| Abstrakt: |
In the present report, we show that progressive growth of the immunogenic C57BL/6J sarcoma, MCA/76‐9, was accompanied by an increase in serum interleukin‐6 (IL‐6) activity. The possible pathways leading to the induction of IL‐6 release by the tumor cells are described. It was shown that macrophage products IL‐lα, IL‐lβ, and to a lesser extent, TNFα, induced the tumor cells in vitro to transcribe the IL‐6 gene and release the gene product. IL‐1 induced significantly more IL‐6 mRNA and bioactivity than TNFα, although both cytokines induced a cumulative increase of bioactivity in the supernates over a period of 24 h. The tumor cells were shown to express receptors for IL‐la, which could be blocked with anti‐IL‐1 receptor antibody. Given the previous reports that tumor‐associated macrophages expressed both IL‐lα/βand TNFα, the data suggest, first, that the mutual interaction of tumor cells and macrophages in situ may contribute to the observed increase in circulating IL‐6 activity, and second, that the release of IL‐6 in vivo may serve to regulate both anti‐tumor immune responses and suppressor mechanisms during significant net loss of body weight, even when tumors reach more than 2 g in weight by 3‐4 weeks of growth (unpublished results). The same tumor cells were routinely grown in vitro in RPMI 1640 medium containing 10% fetal bovine serum (HyClone, Logan, UT), which was routinely tested for endotoxin contamination by the Limulus assay (Whittaker Bioproducts, Walkersville, MD) and shown to be negative at the extinction point of the assay (<0.015 endotoxin units/ml). Mouse serum was obtained at defined intervals after tumor cell implantation by bleeding mice under anesthesia from the brachial plexus. There were four mice in each group for each time point. Lipopolysaccharide (LPS‐Escherichia coliserotype 0111:B4; Sigma Chemical Co., St. Louis, MO) was used to activate macrophages, as described later. The following recombinant proteins and monocloncal antibodies were used: human rIL‐6 (with a specific activity of 106units/mg, Agmen, Thousand Oaks, CA); human rIL‐la (lot IL‐1/87, provided by Dr. Peter Lomedico, Hoffmann‐LaRoche, Nudey, NJ, with a specific activity of 2.5 × 109units/mg and containing 0.125 endotoxin units/ml) and rIL‐lβ (provided by Dr. Hirai, Ot‐ suka Pharmaceutical, Tokushima, Japan, with a specific activity of 4 × 107units/mg; 125I‐labeled IL‐lα (with a specific activity of 2000 Ci/mmol, Amersham, Arlington Heights, IL); murine rTNFa (with a specific activity of 4 × 107units/mg, Genzyme, Boston, MA), or human rTNFα (with a specific activity of 106units/mg, Agmen, Thousand Oaks, CA); rat anti‐mouse IL‐1 receptor type I MAb (IgGl, from Dr. R. Chizzonite, Hoffmann‐LaRoche, Nutley, NJ); purified rat IgG (Sigma), rabbit anti‐mouse IL‐6 MAb (from Dr. Richard Nordan, National Institutes of Health). |
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