Prostaglandins antagonize fibroblast proliferation stimulated by tumor necrosis factor

Tumor necrosis factor (TNF) is known to be a mitogen for human diploid FS-4 fibroblasts. We have shown in an earlier study (Hori et al. (1989) J. Cell. Physiol. 141, 275-280) that indomethacin further enhances the cell proliferation stimulated by TNF. Since indomethacin inhibits the activity of cyclooxygenase, the role of prostaglandins in TNF-stimulated cell growth was examined. Cell growth stimulated by TNF and indomethacin was inhibited by exogenously added prostaglandins (PGE2, PGF2 alpha, and PGD2), among which PGE2 caused the greatest inhibition of cell growth. Treatment of FS-4 cells with 10 ng/ml TNF resulted in the release of prostaglandins (PGE2, 6-keto-PGF1 alpha, PGA2, PGD2, and PGF2 alpha) 2 to 4 fold over that of untreated cells. The amount of all these prostaglandins increased in a time-dependent manner over 6 h after treatment. In both TNF-treated and control cells, PGE2 was released as the predominant prostaglandin. Furthermore, when PGE2 production and DNA synthesis were determined in FS-4 cells treated with increasing doses of indomethacin, these two cellular responses were inversely affected by indomethacin. These data show that prostaglandins induced by TNF antagonize growth stimulatory action of TNF.     

Expression of two distinct cytolytic mechanisms among murine CD4 subsets

A TNF (TNF-alpha and TNF-beta)-sensitive target, L929, and two TNF-resistant targets, P815 and LK were used to compare the cytolytic activity among subsets of CD4+ (Th) clones. Cytolytic activity was induced with either Con A, CD3-mAb, or Ag-pulsed LK cells. Six Th1 clones are strongly cytolytic against all three targets. In contrast, Th2 clones are either noncytolytic or weakly cytolytic. Although there is an apparent correlation between TNF production, killing of L929 cells, and the killing of TNF-resistant targets, an anti-TNF serum (capable of neutralizing both TNF-alpha and TNF-beta) selectively inhibits CD4 clones to lyse L929 cells, whereas the lysis of P815 or LK cells was unaffected. The continuous presence of noncytotoxic levels of Actinomycin D (AcD) and cycloheximide, but not mitomycin C, cyclosporin A (CsA), or cholera toxin (ChT) inhibits the lysis of Ag-pulsed, Ia-bearing LK cells; indicating a requirement for de novo synthesis of RNA and protein for cytolytic activity. Although pretreatment with AcD, CsA, or ChT strongly inhibits production of IL-2, TNF and IFN-gamma, only clones pretreated with AcD lose cytolytic activity against Ag-pulsed, Ia-bearing LK cells. These observations support a model of TNF-independent killing of TNF-resistant targets. The TNF-independent cytolytic activity does not correlate with serine esterase activity released into media upon activation of CD4 clones. Moreover, the effects of metabolic inhibitors on serine esterase release do not correlate with their effects on cytolytic activity. Collectively, the data demonstrate that activated CD4 cells express two distinct cytolytic activities; a TNF (and IFN-gamma)-mediated cytotoxicity and a TNF (and IFN-gamma)-independent cytolytic activity. Both pathways require de novo synthesis of RNA and protein and appear to be independent of granule enzyme release. Only the TNF-independent cytolytic activity is resistant to CsA and ChT inhibition.     

Epidermal growth factor is synergistic with phorbol esters and vasopressin in stimulating arachidonate release and prostaglandin production in renal glomerular mesangial cells.

