Recombinant Human Tumor Necrosis Factor-alpha Research Articles

Synergistic effect of rhTNF-alpha and rhIL-1 alpha in inducing anorexia in rats

To investigate whether there is a synergistic effect of recombinant human tumor necrosis factor-alpha (rhTNF-alpha) and recombinant human interleukin-1 alpha (rhIL-1 alpha) in inducing anorexia, 32 rats with jugular catheters were studied (8 rats/group): controls received normal saline; the IL-1 group received rhIL-1 alpha (10 micrograms/kg); the TNF group received rhTNF-alpha (30 micrograms/kg); and the IL-1 + TNF group received the same concentration of both rhIL-1 alpha+rhTNF-alpha for 3 days; solutions were then switched to normal saline. No significant decrease in light- and dark-phase food intake occurred during infusion of subeffective rhTNF-alpha. Food intake was decreased on the first rhIL-1 alpha infusion day because of a decreased meal number during the dark phase. A significant decrease in food intake occurred with combined infusion of rhIL-1 alpha+rhTNF-alpha because of a reduction in meal number during dark phase and meal size during light phase. Our results show that rhIL-1 alpha and rhTNF-alpha, when administered concurrently, had a synergistic effect in inducing anorexia, suggesting that low concentrations of these cytokines as produced endogenously may have potential effects on other biological functions in vivo.

Stimulation and release of interleukin-1 from peritoneal macrophages of the mouse

Lipopolysaccharide (LPS) caused a concentration-dependent increase of released and cell-associated interleukin-1 (IL-1) in resident peritoneal macrophages from the mouse. LPS was about 30 times more potent at stimulating the level of cell-associated IL-1 than it was at stimulating the release of IL-1. Human recombinant tumour necrosis factor-alpha (TNF-alpha) and the calcium ionophores A23187 and ionomycin induced a concentration-dependent increase of cell-associated IL-1 but failed to cause release of IL-1 at concentrations producing maximal stimulation of cell-associated IL-1. The phorbol ester, 4 beta-phorbol dibutyrate, stimulated the release of IL-1 from mouse macrophages but failed to induce an increase in cell-associated IL-1. Substance P, neurokinin A, neurokinin B, calcitonin gene-related peptide and platelet-activating factor did not increase the released or cell-associated IL-1 in mouse macrophages. These agents also failed to alter released or cell-associated IL-1 stimulated by LPS, 1 microgram ml-1. It appears that a calcium signal is sufficient for the transcription and translation of IL-1 mRNA but does not result in the secretion of biologically active forms of IL-1. Our data also indicate that different intracellular signals may control the release and the cell accumulation of IL-1. We conclude that inflammatory mediators may independently increase either the release of, or the cell accumulation of IL-1.

Circadian dynamics of tumor necrosis factor alpha (cachectin) lethality.

Recombinant human tumor necrosis factor-alpha (TNF-alpha) has demonstrable antitumor activity in transplantable murine tumor models and patients with cancer but is highly toxic to both animals and human beings. The narrow therapeutic index of TNF-alpha has limited its anticancer utility. Toxicity associated with many standard anticancer drugs is highly dependent upon the circadian timing of their administration. The effect of time of day of TNF-alpha administration on lethal toxicity was examined in 238 BALB/c female mice in two studies. Each mouse received a single intravenous injection of human TNF-alpha at one of six equispaced times within the first contiguous 24-h cycle. The probability of dying across all times of day of TNF-alpha treatment was not equal (p < 0.01) and varied up to ninefold. Significant time of day dependence of TNF-alpha toxicity was present over a full order of magnitude of TNF-alpha dose. The frequency of TNF-alpha-induced lethality was greatest and the time to death was most brief when TNF-alpha was administered just before awakening. The survival probability was highest when TNF-alpha was administered in the second half of the daily activity span corresponding roughly to late afternoon and evening hours for human beings. The optimization of TNF-alpha administration timing is a strategy that warrants further investigation for improving the toxic/therapeutic ratio of this important cytokine. From a more fundamental perspective, these data may be essential for achieving a fuller understanding of TNF-alpha in vivo biology.

