Highly purified, Escherichia coli-derived recombinant human tumor necrosis factor (TNF) was labeled with 1251 and employed to determine receptor binding, internalization, and intracellular degradation in murine L929 cells (highly sensitive to the cytotoxic action of TNF) and in diploid human FS-4 cells (resistant to TNF cytotoxicity). 1251-labeled TNF bound specifically to high-affinity receptors on both L929 and FS-4 cells. Scatchard analysis of the binding data indicated the presence of 2200 binding sites per L929 cell and 7500 binding sites per FS-4 cell. The calculated dissociation constants are 6.1 x 10-10 M and 3.2 x 10-10 M for L929 and FS-4 cells, respectively. In both L929 and FS-4 cells, incubation at 370C resulted in a rapid internalization of the bulk of the cell-bound TNF, followed by the appearance of trichloroacetic acid-soluble 125I radioactivity in the tissue culture medium, due to degradation of TNF. Degradation but not cellular uptake of TNF was inhibited in the presence of chloroquine (an inhibitor of lysosomal proteases) in both L929 and FS-4 cells, suggesting that degradation occurs intracellularly, probably within lysosomes. These results show that resistance of FS-4 cells to TNF cytotoxicity is not due to a lack of receptors or their inability to internalize and degrade TNF.Tumor necrosis factor (TNF) was originally described as a protein found in the serum of animals sensitized with Bacillus Calmette-Guerin or Corynebacterium parvum and challenged with bacterial endotoxin (1). TNF is functionally defined as a protein producing hemorrhagic necrosis of some tumors in experimental animals and cytolytic or cytostatic effects in tumor cells in culture. Since the major cellular source of TNF is the macrophage (1-6), TNF is thought to be a mediator of the cytotoxic activity of macrophages against tumor cells (7). Recently, human TNF was purified to homogeneity (8) and cDNA for human TNF was cloned, sequenced, and expressed in Escherichia coli (9-11). As a result of these advances, highly purified recombinant TNF has become available for experimental'studies.One of the most intriguing properties of TNF is the apparent selectivity of its cytotoxic and cytostatic activities. TNF is active against many types of tumor cells, whereas untransformed cells generally remain unaffected (1, 11). The mechanism of this selectivity as well as the mechanisms by which TNF exerts its cytolytic or cytostatic activity are unknown. The action of polypeptide hormones, growth factors, and cytokines is generally initiated by their binding to specific cellular receptors (12). This binding is usually followed by internalization of the receptor-ligand complex, through receptor-mediated endocytosis, and the eventual degradation of the ligand by lysosomal hydrolases (13).In this study we examined the presence of high-affinity receptors on two cell lines-one highly sensitive (L929) and one completely resistant (FS-4)

Certain inflammatory stimuli render cultured human vascular endothelial cells hyperadhesive for neutrophils. This state is transient and reversible, in part because activated endothelial cells secrete a leukocyte adhesion inhibitor (LAI). LAI was identified as endothelial interleukin-8 (IL-8), the predominant species of which is an extended amino-terminal IL-8 variant. At nanomolar concentrations, purified endothelial IL-8 and recombinant human IL-8 inhibit neutrophil adhesion to cytokine-activated endothelial monolayers and protect these monolayers from neutrophil-mediated damage. These findings suggest that endothelial-derived IL-8 may function to attenuate inflammatory events at the interface between vessel wall and blood.