Epidermal growth factor (EGF) is produced in large quantities by the kidney. We identified EGF-binding sites on cultured rat renal glomerular mesangial cells. These cells serve as a model system for the investigation of renal prostaglandin biosynthesis. Since EGF has been shown to modulate phospholipase activity in other cell lines, we studied the ability of EGF to increase arachidonate release and prostaglandin E2 (PGE2) production in mesangial cells. We found that EGF stimulated arachidonate release and PGE2 production in the presence of the Ca2+ ionophore A23187. This stimulation was markedly potentiated by the addition of phorbol myristate acetate (PMA), which activates protein kinase C. However, down-regulation of protein kinase C by prolonged PMA treatment did not block the ability of EGF to stimulate PGE2 production in the presence of A23187. EGF also markedly potentiated the stimulation of PGE2 production by vasopressin, which increases intracellular Ca2+ and activates protein kinase C in these cells. The stimulatory effects of EGF were not the result of prolongation or enhancement of an increase in intracellular Ca2+ produced by ionophore or vasopressin. Furthermore, the synergistic interaction of EGF with PMA and vasopressin occurred despite the fact that these agents markedly decreased EGF binding in mesangial cells, presumably owing to protein-kinase-C-mediated phosphorylation of the EGF receptor. We conclude that there exists a distinct pathway for EGF-stimulated arachidonate release and PGE2 production in rat renal glomerular mesangial cells, which is synergistic with, but not dependent on, activation of protein kinase C. In contrast with long-term mitogenic responses to EGF, this rapid response may allow delineation of the membrane phospholipid changes and signalling steps involved in this aspect of EGF action.     

Phospholipase A2 activity is not involved in the tumor necrosis factor-triggered apoptotic DNA fragmentation in bovine aortic endothelial cells.

Using the arachidonic acid release as a probe of phospholipase A2 activity, we tested the involvement of this enzyme in the TNF-triggered apoptotic cell death in the bovine aortic endothelial cells. We observed that TNF induced a liberation of arachidonic acid from these cells which was comparable to that obtained from the L929 cells. An augmentation of the amount of released arachidonic acid or the reduction of the TNF-stimulated phospholipase A2 activity did not modify the TNF-induced DNA fragmentation in the endothelial cells. We suggest that these events are not required for TNF apoptotic cytotoxicity in the endothelial cells.     

Different interferon-producing capacities of L929 cell sublines and the enhancement of interferon production by priming are controlled pretranslationally

Two sublines of mouse L929 cells designated L929B and L929M were studied. The L929B cells, which displayed a 2-3-fold higher IFN production in response to Sendai virus than that of the L929M cells, had a higher sensitivity to the antiviral and priming effects of IFN and were more resistant to VSV. In good accord with the amount of IFN produced, more translatable IFN mRNA was isolated from the L929B cells. IFN production and IFN mRNA activities were proportionally increased in the IFN-primed cultures of both sublines. Results indicate that both inherent and priming-induced increased-IFN production are based on pretranslational control mechanisms.     

Expression of an exogenous tumor necrosis factor (TNF) gene in TNF-sensitive cell lines confers resistance to TNF-mediated cell lysis.

TNF, a cytokine with cytotoxic activity on a variety of tumor cells, is mainly produced by macrophages; however, some tumor cell types of non-macrophage origin, apparently resistant to TNF-mediated cell lysis, can also produce TNF. It is not clear whether these cells were TNF-resistant a priori or whether protective mechanisms against toxicity of autocrine TNF may be induced in TNF-producing cells. Murine L929sA fibrosarcoma cells, which are highly sensitive to TNF cytotoxicity, were transfected with the neomycin resistance (neor) gene, alone or in combination with the human (h) or the murine (m) TNF gene. All exogenous genes were under control of the constitutive SV40 early promoter. After cotransfection, the number of neor colonies was 10 to 100% as compared with the number of colonies upon transfection with the neor gene alone. An appreciable fraction of these colonies (50-100%) constitutively produced biologically active TNF. mTNF-producing L929 cells were fully TNF resistant, whereas hTNF-producing cells showed partial TNF resistance. Specific TNF binding could not be detected on mTNF-producing L929sA transfectants, whereas hTNF-producing cells showed reduced TNF binding. Apparently, TNF gene expression, even in a priori TNF-sensitive cells, can induce mechanisms to prevent toxicity by both autocrine and exogenous TNF. No TNF resistance was induced by expression of a gene sequence encoding the 9-kDa membrane-bound presequence part of the 26-kDa mTNF proform. Expression of a mutant 26-kDa TNF gene coding for a quasi-inactive mature mTNF induced only weak TNF resistance as compared with the complete resistance obtained after transfection with the wild-type gene. These findings show that the membrane-bound TNF presequence as such is not sufficient for induction of TNF resistance and imply that the active site of mature TNF is involved in modulation of TNF responsiveness upon autocrine TNF production.     

