Interleukin 1 receptor antagonist (IL-1Ra) levels are elevated in the blood of patients with a variety of infectious, immune, or traumatic conditions. To examine whether IL1Ra is produced by liver cells with characteristics resembling an acute-phase protein, human primary hepatocytes isolated from liver biopsies and HepG2 hepatoma cells were stimulated with IL-1 ␤ , IL-6, and TNF ␣ . IL-1Ra was present in the supernatants of both cells, with production significantly enhanced by IL-1 ␤ , and by the combination of IL-1 ␤ and IL-6. The term IL-1Ra refers to two different proteins encoded by the same gene, but generated by alternative splicing of two different first exons. One isoform is secreted (17-kD sIL-1Ra), and the other isoform remains in the cytoplasm (18-kD icIL-1Ra). By Western blot analysis, the supernatants of human hepatoma (HepG2)

The serum complement system is a major mediator of inflammation reactions. Two of the complement proteins, the third (C3) and fifth (C5) components, are precursors of potent phlogistic molecules, C3a and CSa. C5a has potent chemotactic activity and plays an active role in pulmonary inflammation. We present evidence suggesting that several complement proteins, including C5, are synthesized locally in the lung in alveolar type II epithelial cells. Lung tissue from normal mice synthesized and secreted C5 protein similar to the C5 protein in mouse serum, whereas lung tissue from C5-deficient mice did not. Lung tissues from both normal and C5-deficient mice synthesized C3. Rat lung tissue synthesized and secreted C5, as well as C2, C4, C3, and factor B. Cultures of type II cells (95% type II cells, 5% macrophages) regularly synthesized all these proteins. In contrast, cultures of macrophages alone synthesized large amounts of C2 and factor B, and in some experiments C3 and C4, but never C5. The C5 synthesized by the rat cells was slightly larger than serum C5 (200 kD compared with 180 kD) and was not processed to the two-chain molecule seen in serum. Rat lung tissue and purified type II cells contained C5 mRNA with the same molecular mass as the C5 mRNA in rat liver and in mouse lung and liver. Human type II cells also synthesized C5, as well as C2, C4, C3, and factor B. Human pulmonary macrophages synthesized only C2, factor B, and, in some experiments, C3. Synthesis of complement proteins in cells that line the alveolar wall may provide a local source of these proteins for inflammatory responses in the lung. Local synthesis of complement proteins could be regulated independently of the synthesis in the liver.

promoter. Further studies demonstrated that mutual antagonism between the LPS and Raf-1 kinase pathways is not promoter specific, as the same phenomenon is observed in assays using a c-fos enhancer/thymidine kinase promoter/luciferase construct (pc-fos-TK81-luc). Additionally, mutual antagonism with regard to sIL1Ra promoter activity also was observed between the LPS and MEK kinase pathways, indicating that mutual antagonism can occur in more than one MAPK activation pathway.

Expression of the IL-1 receptor antagonist (IL-1ra) can be induced by treatment of monocytes and macrophages with LPS. We have previously demonstrated that the most proximal 294 bp of the human IL-1ra promoter are sufficient for full basal activity and LPS responsiveness. In the present study, we demonstrate the presence of one inhibitory and three positive-acting LPS response elements (LRE) within this proximal 294-bp IL-1ra promoter fragment. By both 5'-deletional analysis and heterologous promoter studies, an element between -294 and -250 was found to mask the LPS response. By 5'-deletional analysis and heterologous promoter experiments, two positive-acting LRE were identified between -250 and -200 (LRE3) and -200 and -148 (LRE2) which exhibited cooperativity in that neither element alone was active. Furthermore, LRE2 also cooperated with a more proximal site between -148 and -31 (LRE1), which also was not active alone. LRE1 was identified as an NF-kappa B-binding site. Site-directed mutagenesis of this site, located between -93 and -84, resulted in a > 50% decrease in the LPS responsiveness of the 294-bp promoter. By electrophoretic mobility shift assays, with or without specific antisera to members of the rel/NF-kappa B family, the complex binding to LRE1 was shown to contain primarily NF-kappa B1/p50 and lesser amounts of RelA/p65. These results indicate that the net activation of the human IL-1ra promoter in response to LPS involves the functional interaction of at least four cis-acting DNA elements within the proximal 294 bp.

The potential mechanisms involved during the faster release of [125I]-iodo-2'-deoxyuridine (125IdUrd)-labeled target sarcoma cells in the presence of normal C57BL/6J peritoneal macrophages were investigated. Maximum (= 90%) "spontaneous" release of 125I from target cells cultured alone occurred over a period of about 10 days. However, after about 3 days, confluent sheets of target cells developed. In the presence of normal macrophages, 90% of the 125I was released between 3 and 7 days, again with the formation of confluent sheets of target cells. This enhanced 125I release was not influenced by increasing the relative concentration of IdUrd using the nonradioactive isotope 127IdUrd. Established mechanisms of target cell destruction were investigated but no evidence was found for the involvement of superoxide anion, hydrogen peroxide, or regulation by prostaglandin synthesis. The macrophage-mediated effect was abrogated by incorporating hydrocortisone-acetate (10(-7) to 10(-4) M) into the culture medium but this did not affect target cell proliferation. The use of serum-free culture medium suggested that macrophages secreted a soluble mediator that was not derived from or dependent on the presence of fetal bovine serum. In addition, macrophage-conditioned medium was able to induce the faster 125I release. The failure to precipitate with 20% trichloroacetic acid the 125I released from target cells cultured in the presence of macrophages indicated that the radioactive component had been separated from the precipitable DNA. The data are discussed in light of two possible hypotheses: that macrophages recognized subtle changes in IdUrd-labeled cells and exacerbate radiotoxicity, and that the faster release reflected proliferative death caused by stimulated growth.