Proinflammatory cytokines (interleukin-1 beta and tumor necrosis factor-alpha) down regulate synthesis and secretion of thrombospondin by human endothelial cells.

We examined the effects of proinflammatory cytokines on the expression of two extracellular matrix proteins, e.g., thrombospondin (TSP) and fibronectin (FN) b cultured human umbilical vein endothelial cells (HUVECs). Treatment of HUVECs with human recombinant interleukin-1 beta (IL-1 beta) or human tumor necrosis factor-alpha (TNF-alpha) caused a time-and dose-dependent decline in TSP production whereas FN production was not modified. At low concentrations, IL-1 beta and TNF-alpha in combination ha a greater effect than either agent alone. Interferon-gamma (IFN-gamma) was without effect. The decline in TSP synthesis resulted in a decreased secretion of this glycoprotein into the extracellular matrix. Endothelial cell monolayers cultured on porous filters were used to study the polarity of TSP secretion. Approximately two thirds of the synthesized protein was secreted to the apical side medium and one third to the basal side medium and both types of secretion were inhibited to a similar extent by cytokine treatment. Immunoprecipitation experiments revealed no apparent degradation of secreted TSP, either in the apical or in the basal compartment. Treatment of HUVECs with lL-1 beta, either alone or in combination with TNF-alpha, had no significant effect on the steady-state TSP mRNA levels, suggesting a posttranscriptional regulation. Our results indicate that IL-1 beta decreasing TSP deposition and suggest different regulatory mechanisms for the expression of various secreted proteins by endothelial cells.

Tumor necrosis factor and interleukin-1 synergy in the context of malaria pathology.

Reports linking human malarial illness and pathology with serum tumor necrosis factor (TNF) levels are now common, although the association is not always precise. Possible reasons for this discrepancy include the reported variation in levels of interleukin-1 (IL-1), a cytokine known to synergize with TNF. We have examined the extent of synergy between recombinant human TNF and either recombinant human IL-1 alpha or recombinant human IL-1 beta in producing hypoglycemia and increasing plasma levels of nitric oxide in malaria (Plasmodium vinckei)-infected CBA mice. Very low concentrations of either IL-1 alpha or IL-1 beta, with negligible effects on their own, greatly enhanced the effectiveness of TNF in bringing about these changes. In particular, synergy in generating nitric oxide, a mediator argued to induce cerebral malaria, was profound. Thus, variation in generation of IL-1 during infection provides one explanation for the poor correlation sometimes encountered between serum TNF levels and clinical condition.

Influence of tumour necrosis factor-alpha on the expression of Fc IgG and IgA receptors, and other markers by cultured human blood monocytes and U937 cells.

The expression of Fc receptors for IgG (Fc gamma R) and IgA (Fc alpha R) and of various other antigens on the human monocytic cell line U937 and peripheral blood monocytes, under stimulation with human recombinant tumour necrosis factor-alpha (TNF-alpha) and other cytokines, was investigated by flow cytometry. TNF-alpha, as well as interferon-gamma (IFN-gamma) or interleukin-6 (IL-6) had a significant up-regulating effect on U937 expression of Fc gamma RI/CD64. Furthermore, the action of TNF-alpha was augmented by IL-6, and more evidently by IFN-gamma. IFN-alpha alone had only a marginal effect, but was able to increase the TNF-alpha-driven Fc gamma RI expression. In contrast to U937 cells, TNF-alpha did not enhance significantly Fc gamma RI expression on human monocytes. Interestingly, on both U937 cells and monocytes, Fc alpha R was augmented markedly by TNF-alpha. Furthermore, TNF-alpha induced the expression of HLA-DR and HLA-DP antigens on monocytes and U937 cells. The expression of Fc gamma RII/CD32, FC gamma RIII/CD16, CD14, complement receptor type 1 (CR1/CD35), CR4 (CD11c/CD18), and MHC class-I antigens, was not influenced significantly by TNF-alpha. The results of this study show that TNF-alpha may act on human mononuclear phagocytes, alone or in combination with other cytokines, by modulating the expression of various cell-surface antigens.

Differential induction of metallothionein synthesis by interleukin-6 and tumor necrosis factor-alpha in rat tissues.