Signal transduction by tumor necrosis factor alpha is mediated through a guanine nucleotide-binding protein in osteoblast-like cell line, MC3T3-E1.

Transmembrane signalling mechanisms of tumor necrosis factor alpha (TNF alpha) were examined with special reference to the involvement of G-protein, in intact and permeabilized murine osteoblast-like cells. TNF alpha stimulated the release of 3H radioactivity from intact cells labeled with [3H]arachidonic acid within 10 min in a dose dependent manner and the production of lyso forms of phospholipids, an event presumably mediated through the activation of phospholipase A2. Production of cAMP and inositol 1,4,5-trisphosphate was not affected by TNF alpha. Pretreatment of the cells with pertussis toxin inhibited the liberation of [3H]arachidonate. GTP gamma S (guanosine 5'-3-O-(thio)triphosphate) reduced the binding affinity of [125I]TNF alpha to beta-escin-permeabilized cells. The addition of TNF alpha together with an unhydrolyzable analog of GTP, GTP gamma S, to the beta-escin-permeabilized cells prelabeled with [3H]arachidonic acid led to a release of the 3H radioactivity. The production of prostaglandin E2 (PGE2) was markedly stimulated by TNF alpha in a dose over 100 ng/ml, with a latent time of about 3 h, and the stimulation was abolished by pretreatment with pertussis toxin. The time and dose requirements for this process differed from those for the possible activation of phospholipase A2, thereby indicating that other process(es) in addition to the activation of phospholipase A2 may be responsible for the enhanced production of PGE2. The activity of cyclooxygenase (i.e. the combined activities of prostaglandin endoperoxide syntase and PGH2-PGE2 isomerase) was stimulated by TNF alpha with much the same time and dose requirements as for the production of PGE2, and the activation was found to be due to the increased amount of the enzyme, as assessed by a Western blot analysis with anti-cyclooxygenase antibody. This process was also sensitive to pertussis toxin. Therefore, receptors for TNF alpha in MC3T3-E1 cells apparently couple to G-protein sensitive to pertussis toxin and the coupling regulates the activations of phospholipase A2 and the de novo synthesis of cyclooxygenase.     

Hydrocortisone inhibits prostaglandin production but not arachidonic acid release from cultured macrophages

We have investigated the action of hydrocortisosone on arachidonic acid mobilisation in cultures of mouse peritoneal macrophages, mouse L929 cells and the mouse macrophage-like cell line RAW264. Hydrocortisone inhibits both arachidonic acid release and prostaglandin production by L929 cells. However, prostaglandin production by macrophages or RAW264 cells is inhibited with a concomitant stimulation rather than inhibition of arachidonic acid release. These data suggest that hydrocortisone acts at the level of phospholipase activity in fibroblasts but at a later stage of prostanoid production in macrophages.     

Production of interleukin-1 and tumor necrosis factor by cisplatin-treated murine peritoneal macrophages

