Tumour necrosis factors, TNF-alpha and TNF-beta (previously called lymphotoxin), are the products of activated monocytes and lymphocytes, respectively, and both have recently been purified, sequenced and cloned by recombinant DNA methods, revealing 35% identity and 50% homology in the amino-acid sequence. Both proteins have been found to be specifically toxic to many tumour cells. Furthermore, it has been reported that various interferons are synergistic with TNF for anti-tumour effects in vitro, while activities attributed to the two proteins have also been shown to necrotize various tumours in vivo. We have now prepared 125I-labelled highly purified recombinant human TNF-alpha to study in detail its binding to the human cervical carcinoma cell line ME-180. Our results indicate that there is a single class of specific high-affinity receptors for TNF on this cell line which has a Kd of about 0.2 nM and an average of 2,000 receptor sites per cell. The binding of labelled TNF-alpha to these cells can be inhibited by both TNF-alpha and TNF-beta but not by gamma-interferon (IFN-gamma). However, preincubation of cells with IFN-gamma increases the total number of TNF receptors two to threefold without any significant change in the affinity constant. This is the first report that TNF-alpha and -beta share a common receptor and that the receptors can be up-regulated by interferon. Our results may explain previous observations regarding similar biological activities observed for these two cytotoxic proteins and also their synergistic action with interferons.

A human myelomonocytic cell line, U937, produced an interleukin-1 (IL-1) receptor antagonist protein (IRAP) which was purified and partially sequenced. A complementary DNA coding for IRAP was cloned and sequenced. The mature translation product of the cDNA has been expressed in Escherichia coli and was an active competitive inhibitor of the binding of IL-1 to the T-cell/fibroblast form of the IL-1 receptor. Recombinant IRAP specifically inhibited IL-1 bioactivity on T cells and endothelial cells in vitro and was a potent inhibitor of IL-1 induced corticosterone production in vivo.

The ATP-sensitive, inwardly rectifying K+ channel, ROMK, has been suggested to be the low-conductance ATP-sensitive K+ channel identified in apical membranes of mammalian renal thick ascending limb (TAL) and cortical collecting duct (CCD). Mutations in the human ROMK gene (KIR1.2) have been identified in kindreds with neonatal Bartter’s syndrome. In the present study, we generated polyclonal antibodies raised against both a COOH-terminal (amino acids 252–391) ROMK-maltose binding protein (MBP) fusion protein and an NH2-terminal (amino acids 34–49) ROMK peptide. Affinity-purified anti-ROMK COOH-terminal antibody detected the 45-kDa ROMK protein in kidney tissues and HEK-293 cells transfected with ROMK1 cDNA. The antibody also recognized 85- to 90-kDa proteins in kidney tissue; these higher molecular weight proteins were abolished by immunoabsorption with ROMK-MBP fusion protein and were also detected on Western blots using anti-ROMK NH2-terminal antibody. Immunofluorescence studies using anti-ROMK COOH-terminal antibody showed intense apical staining along the loop of Henle and distal nephron; staining with preimmune and immunoabsorbed serum was negative. When colocalized with distal nephron markers [the thiazide-sensitive cotransporter (rTSC1), the bumetanide-sensitive cotransporter (rBSC1), the vacuolar type H+-ATPase, and neuronal nitric oxide synthase (NOS I)], the ROMK protein was found primarily at the apical border of cells in the TAL, macula densa, distal convoluted tubule, and connecting tubule. Within the CCD, the ROMK protein was expressed in principal cells and was absent from intercalated cells. The tubule localization and polarity of ROMK staining are consistent with the distribution of ROMK mRNA and provide more support for ROMK being the low-conductance K+secretory channel in the rat distal nephron.

The effect of a variety of cytokines on lipid metabolism in 3T3 Li mouse fibroblasts and adipocytes was studied. Uptake of [3H]acetate by adipocytes and heparinreleasable lipoprotein lipase activity was inhibited after treatments of the cells with picomolar concentrations of recombinant human tumor necrosis factor a (rHuTNF-a), human tumor necrosis factor 13 (rHuTNF-f, also called lymphotoxin), murine interferon-y (rMuIFN-y), and a human hybrid interferon-a [rHuIFN-a2/aj (Bgl I)]. Recombinant human interferon-y (rHuIFN-y), natural human colony-stimulating factor (HuCSF), and human interleukin 2 (HuIL-2) had no effect. Similar though less-marked suppression of [3H~acetate uptake by cytokines was seen in 3T3 Li fibroblasts. Cytokines inhibited the incorporation of [3H]acetate into both membrane and storage lipids in the adipocytes. In addition to blocking lipid uptake and synthesis, rHuTNF-a and -(3, and rMuIFN-y stimulated the release of free fatty acid into the medium from adipocytes. Binding studies suggest that rHuTNF-a and rHuTNF-P compete for the same cell-surface receptor on 3T3 Li adipocytes, while rMuIFN-y binds to a separate receptor.The binding of rTNF-a to both adipocytes and fibroblasts can be significantly enhanced by preexposure of the cells to rMuIFN-y. There appear to be both high-and low-affinity receptors for rHuTNF-a on adipocytes, whereas fibroblasts exhibit a single class of high-affinity receptors. These results suggest that a variety of structurally distinct cytokines possess lipid mobilization activity, which may be of critical importance to the host in defense against infection or malignancy.The invasion of the body by viruses, bacteria, or parasites usually elicits an integrated host response that kills and removes the infectious agents and provides immunity against future challenges. Much of the host response is modulated by a class of inducible proteins called cytokines (1-3). It is now clear that there are many distinct host cytokines that can be produced by a wide variety of cell types, including epithelial cells, fibroblasts, tumor cells, and particularly lymphocytes (lymphokines) and macrophages (monokines) (4-6). Tumor necrosis factors (TNFs), interleukins (ILs), and a-, ,B, and -interferons (IFN-a, -(3, and -y) are representative and well-characterized cytokines. Although there is considerable structural and functional heterogeneity among these families of cytokines, they all appear to have roles in the regulation of host immunity and inflammation (7-12). When administered parenterally at high doses they can be toxic, and many of the symptoms associated with the common cold and influenza such as fever, nausea, and muscle aches can be induced by the administration of pure cytokines (13,14). Unraveling the sequence of cytokine actions that occur during the host response to infection is very complex because (i) cytokines regulate the production of one another (15-17), (ii) an individual cell may produce many different cytokines (18, 19), and (iii) different cytokines may hav...

Functional characterization of oncogene products that induce cellular transformation has progressed rapidly in recent years. However, less is known about the mechanism(s) by which the transformed cells may escape destruction by host immune defenses and form tumors. A recently described oncogene that has an important association with aggressive human breast carcinoma is "HER2," for human epidermal growth factor receptor 2. The oncogene has also been called NGL and human c-erbB-2 (ERBB2). In this paper we show that amplification of HER2 oncogene expression can induce resistance of NIH 3T3 cells to the cytotoxic effects of recombinant tumor necrosis factor alpha (rTNF-alpha) or macrophages. Resistance is accompanied by an increased dissociation constant for rTNF-alpha binding to high-affinity receptors on the HER2-transformed NIH 3T3 cells. The resistance phenotype is independent of transformation since NIH 3T3 cells transformed by the activated human homologue of the Harvey-ras oncogene (HRAS) retain high-affinity binding sites for rTNF-alpha as well as sensitivity to its cytotoxic effects. These results suggest that HER2 may potentiate tumorigenesis by inducing tumor cell resistance to host defense mechanisms.