Metallothionein (MT) synthesis induced by the inflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF), was studied in vivo. Administration of recombinant human IL-6 or TNF to rats caused the acute phase responses including rapid decreases in plasma zinc (Zn), and increases in plasma copper (Cu) and ceruloplasmin. Hepatic concentration of MT-I, one of MT isoforms, began to increase within 3 h after the injection of IL-6 or TNF. In IL-6-treated rats, MT-I concentration in liver reached a maximum level at 12 h and decreased with a transient rebound, whereas, in TNF-treated rats, a high level of MT-I lasted for about 48 h. MT-II, the other MT isoform, was induced more than MT-I in liver by both cytokines. MT-I was also induced in lung and heart by TNF, but little by IL-6. The data suggest that IL-6 may be responsible for MT synthesis in liver, whereas TNF may be responsible not only in liver but also in lung and heart. Furthermore plasma concentration of MT did not always reflect the enhanced concentration of MT by TNF and IL-6 in liver, suggesting involvement of many factors influencing plasma MT levels. The interrelation between IL-6 and TNF for MT synthesis has also been discussed.

Cytotoxic manifestations of the interaction between hyperthermia and TNF: DNA fragmentation.

The relationship of DNA fragmentation to the greatly enhanced cytotoxicity seen in vitro against tumour cells when recombinant human tumour necrosis factor-alpha (TNF-alpha) is combined with hyperthermia was investigated. The TNF-alpha-sensitive L929 and -resistant EMT6 cells were treated with 8.8 and 16 ng of TNF-alpha per ml, respectively, and then heated at 40.5 degrees C for 24 h (L929) or at 43 degrees C for 1 h (L929) or 1.5 h (EMT-6) beginning 1 h later. For both cell lines at both temperatures, the addition of heating to the TNF-alpha treatment significantly decreased viability and increased DNA fragmentation at earlier time points than seen with either TNF-alpha or heat alone. DNA fragmentation was further studied using agarose gel electrophoresis to examine the size distribution of the DNA fragments and the ability of intracellular calcium buffering agents BAPTA and quin-2 to inhibit fragmentation. At 4.5 h after L929 cells were treated with TNF-alpha at 43 degrees C, the size distribution of DNA fragments more closely resembled the oligonucleosome sized apoptotic DNA fragmentation, as seen in irradiated rat thymocytes, than the spectrum of DNA fragments seen in necrotic fragmentation. However, while BAPTA and quin-2 inhibited the calcium-dependent apoptotic fragmentation seen in thymocytes they did not inhibit the DNA fragmentation in L929 cells. In addition, the loss of membrane integrity in both L929 and EMT-6 cells preceded or approximated the appearance of DNA fragmentation, whereas loss of membrane integrity usually follows DNA fragmentation in apoptosis. However, morphological studies showed that apoptotic bodies were present in L929 cell cultures treated with TNF-alpha and heat, and were distinguishable from necrosing cells. We conclude that both types of DNA fragmentation are operant in some cell lines exhibiting a cytotoxic response to TNF-alpha and heat treatments, and that increased fragmentation reflects the greatly enhanced cytotoxic interactions seen with combination treatments in those cells.

Cytokines decrease apolipoprotein accumulation in medium from Hep G2 cells.

Cytokines, important biochemical mediators of inflammation, cause a rapid fall in the plasma concentration of cholesterol in vivo. One mechanism by which cytokines may cause acquired hypocholesterolemia is by decreasing the hepatic synthesis and secretion of apolipoproteins. To test this hypothesis, we incubated Hep G2 cells with human recombinant tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6. Each of the cytokines resulted in a dose-related reduction in the concentrations of apolipoprotein (apo) A-I, apoB, and lecithin:cholesterol acyltransferase (LCAT) activity in the medium after 24 hours of incubation. The effect of cytokines on apolipoprotein accumulation was not affected by preincubation of Hep G2 cells with fatty acids. Cytokines decreased the concentration of cellular apoA-I mRNA in a dose-related fashion but did not affect cellular concentrations of apoB mRNA. The concentrations of triglyceride and cholesterol were also reduced in the medium of cells incubated with cytokines. Total cell sterol synthesis rates were calculated by [14C]acetate incorporation. Cells incubated with interleukin-6 had a 31% increase in sterol synthesis rate but a 41% decrease in sterol secretion. These data suggest that these cytokines can decrease the hepatic synthesis and/or secretion of apolipoproteins and that this may explain, in part, the acquired hypocholesterolemia seen during acute and chronic inflammation.