Supernatants collected from cisplatin-treated macrophages demonstrated enhanced cytotoxicity against actinomycin-D-treated L929 cells and also enhanced the thymocyte proliferation in response to concanavalin A, showing that cisplatin-treated macrophages release interleukin-1 (IL-1) and tumor necrosis factor (TNF) into the culture supernatant. The supernatant collected from untreated macrophages showed little TNF and IL-1 activity. The release of TNF and IL-1 was observed to be dependent on the dose and duration of cisplatin treatment. Medium alone containing cisplatin did not enhance thymocyte proliferation and had little cytotoxic effect on actinomycin-D-treated L929 cells. Cisplatin-treated macrophage culture supernatants were chromatographed over a Superose 12 column on an FPLC system. TNF activity eluted in two major peaks with apparent molecular weights of 50-55 and 15-20 kilodaltons, respectively. The kinetics of IL-1 release was also studied. Maximum production and release of IL-1 were observed up to 24 h after cisplatin treatment and then gradually declined. Freeze-thaw lysates of cisplatin-treated macrophages also showed enhanced IL-1 activity. Paraformaldehyde (PFA)-fixed cisplatin-treated macrophages showed significantly enhanced cytotoxic activity against L929 cells as compared to PFA-fixed untreated macrophages. PFA-fixed cisplatin-treated macrophages also enhanced thymocyte proliferation. These results suggest that cisplatin treatment of murine macrophages also results in increased expression of membrane-associated IL-1 and TNF activity.     

Mass quantitation of agonist-induced arachidonate release and icosanoid production in a fibrosarcoma cell line. Effect of time of agonist stimulation, amount of cellular arachidonate, and type of agonist

The mass of total arachidonate released from phospholipids upon agonist stimulation of the cell and the fraction of released arachidonate which is converted to icosanoids are two parameters of arachidonate metabolism which have been difficult to quantitate because the mass of arachidonate released upon cell stimulation is very low. We have been able to quantitate both of these parameters under a variety of experimental conditions using a unique essential fatty acid-deficient mouse fibrosarcoma cell line (EFD-1), which when repleted with arachidonate, produces prostaglandin E2 (PGE2). Because there is no endogenous pool of arachidonate in these cells, the specific activity of exogenous arachidonate does not change upon incorporation into cells, an advantage which permits mass determination of very small quantities of arachidonate directly from radioactive counts. EFD-1 cells were incubated with various concentrations of [14C]arachidonate (for release studies) or unlabeled arachidonate (for PGE2 radioimmunoassays) for 24 h and then stimulated with bradykinin. The time courses for arachidonate release and PGE2 production demonstrated that free arachidonate was rapidly converted to PGE2 with plateau levels attained for both parameters within 240 s of agonist exposure for 2 microM and for 10 microM arachidonate-repleted cultures. There was a linear relationship (r = 0.94) between the mass of arachidonate in the cell and the mass of arachidonate released upon stimulation, up to a cellular concentration of 11 nmol of arachidonate/10(6) cells, a concentration 10-20% above normal for the parent mouse fibrosarcoma cell line (HSDM1C1) which is not essential fatty acid-deficient. Importantly, the percent of released arachidonate which was converted to PGE2 decreased from 90 to 15% with increasing concentrations of cellular arachidonate, because PGE2 production plateaued at greater than or equal to 6 nmol of arachidonate/10(6) cells, but total arachidonate release continued to rise. Finally, we demonstrated that agonist stimulation with thrombin, A23187, and bradykinin all showed the same percent conversion of released arachidonate to PGE2, implying that the determination of this fraction is not a function of the mechanism of release. These studies with our unique cell line indicate that, when the concentration of arachidonate in the cell is not elevated above amounts normally found in our HSDM1C1 cell line, released arachidonate is rapidly and almost quantitatively converted to PGE2, independent of the agonist used to stimulate the cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ras signaling in tumor necrosis factor-induced apoptosis.

