IL-1 induces the production of inflammatory mediators, proteolytic enzymes, and other cytokines that are involved in the joint destruction and inflammation of rheumatoid arthritis (29). IL-1 also modifies connective tissue and bone metabolism through the induction and type switch of collagen and the induction of bone resorption (21).The proIL-113 gene is normally not transcribed in competent cells until activated by stimuli such as lipopolysaccharide (LPS) or phorbol 12-myristate acetate (PMA) or by 17-kDa IL-11 protein (9,13,14 with LPS, IL-1P mRNA is rapidly and transiently transcribed in the absence of protein synthesis (15), suggesting the activation of preexisting transcription factors. Our previous studies revealed the importance of promoter sequences immediat...

Interleukin 1 (IL-1) induces the synthesis of kappa immunoglobulin light chains and the expression of surface immunoglobulin in the murine pre-B-cell line 70Z/3 (J. G. Giri, P. W. Kincade, and S. B. Mizel, J. Immunol. 132:223-228, 1984). In the present study, we found that these effects of IL-1 are mimicked by cyclic AMP (cAMP) analogs and cAMP-elevating drugs. The induction of kappa immunoglobulin light-chain gene expression by IL-1 was associated with an increase in intracellular cAMP levels. Incubation of 70Z/3 cells with IL-1 or cAMP resulted in the activation of the kappa immunoglobulin enhancer, as detected by the induction of chloramphenicol acetyltransferase (CAT) in cells transfected with a kappa enhancer-CAT expression plasmid. In contrast, CAT plasmids lacking a kappa immunoglobulin enhancer were inactive in the presence of IL-1 or cAMP. Furthermore, IL-1 and cAMP analogs and inducers were found to induce the activation of a NF-kappa B-like DNA-binding protein that exhibited specificity for the kappa immunoglobulin enhancer. These results suggest that cAMP may play an important role as a second messenger for IL-1 in the induction of kappa immunoglobulin light-chain synthesis in pre-B cells via the activation of a DNA-binding protein that is similar or identical to NF-kappa B.

We demonstrated that interleukin 1 (IL-1), a potent peptide mediator in immune and inflammatory responses, stimulates the synthesis of cAMP in a variety of IL-i-responsive cell targets. We also showed that cAMP analogs and cAMP-inducing agents can replace IL-1 in the induction of interleukin 2 receptors on lymphocytes as well as in phytohemagglutinin-induced murine thymocyte proliferation. By use of IL-1 and the cAMP-inducer, forskolin, a direct correlation between the induced level of cAMP and the degree of lymphocyte interleukin 2 receptor expression or thymocyte proliferation was established. Our results indicate that cAMP may be an important intracellular second messenger for IL-1.Interleukin 1 (IL-1), a peptide hormone produced by macrophages as well as several other cell types, stimulates the growth and differentiation of numerous cell types including lymphocytes, neutrophils, fibroblasts, synovial cells, osteoclasts, hepatocytes, and adipocytes (1-4) as well as being a potent fever inducer (5). In addition, IL-1 has been suggested to play an important role in the pathophysiology of a variety of chronic inflammatory diseases (6, 7).Within the last few years, tremendous progress has been made in our understanding of the biological and biochemical properties of IL-1. Two forms of IL-1, termed a and ,B, were cloned, sequenced, and expressed (8-12), and the receptor for IL-1 was characterized (13-15). However, the events that occur after the interaction of IL-1 with its receptor have not been elucidated. Several studies have shown that IL-1 can induce the expression of interleukin 2 receptors (IL-2R) on lymphocytes (16,17). Recently, we reported that YT cells, a human natural killer-like cell line, possess IL-1 receptors and also increase their expression of IL-2R in response to (18).cAMP Assay. Cells were incubated in the presence or absence of IL-1 and drugs at 37°C for varying periods. The incubations were stopped by the addition of ice-cold C13CCOOH (final concentration, 6%). The cells were then sonicated in the C13CCOOH, and the particulate material was removed by centrifugation. The Cl3CCOOH-soluble fraction was extracted once with ether before measurement of cAMP content using an RIA kit (Amersham/Searle). Cell membranes were prepared from the various cell types by hypotonic lysis in 10 mM Tris-HCl, pH 7.5, containing aprotinin (10 ,g/ml), pepstatin (1 ,ug/ml), leupeptin (1 ,ug/ml), and Na-p-tosyl-L-lysine chloromethyl ketone (50 ,ug/ml) at 4°C (30 min on ice followed by Dounce homogenization). The homogenate was centrifuged at 500 x g for 5 min at 4°C. The supernatant was recovered and recentrifuged. The nucleifree supernatant was then layered over 0.25 M sucrose and centrifuged at 15,000 x g for 30 min at 4°C. The membranecontaining pellet was resuspended in 20 mM Hepes, pH 7.2, and washed several times before use. The washed membranes were suspended in a buffer containing 20 mM Hepes, pH 7.2/50 ,uM ATP/0.3% bovine serum albumin/10 mM MgCl2/pyruvate kinase (5 ,g/ml)/4.5 mM phosphoenolpyruvat...