Blood-brain barrier changes following intracerebral injection of human recombinant tumor necrosis factor-alpha in the rat.

Human recombinant tumor necrosis factor-alpha (rTNF-alpha) was administered to normal Fischer 344 rats by stereotaxic intracerebral (IC) injection. Animals receiving a single injection of either 6 x 10(4) U rTNF-alpha or an equivalent volume of excipient (vehicle) in their right parietal lobe. In order to demonstrate any effects rTNF-alpha might have on the blood-brain barrier (BBB), two studies were conducted, one employing exogenous horseradish peroxidase (HRP,44 kD) as a tracer of BBB permeability and the other using endogenous IgG (150 kD). Rats given rTNF-alpha showed transitory BBB permeability to HRP by 24 hours post-injection; this BBB compromise was determined to be no longer than 60 hours. In the other study, IgG was seen to cross the BBB by 48 hours post-rTNF-alpha injection. Alternatively, rats injected IC with excipient showed only limited BBB opening as a result of injection-induced trauma. We conclude that human rTNF-alpha, injected IC into normal rats triggers a temporary breakdown in BBB integrity which begins sometimes between 12 and 24 hours post-injection, is large enough to permit macromolecules of at least 150 kD to pass, and resolves by 72 hours post-injection.

A comparison of the Antileukaemic Effects of Recombinant Human Tumour Necrosis Factor-α and its Muteins on Leukaemia L1210 and Leukaemia P388 in Mice

We investigated the influence of recombinant human tumour necrosis factor alpha (TNF-α) and its derivatives termed muteins III, V, VI—in which the first 3 to 7 amino acids of native TNF-α have been replaced—on the survival time of mice inoculated with leukaemia L1210 or leukaemia P338. TNF-α prolonged the survival of mice with leukaemia L1210 but did not have any therapeutic activity in leukaemia P388-bearing mice. Muteins-treated mice with leukaemia P388 lived longer than animals receiving TNF-α, while those inoculated with leukaemia L1210 did not show any significant prolongation of life compared with the TNF-α treated group. The results presented in this report indicate that the antileukaemic activity of TNF-α is governed at least in part by the nature of the N-terminal amino acids.

Effect of cytokines and prostaglandins on the growth of chick retinal pigment epithelial cells.

The effect of cytokines and prostaglandins on the growth of chick retinal pigment epithelial (RPE) cells was studied in vitro. The growth of RPE cells was evaluated by [3H]-thymidine uptake. Human recombinant interleukin-1 beta, interferon-beta and tumor necrosis factor-alpha stimulated the growth of RPE cells at 50-200 units/ml. Prostaglandins such as PGE1, PGE2 and PGF2 alpha suppressed RPE cell growth at 10(-5) approximately (-6)M. Indomethacin, an inhibitor of cyclooxygenase, did not affect the spontaneous RPE cell growth at 10(-7) approximately (-9) M. However, 10(-7) M indomethacin enhanced RPE cell growth stimulated by cytokines synergistically. The addition of 10(-6) M PGF2 alpha suppressed the enhanced growth of RPE cells which was induced with cytokines and indomethacin. Furthermore, RPE cells stimulated with cytokines produced PGE2. These results suggest that RPE cells produce prostaglandins which have a negative regulatory role in RPE cell growth.