Tumor necrosis factor (TNF) exerts cytotoxicity on many types of tumor cells but not on normal cells. The molecular events leading to cell death triggered by TNF are still poorly understood. Our previous studies have shown that enforced expression of an activated H-ras oncogene converted non-tumorigenic, TNF-resistant C3H 10T1/2 fibroblasts into tumorigenic cells that also became very sensitive to TNF-induced apoptosis. This finding suggested that Ras activation may play a role in TNF-induced apoptosis. In this study we investigated whether Ras activation is an obligatory step in TNF-induced apoptosis. Introduction of two different molecular antagonists of Ras, the rap1A tumor suppressor gene or the dominant-negative rasN17 gene, into H-ras-transformed 10TEJ cells inhibited TNF-induced apoptosis. Similar results were obtained with L929 cells, a fibroblast cell line sensitive to TNF-induced apoptosis, which does not have a ras mutation. While Ras is constitutively activated in TNF-sensitive 10TEJ cells, TNF treatment increased Ras-bound GTP in TNF-sensitive L929 cells but not in TNF-resistant 10T1/2 cells. Moreover, RasN17 expression blocked TNF-induced Ras-GTP formation in L929 cells. These results demonstrate that Ras activation is required for TNF-induced apoptosis in mouse fibroblasts.     

Modulation of tumor necrosis factor-alpha cytotoxicity in L929 cells by bacterial toxins, hydrocortisone and inhibitors of arachidonic acid metabolism

L929 cells were incubated with tumor necrosis factor-alpha (TNF-alpha) in the presence or absence of various inhibitors of arachidonic acid metabolism. The addition of either hydrocortisone or nordihydroguaiaretic acid (NDGA) decreased the cytotoxic effect of TNF-alpha but exogenously added arachidonate or linoleate, indomethacin and eicosatetraynoic acid (ETYA) were without effect. While it was found that TNF-alpha stimulated arachidonic acid release, no metabolites of this fatty acid could be evidenced. Cytotoxicity of TNF-alpha could also be decreased by the addition of either cholera or pertussis toxin. These results suggest that a GTP-binding protein is involved in the cytotoxic action of TNF-alpha. Arachidonic acid, released possibly by a phospholipase A2, might also play a role, but probably not via its conversion to known metabolites.     

Tumor necrosis factor-mediated cytotoxicity involves ADP-ribosylation

The mechanism of TNF-mediated cytotoxicity was studied in several cell lines, including L929 murine fibroblasts. TNF caused a time- and dose-dependent increase of ADP-ribosylation in L929 target cells parallel to cell death. During the course of TNF-mediated cytotoxicity in the presence of actinomycin D, an increase in ADP-ribosylation became apparent between 4 and 6 h after exposure to TNF. Intracellular NAD+ and ATP levels decreased parallel to but not preceding cell death. Two inhibitors of ADP-ribosylation, namely 3-aminobenzamide and nicotinamide, prevented TNF-mediated cytotoxicity. Another target, the human cervical carcinoma cell line ME-180, showed an increase in ADP-ribosylation when treated with TNF, and the cytotoxic action of TNF on this target cell was inhibited by these two inhibitors. In the absence of actinomycin D, treatment of L929 cells with TNF also increased ADP-ribosylation, and the cytotoxic action of TNF was inhibited by nicotinamide. These results indicate that ADP-ribosylation may be involved in the TNF-mediated cytotoxic reaction.     

Metaxin is required for tumor necrosis factor-induced cell death

We used retrovirus insertion-mediated random mutagenesis and tumor necrosis factor (TNF) selection to generate TNF-resistant lines from L929 cells. The metaxin gene, which encodes a protein located on the outer membrane of mitochondria, was identified to be the gene disrupted in one of the resistant lines. The requirement of metaxin in TNF-induced cell death of L929 was confirmed by the restoration of TNF sensitivity after ectopic reconstitution of metaxin expression. Analysis of the cell death induced by other stimuli revealed that metaxin deficiency-mediated death resistance was selective to certain stimuli. Studies using deletion mutants of metaxin showed that mitochondrial association of metaxin is required for the function of metaxin. Over-expression of truncated metaxin lacking the mitochondria anchoring sequence mimicked metaxin deficiency in wild-type cells. Interfering with metaxin prevented TNF-induced necrotic cell death in L929 cells and apoptosis in MCF-7 cells. Our work has thus defined a novel component in the death pathway used by TNF and some other death stimuli.     

