Molecular Regulation of Monocyte Chemoattractant Protein-1 Expression in Pancreatic -Cells Burak Kutlu, 1 Martine I. Darville, 1 Alessandra K. Cardozo, 1,2 and De ´ cio L. Eizirik 1 Pancreatic -cells are selectively destroyed during the course of type 1 diabetes. In the early stages of the disease, inflammatory infiltrates of mononuclear cells, containing predominantly monocytes and T-cells, are present in the islets (insulitis). Chemokines, such as monocyte chemoattractant protein-1 (MCP-1), play a key role in the recruitment and activation of these immunocytes. We have previously described cytokine- induced MCP-1 gene expression in human and rat pan- creatic islets. In the present study, the transcriptional regulation by cytokines of the rat MCP-1 gene in fluo- rescence-activated cell sorting–purified rat -cells, in- sulin-producing INS-1E cells, and RINm5F cells was investigated. Transient transfections with luciferase- reporter constructs identified an interleukin (IL)-1– responsive enhancer region between –2,180 bp and 2,478 bp. Mutation of either of the two nuclear factor (NF)-B sites present in this region abrogated IL-1– induced MCP-1 promoter activity. Binding of NF-B to the two sites was shown in vitro by gel shift assays, while supershift assays revealed the presence of p65/ p50 heterodimers and p65 homodimers. In vivo binding of NF-B was confirmed by chromatin immunoprecipita- tion assay. Blocking of NF-B activation in cytokine- exposed primary -cells by an adenovirus overexpressing a nondegradable form of IB or by pyrrolidine dithio- carbamate decreased IL-1–induced MCP-1 mRNA ex- pression. We conclude that NF-B plays an important role for MCP-1 expression in -cells. This transcription factor may be an interesting target for ex vivo gene therapy before islet transplantation. Diabetes 52: 348 –355, 2003 T here is strong evidence that type 1 diabetes develops as a consequence of autoimmunity, leading to -cell destruction (1). In the early stages of insulitis, activated macrophages and T-cells are attracted to the islets and produce cytokines and free radicals, which contribute to -cell dysfunction and death (2,3). The mechanisms regulating the attraction of monocytes and T-cells to the islets remain to be clarified. We have recently described that human and rat islets, and islets isolated from prediabetic NOD mice, express the chemokine MCP-1 (4,5). Subsequent studies by other groups confirmed that human islets produce and secrete biologically active MCP-1 (6). Importantly, MCP-1 released by pancreatic islets plays a relevant role for the clinical outcome of islet allografts in type 1 diabetic patients, as suggested by the observation that high MCP-1 secretion is negatively correlated with successful engraft- ment and long-lasting insulin independence (6). These intriguing observations suggest that MCP-1 and other chemokines produced by the cytokine-exposed islet cells (7,8) are involved in the recruitment and activation of immune cells during both insulitis and islet graft destruction. MCP-1 belongs to the C-C family of chemokines (9 –11) and is produced by diverse cell types in response to cytokines, oxidized LDL, and bacterial lipopolysaccharide (12–14). The differential induction and binding of activated transcription factors to the promoter region of the MCP-1 gene provides a critical regulatory step, allowing expres- sion of the chemokine in a cell- and stimulus-specific manner (15). MCP-1 attracts monocytes (16), T-cells (17), and natural killer cells (18) to the site of inflammation, and it also regulates the release of arachidonic acid and the respiratory burst by monocytes (19,20). Local release of MCP-1 probably contributes to the inflammatory response in rheumatoid arthritis, atherosclerosis, glomerular dis- eases, and pulmonary granulomatous vasculitis (21–27). Of note, transgenic mice expressing MCP-1 under control of the insulin promoter develop an intense insulitis (28). In vivo footprinting in murine fibroblasts showed that tumor necrosis factor (TNF)- induces site occupancy in both proximal and distal regions of the MCP-1 promoter (29). The distal region is located more than 2.2 kb up- stream of the transcription start site and is required for induction of MCP-1 expression by TNF- and IL-1 in mouse fibroblasts (30) and in human mesangial cells (31). This enhancer region contains two binding sites for NF-B that are conserved in the human, mouse, and rat MCP-1 genes (30,32). Moreover, in the human gene a proximal NF-B site cooperates with an adjacent AP-1 site for induction by TNF- or IL-1 (33). In human osteoblastic cells interferon (IFN)- stimulates MCP-1 transcription through a complex element in the promoter region, namely an IFN response-inhibitory sequence (IRIS) adja- cent to an IFN- activation site (-activated sequence [GAS]) (34). This element is conserved in the human, mouse, and rat genes (35). It is noteworthy that IFN- does not stimulate MCP-1 expression in rat -cells or human From the 1 Laboratory of Experimental Medicine, Universite ´ Libre de Brux- elles, Brussels, Belgium; and the 2 Gene Expression Unit, Diabetes Research Center, Vrije Universiteit Brussel, Brussels, Belgium. Address correspondence and reprint requests to De ´ cio L. Eizirik, Labora- tory of Experimental Medicine, Universite ´ Libre de Bruxelles, Route de Lennik, 808, B-1070, Brussels, Belgium CP 618. E-mail: deizirik@ulb.ac.be. Received for publication 25 July 2002 and accepted in revised form 13 November 2002. ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assay; FACS, fluorescence-activated cell sorting; GAS, -activated sequence; IFN, interferon; IL, interleukin; IRIS, IFN response-inhibitory sequence; MCP-1, monocyte chemoattractant protein-1; NF, nuclear factor; OD, optical density; PDTC, pyrrolidine dithiocarbamate; TNF, tumor necrosis factor. 348 DIABETES, VOL. 52, FEBRUARY 2003