Respective effects of soluble interleukin‐1 receptor and tumour necrosis factor receptor on IL‐1 and TNF‐α‐induced DNA synthesis of common acute lymphoblastic leukaemia blasts in vitro

This study investigates the capacity of human recombinant interleukin-1 alpha (IL-1 alpha), IL-1 beta and tumour necrosis factor-alpha (TNF-alpha) to induce DNA synthesis of highly purified blasts from nine adult common acute lymphoblastic leukaemias (cALL) in 7 d liquid culture. IL-1 alpha, IL-1 beta and TNF-alpha stimulated 3H-TdR uptake in leukaemic blasts in a dose-dependent fashion. The IL-1-induced DNA synthesis of cALL cells could not be prevented by the addition of neutralizing antibodies against IL-3, GM-CSF, IL-6 or TNF-alpha. Similarly, the TNF-alpha-stimulated 3H-TdR incorporation of leukaemic blasts was not affected by the addition of antibodies towards IL-1 alpha, IL-1 beta, IL-3, GM-CSF or IL-6. These observations suggest that IL-1 as well as TNF-alpha stimulated growth could not be attributed to the endogenous production of factors, corresponding to the antibodies used in these experiments. Both IL-1 as well as TNF-alpha mediate their action through interaction with specific cell surface receptors. Recently two distinct types of IL-1 receptors (IL-1-Rs), IL-1R(p80) and IL-1-R(p65), as well as two distinct types of TNF-receptors (TNF-Rs), TNF-R (p55) and TNF-R (p75) have been identified. Both types of TNF-Rs exist also in soluble forms (sTNF-Rs), while soluble IL-1-Rs (sIL-1-Rs) have not yet been found naturally. In this study we show that sIL-1-R as well as sTNF-R modulate the effects of their corresponding cytokine in a dose-dependent bimodal fashion; at lower concentrations they augmented while at higher concentrations they inhibited the cytokine-stimulated DNA synthesis of cALL blasts in vitro. It may therefore be concluded from this study that soluble receptors for both IL-1 and TNF, at least in vitro, are functional and interfere with their corresponding cytokine bioactivity.

Modulation of antibody-mediated glomerular injury in vivo by interleukin-6

We have shown previously that pretreatment with small doses of bacterial lipopolysaccharide (LPS), human recombinant interleukin-1 beta (hrIL-1 beta) and human recombinant tumor necrosis factor-alpha (hrTNF) increase injury in the heterologous phase of nephrotoxic nephritis (NTN). All three pretreatments induce synthesis of interleukin-6 (IL-6) which in some systems down-regulates synthesis of IL-1 and TNF. We have now investigated the influence of IL-6 on injury in both heterologous and autologous phases of NTN in rats. Injection of hrIL-6 in doses sufficient to induce hepatic synthesis of acute phase proteins (assessed by plasma alpha 2-macroglobulin concentration) had no effect on glomerular injury in the heterologous phase of NTN (albuminuria in NTAb alone 9 +/- 6; LPS/NTAb 34 +/- 10 and IL-6/NTAb 2 +/- 1 mg/24 hr, P < 0.001, Wilcoxon test). In contrast, IL-6 pretreatment partially abrogated the effect of LPS on albumin excretion (NTAb 4 +/- 2; LPS/NTAb 85 +/- 11 and IL-6/LPS/NTAb 32 +/- 6 mg/24 hr, P < 0.002), percentage of glomerular capillary thrombi (3 +/- 1%; 39 +/- 8%; and 6 +/- 1%, P < 0.001) and glomerular neutrophil infiltrate (29 +/- 3; 58 +/- 5; and 34 +/- 2 neutrophils/50 glomeruli in section, P < 0.001, respectively) at 24 hours. The effect of IL-6 was also evident four hours after induction of nephritis and was associated with a marked reduction in glomerular concentration of mRNA for IL-1 beta and TNF, without change in that of tubulin. Serum TNF concentrations were also significantly reduced at four hours in IL-6 treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of tumor necrosis factor on inward potassium current and cell morphology in cultured human oligodendrocytes

The effects of recombinant human tumor necrosis factor-alpha (rhTNF-alpha) on inward rectifier potassium [K(IR)] currents and on cell morphology have been studied in cultured human oligodendrocytes. Cell-attached patches were used to isolate and record unitary currents through an inward rectifier K+ channel with a conductance of 23 pS. In control oligodendrocytes the mean open times showed an exponential dependence on patch potential with an e-fold decrease over a patch hyperpolarization of 28 mV. Treatment of oligodendrocytes with rhTNF (at 250 ng/ml for 24-48 h) had significant actions to diminish the mean open times of K(IR) relative to control values. At cell resting potential the mean open times were reduced by 60% after rhTNF application; the amplitudes of unitary currents or the extrapolated zero-current potentials were not significantly changed by the cytokine. The rhTNF treatments were not cytotoxic to cultured human oligodendrocytes; however, in some experiments rhTNF caused evident retraction of cell processes.