Transduction of a signal for arachidonic acid metabolism by untriggered CSF-1 receptor induces an opposite effect to that induced by CSF-1 receptor and its ligand: separate regulation of phospholipase A2 and cyclooxygenase by CSF-1 receptor/CSF-1.

The mouse hematopoietic cell line, 32D, was transfected with c-fms, which encodes for the CSF-1 receptor, a tyrosine kinase (TK). In the absence of CSF-1, transfected cells show moderate levels of arachidonic acid (AA) release and produce a substantial amount of prostaglandin E2 (PGE2) in comparison with the original cell line. Exposure of transfected cells to CSF-1, while inducing a substantial increase in arachidonate release, nevertheless resulted in inhibition of PGE2 production. Addition of ST638, a tyrosine kinase inhibitor, to cells transfected with c-fms in the absence of CSF-1 inhibited PGE2 production within 10-60 min. Its addition to the same cells in the presence of CSF-1 induced an opposite effect, but required longer treatment (24 h). In either cell type, AA release was not affected by this agent. These data indicate that CSF-1 may regulate cyclooxygenase activity. The different effect of CSF-1 receptor on PGE2 production in the presence or absence of CSF-1 and the opposite effect of a tyrosine kinase inhibitor on PGE2 suggest that both the receptor alone or the receptor-ligand complex may transduce an active, but different, signal through tyrosine phosphorylation. CSF-1 receptor and CSF-1 may exert separate, but related, effects on phospholipase A2 and cyclooxygenase activity which, in concert, or along with other tyrosine kinases, regulate prostaglandin production.     

Interleukin 1 and tumor necrosis factor potentiate angiotensin II- and calcium ionophore-stimulated prostaglandin E2 synthesis in rat renal mesangial cells

In resting mesangial cells, angiotensin II and the calcium ionophore A23187 stimulated prostaglandin E2 (PGE2) formation. After pretreatment with interleukin 1 beta (IL-1 beta) or tumor necrosis factor alpha (TNF alpha), which are themselves potent stimuli for PGE2 synthesis, mesangial cells displayed an amplified response to angiotensin II and A23187. The cytokine-induced effects occurred in a time- and dose-dependent manner and were attenuated by actinomycin D, cycloheximide and dexamethasone. IL-1 beta and TNF alpha treatment also increased the amount of arachidonic acid released after stimulation of cells with angiotensin II and A23187. In addition, IL-1 beta but not TNF alpha treatment augmented the formation of PGE2 from exogenous arachidonic acid by mesangial cells. Furthermore, the conversion of prostaglandin H2 to PGE2 was not changed by IL-1 beta and TNF alpha. These results suggest that IL-1 beta and TNF alpha exert a priming effect on PGE2 production in mesangial cells.     

Tumour necrosis factor induced an early release of superoxide and a late mitochondrial membrane depolarization in L929 cells. Increase in the production of superoxide is not sufficient to mimic the action of TNF

Tumour necrosis factor alpha (TNF) cytotoxicity is mediated, at least in part, by oxidative stress. One of the post-receptor events shortly after the addition of TNF is the generation of the superoxide anion (O2-*). In the present study, we attempted to examine the role of O2-* in the regulation of mitochondrial membrane potential (Delta(Psi)m) and the release of cytochrome c (cyto c) in L929 cells after stimulation with TNF. Challenge of cells with TNF (50 ng/ml) resulted in an early (30 min after the addition of TNF) increase in the production of O2-*. The use of mitochondrial electron transport chain inhibitors such as antimycin A and rotenone could, respectively, potentiate or suppress the TNF-mediated release of O2-* and cytotoxicity. TNF also induced a late (>3 h after the addition of TNF) depolarization in the Delta(Psi)m. Reduction in the release of O2-* by rotenone (50 microM) or thenoyltrifluoroacetone (250 microM) suppressed both the TNF-mediated Delta(Psi)m depolarization and cyto c release. However, increase in the production of O2-* by antimycin A (25 microM) only slightly enhanced the TNF effect in altering the Delta(Psi)m and the release of cyto c. Treating cells with antimycin A alone could not induce a reduction in Delta(Psi)m nor a release of cyto c. Taken together, our results indicate that TNF induced damage in mitochondria in L929 cells. Our data also show that an increase in the production of O2-* was important in the TNF cytotoxicity, but was not sufficient to mimic the action of TNF.     

