In mammals, several well-defined metabolic changes occur during infection, many of which are attributable to products of the reticuloendothelial system. Among these changes, a hypertriglyceridaemic state is frequently evident, resulting from defective triglyceride clearance, caused by systemic suppression of the enzyme lipoprotein lipase (LPL). We have found previously that macrophages secrete the hormone cachectin, which specifically suppresses LPL activity in cultured adipocytes (3T3-L1 cells). When originally purified from RAW 264.7 (mouse macrophage) cells, cachectin was shown to have a pI of 4.7, a subunit size of relative molecular mass (Mr) 17,000 and to form non-covalent multimers. A receptor for cachectin was identified on non-tumorigenic cultured cells and on normal mouse liver membranes. A new high-yield purification technique has enabled us to determine further details of the structure of mouse cachectin. We now report that a high degree of homology exists between the N-terminal sequence of mouse cachectin and the N-terminal sequence recently determined for human tumour necrosis factor (TNF). Purified cachectin also possesses potent TNF activity in vitro. These findings suggest that the 'cachectin' and 'TNF' activities of murine macrophage conditioned medium are attributable to a single protein, which modulates the metabolic activities of normal as well as neoplastic cells through interaction with specific high-affinity receptors.

A cytokine that can synergize with interleukin 2 to activate cytotoxic lymphocytes was purified to homogeneity. The protein, provisionally called cytotoxic lymphocyte maturation factor (CLMF), was isolated from a human Blymphoblastoid cell line that was induced to secrete lymphokines by culture with phorbol ester and calcium ionophore. The purification method, utilizing classical and high-performance liquid chromatographic techniques, yielded protein with a specific activity of 8.5 x 107 units/mg in a T-cell growth factor assay. Analysis of the purified protein by sodium dodecyl sulfate/polyacrylamide gel electrophoresis demonstrated that CLMF is a 75-kDa heterodimer composed of disulfide-bonded 40-kDa and 35-kDa subunits. Determination of the N-terminal amino acid sequences of the two subunits revealed that both subunits are not related to any previously identified cytokine. Purified CLMF stimulated the proliferation of human phytohemagglutinin-activated lymphoblasts by itself and exerted additive effects when used in combination with suboptimal amounts of interleukin 2. Furthermore, the purified protein was shown to synergize with low concentrations of interleukin 2 in causing the induction of lymphokine-activated killer cells.The potential utility of cytokines in the treatment of neoplasia and as immunoenhancing agents has recently been demonstrated in studies using human recombinant interleukin 2 (rIL-2) (1-6). However, the clinical use of rIL-2 has been complicated by the serious side effects that it may cause (2, 3). One approach to improving the efficacy of cytokine therapy while reducing toxicity is to use two or more cytokines in combination. For example, synergistic antitumor activity has been shown to result when rIL-2 is administered to tumor-bearing mice together with recombinant interferon a (rIFN-a) (7,8) or with recombinant tumor necrosis factor a (rTNF-a) (9). The antitumor effects of rIL-2 are thought to be mediated by host cytotoxic effector lymphocytes, which are activated by rIL-2 in vivo (10). rIFN-a (11) and rTNF-a (12, 13) have been shown to synergize with rIL-2 in activating cytotoxic effector cells in vitro as well as to exert synergistic antitumor effects when given in combination with rIL-2 in vivo (7-9). Hence, a cytokine was sought that could synergize with rIL-2 to activate cytotoxic lymphocytes in vitro and thus might also have utility as an antitumor agent when administered in combination with rIL-2 in vivo.Previously we demonstrated that IL-2-depleted lymphokine-containing cell supernatant solutions from cultures of human peripheral blood lymphocytes activated with phytohemagglutinin (PHA) or in mixed lymphocyte cultures contained such a factor, provisionally called cytotoxic lymphocyte maturation factor (CLMF) (14, 15). However, the quantities of human CLMF produced by peripheral blood lymphocytes were too low to permit its purification to homogeneity. Therefore, human lymphoid cell lines were screened for the production of cytokines that could synergize with rIL-2 to a...