A colorimetric assay for trypanosome viability and metabolic function

We have adapted a tetrazolium salt (MTT) colorimetric cytotoxicity assay to the assessment of viability and metabolic function in cultured African trypanosomes. Trypomastigotes of Trypanosoma congolense and T. brucei rhodesianse were harvested from the blood of parasitemic rats and cultured under axenic conditions that support trypanosome viability and growth. Analysis of serial dilutions of these bloodstream forms indicated that the assay could detect 10 4 parasites. To assess the effect of lymphoid cytokines on trypomastigote viability, 10 5 freshly harvested parasites were cultured with a wide range of dilutions of human recombinant IL-1, IL-3, IL-6, interferon-gamma (IFNγ) or tumor necrosis factor-alpha (TNFα), or bovine recombinant IFNγ or TNFα for 24, 48 or 96 h. These cytokines had no apparent growth enhancing or inhibitory effect on the trypomastigotes compared with growth in supplemented medium alone. This assay has several advantages over traditional counting methods, including increased sensitivity and rapid, repeatable quantitation. This adaptation of the MTT colorimetric assay should be useful in screening drugs and host-derived factors for growth-modulating effects on trypanosomes and other extracellular protozoan parasites.

Analysis of the Mechanism of Ultraviolet (UV) B Radiation – Induced Prostaglandin E2 Synthesis by Human Epidermoid Carcinoma Cells

Stimulation of cultured human keratinocytes with interleukin (IL)-1 alpha is known to elicit prostaglandin (PG) E2 release. Ultraviolet (UV) B radiation induces keratinocyte PGE2 and cytokine production. The present study deals with the autocrine roles of UVB-induced, keratinocyte-derived cytokines IL-1 and tumor-necrosis-factor (TNF) alpha and their corresponding receptor molecules for UVB-induced PGE2 release. In vitro exposure of transformed human keratinocytes (KB cells) induced PGE2 production five- to eightfold. This increase was inhibited by 70%, if irradiated cells were cultured in presence of monoclonal antibody (MoAb) M4, which blocks IL-1 effects by binding to the type 1 IL-1 receptor (IL-1R). In contrast, MoAb M22, which blocks the type 2 IL-1R, had no significant effects. Addition of recombinant human TNF alpha to unirradiated KB cells resulted in five- to eightfold increased PGE2 synthesis, and this increase could be mimicked by stimulation of KB cells with MoAb htr-9, which exerts TNF alpha-like bioactivity by binding to the 55-kD TNF receptor (TNFR). UVB-induced PGE2 synthesis was blocked by 50% in the presence of neutralizing anti-TNF alpha-Ab, and was completely inhibited by addition of both anti-TNF alpha-Ab and MoAb M4. To elucidate a possible regulatory intracellular step in PGE2 synthesis, specific cyclooxygenase activity in KB cells was determined. Following UVB treatment, cyclooxygenase activity increased twofold, but remained unaltered, if irradiated KB cells were cultured in the presence of anti-TNF alpha-Ab plus MoAb M4. These studies indicate that keratinocyte-derived TNF alpha and IL-1 together mediate UVB-induced PGE2 release via specific cell surface receptors, and that one intracellular mechanism is an increased prostanoid-synthesizing capacity of irradiated cells.

Role of hypothalamic interleukin-6 and tumor necrosis factor-alpha in LPS fever in rat.