Although tumor necrosis factor (TNF) and interleukin 1 (IL-1) affect many cell functions, the molecular mechanisms of TNF and IL-1 action are not understood. Our present study shows that exposure of human FS-4 fibroblasts to TNF or IL-1 caused a rapid accumulation of intracellular cAMP and an increase in protein kinase activity. Intracellular cAMP levels peaked 3-5 min after the addition of TNF or IL-1 and returned to basal level by 15 min. Increased phosphorylation of histone 1H1-B protein was demonstrated with extracts prepared from TNF-or IL-i-treated cells, suggesting an increase in cAMP-dependent protein kinase activity. No evidence was obtained for protein kinase C activation in TNFtreated FS-4 cells. TNF, IL-1, and forskolin all stimulated interleukin 6 (IL-6) mRNA levels in FS4 cells. The protein kinase inhibitor H-8, inhibiting preferentially cAMPdependent kinase activity, reduced forskolin-stimulated IL-6 mRNA induction more strongly than TNF-or IL-i-driven IL-6 mRNA induction. These results suggest that activation of cAMP-dependent protein kinase by TNF and IL-1 is important in some actions of these cytokines. In addition, our data on IL-6 induction by TNF and IL-1 suggest that other, yet unidentified, signal transduction mechanisms contribute to TNF and IL-1 actions on gene expression in human fibroblasts.The cytokines tumor necrosis factor (TNF) and interleukin 1 (IL-1) mediate a variety of actions important in host defense, inflammation, and autoimmunity (reviewed in refs. 1-3). Although unrelated to each other in their amino acid sequences and known to recognize separate receptors, TNF and IL-1 are surprisingly similar in many of their actions (reviewed in ref.3). In cultured cells, TNF and IL-1 were shown to cause activation of the same genes-e.g., both induce IL-1 synthesis in monocytes or endothelial cells (4, 5), both cause a similar enhancement of c-fos and c-myc protooncogene mRNA levels in human fibroblasts (6), and both induce interleukin 6 (IL-6) (7,8). [IL-6 is also known as interferon-p2 (9), 26-kDa protein (10), B-cell stimulatory factor 2 (11), and hepatocyte stimulatory factor (12).] The similarity in the actions of TNF and IL-1 suggests that they activate similar intracellular pathways, but virtually nothing is known about the molecular mechanisms of their actions.The increase in IL-6 mRNA levels by TNF or IL-1 in human fibroblasts is demonstrable within 1 hr, and it is accompanied by an increase in the rate of IL-6 mRNA transcription (13). In a recent paper, we showed that IL-6 is rapidly induced by various treatments that enhance intracellular cAMP levels and we suggested that the induction of by TNF and IL-1 also may involve the cAMP pathway (14).In this paper, we show that both TNF and IL-1 cause a rapid and transient increase in intracellular cAMP and an increase in protein kinase activity in human fibroblasts. Results Research, Osaka, Japan). Recombinant E. coli-derived human IL-la (3 x 107 units/ mg) was obtained from P. Lomedico (Hoffmann-La Roche).The plasmid P-...

A complete experimental format is given for the reconstitution of human hemoglobin from the separated heme-free a-and -globin chains (a', ft') and hemin, by two alternative routes. Based on their oxygen binding properties, the reaction of the ferri-forms with reducing agent, and the response of the oxygen binding curves to pH variation and to the addition of the a losteric effector 2,3-diphosphoglycerate, the molecules are native. One reconstitution route uses direct addition of hemin to the separated globin chains with production of the separated subunits, which can then be recombined and reduced. This procedure occasions losses by precipitation in the heme-addition step except at hig dilutions, and the yields are low. In the second pathway, either globin chain is mixed with the complementary untreated subunit to form the half-filled (with heme) intermediates, which combine stoichiometrically with hemin. No precipitation accompanies these reactions. For a-globin, the yield is about 50% because of incomplete combination with the heme-containing P chain. For -globin, the yield is better than 70%. It is suggested that experiments intended to test either globin chain should use the second route in preparation for structural or functional comparisons with native hemoglobin.In two previous papers (1, 2), we described the preparation and properties of isolated heme-free human globin chains (a", a°) and of two kinds of reassembly intermediates in which both kinds of chains are present but only half of the heme sites are filled (HFa and HFft molecules). The first paper demonstrated that the separated globin chains near neutral pH have different conformations, a" being substantially more disordered than a", in contrast to the separated heme-containing subunits, which are folded similarly, and in contrast also to the heme-free chains when bound together in dimeric apohemoglobin (a°,B°). Moreover, it was shown that, once separated, the globin chains do not readily recombine at neutral pH and accessible temperatures to form apohemoglobin. The second paper demonstrated that if either chain contains a heme, then the complementary chains do specifically recombine. During such a recombination, the disordered a-globin is refolded to the conformation it possesses in apohemoglobin. The coupled binding and refolding was termed an alloplex interactiont, to emphasizethe extensive refolding induced by a neighboring subunit.In future papers on this subject, the kinetics and mechanism of the alloplex interaction and the equilibrium of the HFa and HFB intermediates will be reported. The present paper shows that human hemoglobin can be reconstituted from separated globin chains and hemin by two different routes and that the reconstituted hemoglobin is similar or identical to native hemoglobin with respect to cooperativity in oxygen binding, the Bohr effect, and response to the allosteric effector 2,3-diphosphoglycerate (P-glycerate).In the course of our work on this problem, we found it necessary to modify several of our previous...