Dexamethasone inhibits the cytotoxic activity of tumor necrosis factor

Effect of dexamethasone (DEX) on the cytotoxic activity of tumor necrosis factor (TNF) was examined using murine fibroblast cell line (L929 cells). DEX protected cells from the cytotoxic action of TNF. Protection of cytotoxic action was apparent when cells were pre-treated with DEX for 12h and no protection was observed in the presence of cycloheximide. These results suggested that de novo synthesis of new proteins was required for DEX-mediated protection. Moreover, prolonged simultaneous treatment with TNF and DEX resulted in the enhancement of cell growth, suggesting that TNF acted as a growth factor when cells were protected from the cytotoxic action of TNF. These results suggested that the signal transduction system for fibroblast growth enhancing and cytotoxic action of TNF were different from each other and that the interaction between TNF and glucocorticoids may play a modulating role in some inflammatory processes in vivo.     

Decreased prostaglandin production by cholesterol-rich macrophages

The regulation of prostaglandin production by macrophages enriched in cholesterol was examined. Mouse peritoneal macrophages were incubated for 18 h with 25 micrograms/ml of human acetyl-LDL (low density lipoprotein) and trace amounts of labeled arachidonic acid. After cholesterol enrichment, the cells were incubated with phorbol 12-myristate 13-acetate (PMA), calcium ionophore, or zymosan to stimulate endogenous arachidonic acid metabolism. A high performance liquid chromatography profile of the eicosanoids released revealed no qualitative differences between unmodified and modified macrophages. Cholesterol-rich cells, however, released less prostacyclin (PGI2) and prostaglandin E2 (PGE2) compared to unmodified cells, and products from the lipoxygenase pathway became the predominant metabolites. A decrease in the synthesis of PGI2 and PGE2 by cholesterol-rich macrophages was confirmed by radioimmunoassay and radiolabeled experiments. The activity of prostaglandin synthetase was modestly increased in the cholesterol-modified macrophages compared to controls. As an estimation of phospholipase activity, the release of labeled arachidonic acid from membrane phospholipids, however, was significantly decreased in cholesterol-rich macrophages. The phosphatidylinositol fraction was particularly resistant to arachidonate release in response to calcium ionophore and PMA in the modified cells. The measurement of membrane phospholipid fatty acid composition before and after calcium ionophore supported the observation that less arachidonate was released by cholesterol-enriched cells in response to the ionophore. Based on these observations, we propose that prostaglandin synthesis from endogenous arachidonate stores is decreased in the cholesterol-rich macrophage. A decrease in agonist-induced activation of the phospholipase activity is proposed as a mechanism for this effect.     

Comparative studies of the biological activities of human tumor necrosis factor and its derivatives

Biological activities of human tumor necrosis factor (TNF) and its derivatives were compared. In cytotoxicity assay with L929 cells, one derivative, designated as TNF(Asn), showed significantly lower activity than any other TNF examined. In binding assay, this derivative was also shown to have lower affinity for TNF receptors on L929 cells, suggesting that the cytotoxic activity of TNFs on L929 cells correlates with their affinity for receptors. We also found that the cytotoxic activity of TNF on A673 cells and its inhibitory effect on lipoprotein lipase were parallel with the cytotoxic activity on L929 cells, but the growth-enhancing activity on FS-4 cells and the cytotoxic activity on endothelial cells were not. It was also shown that TNF(Asn) had lower affinity than any other TNF for receptors on these target cells tested. These results suggested that there might be at least two types of cellular responses to TNF; one might correlate with the receptor-binding affinity of TNFs and the other not.