The purpose of this study was to determine, using push-pull perfusion, the levels of interleukin (IL)-1-like, IL-6-like, and tumor necrosis factor-alpha (TNF)-like activity in the anterior hypothalamus during lipopolysaccharide (LPS)-induced fever in rats. Additionally, slow anterior hypothalamic infusions of human recombinant IL-6 (hrIL-6) or TNF (hrTNF) for several hours were performed to determine possible febrile effects of these two cytokines. Artificial cerebrospinal fluid (aCSF) was infused as a control. Samples of cerebrospinal fluid were collected 60 min before and 60, 180, 300, and 420 min after the intraperitoneal injection of LPS. A control group was injected intraperitoneally with saline. The core temperature (measured by biotelemetry) of LPS-injected rats was significantly higher (P < 0.05) than the temperature of the rats injected with saline at 180, 300, and 420 min after the injection. The average postinjection IL-6 levels were significantly higher (P < 0.05) in the LPS-injected group. TNF was significantly higher (P < 0.05) than the baseline only at 180 min. There were no changes in levels of IL-1-like activity. Infusion of hrIL-6 at a level similar to the peak IL-6 level measured during LPS-induced fever resulted in a slowly developing and long-lasting increase in core temperature. Infusion of hrTNF at a level corresponding to the peak TNF level measured during LPS-induced fever did not induce a significant increase in core temperature. These results support the hypothesis that elevated hypothalamic concentrations of IL-6 are involved in the induction of fever elicited by peripheral (intraperitoneal) injection of LPS.

Evaluation of antitumor effect of tumor necrosis factor in terms of protein metabolism and cell cycle kinetics.

To determine the significance of protein breakdown in regulating tumor growth and to better understand the antitumor mechanism of tumor necrosis factor in vivo, we measured the effects of a 6-h constant intravenous infusion of human recombinant tumor necrosis factor-alpha (rHuTNF) on tumor protein metabolism and cell cycle kinetics in rats bearing the Walker-256 carcinosarcoma. Protein metabolism was investigated with the use of [14C]leucine infusion; estimates of tumor cell cycle kinetics were obtained in vivo by use of 5-bromo-2'-deoxyuridine (BrdUrd) pulse labeling and bivariate BrdUrd/DNA analysis by flow cytometry. Reduction in tumor growth by rHuTNF was associated with a dose-dependent increase in tumor proteolysis but no change in tumor protein synthesis. At the cellular level, rHuTNF had a significant cytostatic effect on G2/M cells and caused a marked decrease in the fraction of cells capable of BrdUrd uptake. Release of BrdUrd, an indicator of cell death, was noted in only 7.5% of tumor cells labeled at the beginning of rHuTNF infusion. These results suggest that either tumor protein breakdown may influence cell cycle activity by regulating cytoplasmic protein mass or that tumor proteolysis may be a compensatory mechanism for limiting cytoplasmic size when cellular division is interrupted suddenly.

Effect of recombinant human cytokines on porcine neutrophil function

The activity of four recombinant human cytokines on porcine neutrophils was evaluated. Porcine neutrophils were treated with varying doses of recombinant human tumor necrosis factor-alpha (rHu-TNF), interferon-gamma (rHu-IFN), interleukin-8 (rHu-IL-8), or granulocyte-macrophage colony-stimulating factor (rHu-GM-CSF). The function of treated neutrophils was compared with that of non-treated controls in the following assays: antibody-independent neutrophil cytotoxicity (AINC), antibody-dependent cell-mediated cytotoxicity (ADCC), iodination, Staphylococcus aureus ingestion, cytochrome C reduction, random migration, and chemotaxis. Treatment with rHu-TNF produced significant ( P < 0.05) depression of neutrophil random migration (2.5, 25, and 250 ng ml −1 rHu-TNF) and iodination (250 ng ml −1) and a near significant ( P = 0.08) depression in ADCC (250 ng ml −1). Treatment with 25 000 U ml −1 of rHu-IFN caused a significant increase in AINC. At lower doses of rHu-IFN, there was a trend (0.05 < P ≤ 0.08) toward depression of AINC (250 U ml −1) and ADCC (25 U ml −1) and enhancement of iodination (250 U ml −1). Treatment with 50 ng ml −1 of rHu-IL-8 caused a near significant increase ( P = 0.06) M in AINC. There were no significant differences noted when porcine neutrophils were treated with rHu-GM-CSF (2.5–2500 U ml −1). No synergism was noted between rHu-TNF and rHu-IFN.