Binding of antibodies to effector cells by way of receptors to their constant regions (Fc receptors) is central to the pathway that leads to clearance of antigens by the immune system. The structure and function of this important class of receptors on immune cells is addressed through the molecular characterization of Fc receptors (FcR) specific for the murine immunoglobulin G isotype. Structural diversity is encoded by two genes that by alternative splicing result in expression of molecules with highly conserved extracellular domains and different transmembrane and intracytoplasmic domains. The proteins encoded by these genes are members of the immunoglobulin supergene family, most homologous to the major histocompatibility complex molecule E beta. Functional reconstitution of ligand binding by transfection of individual FcR genes demonstrates that the requirements for ligand binding are encoded in a single gene. These studies demonstrate the molecular basis for the functional heterogeneity of FcR's, accounting for the possible transduction of different signals in response to a single ligand.

Vacuolar H+-ATPases function in generating protonmotive force across the membranes of organelies connected with the vacuolar system of eukaryotic cells. This family of H+-ATPases is distinct from the two other families of H+-ATPases, the plasma membrane-type and the eubacterialtype. One of the subunits of the vacuolar H+-ATPase binds N,N'-dicyclohexylcarbodiimide (DCCD) and has been implicated in the proton-conducting activity of these enzymes. We have cloned and sequenced the gene encoding the DCCDbinding protein (proteolipid) of the H+-ATPase of bovine chromaffim granules. The gene encodes a highly hydrophobic protein of 15,849 Da. Hydropathy plots revealed four transmembrane segments, one of which contains a glutamic residue that is the likely candidate for the DCCD binding site. Sequence homology with the vacuolar proteolipid and with the proteolipids of eubacterial-type H+-ATPases was detected. The proteolipids from Escherichia coli, spinach chloroplasts, and yeast mitochondria matched better to the NH2-terminal part of the vacuolar protein. The proteolipids of bovine mitochondria and Neurospora mitochondria matched better to the COOHterminal end of the vacuolar proteolipid. These findings suggest that the proteolipids of the vacuolar H+-ATPases were evolved in parallel with the eubacterial proteolipid, from a common ancestral gene that underwent gene duplication.Proton-transporting ATPases (H+-ATPases) play a crucial role in biological energy transduction (1). These ion pumps can be classified into three main families: plasma membranetype, eubacterial-type, and vacuolar-type enzymes (2-5). The plasma membrane-type enzyme is present in the plasma membrane ofplants, fungi, and acid-secreting gastric vesicles (6, 7). The catalysis of these enzymes involves a phosphoenzyme intermediate. The gene coding for this 100-kDa protein in yeast and Neurospora crassa has been cloned and sequenced (8)(9)(10).The eubacterial-type enzyme occurs in chloroplasts, mitochondria and bacteria and .operates without a phosphoenzyme intermediate (11)(12)(13). These enzymes are composed of two distinct structures, a membrane sector, which is hydrophobic, and a catalytic sector, which is hydrophilic in nature. The function of the membrane sector is to conduct protons across the membrane. This sector is composed of three or more polypeptides, one of which is an N,N'-dicyclohexylcarbodiimide (DCCD)-binding protein (proteolipid of about 8 kDa) that is involved in the proton conduction (14-16). The catalytic sector, which is the site of the ATPase reaction, can be readily separated from the membrane by EDTA treatment or by applying mechanical force (17).The vacuolar-type enzyme is present in organelles connected with the vacuolar system of eukaryotic cells and pumps protons without the involvement of a phosphoenzyme intermediate (2-5). These H+-ATPases are composed of several polypeptides, and to our knowledge none of these have been sequenced. A 16-kDa subunit resembles the eubacterial proteolipids in that it binds DCCD and is s...

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