Tumour necrosis factor α production by rat blood and itsex vivo pharmacological modulation

Rat blood was investigated as a suitable test system for the discovery of inhibitors of tumour necrosis factor (TNF) biosynthesis. Lipopolysaccharide (LPS) caused a concentration- and time-dependent stimulation of TNF production by heparinised rat blood with peak levels (1000–5000 U/ml; L929 bioassay) at 6h. Bioactive material was neutralised with a polyclonal rabbit anti-murine TNF antibody which cross-reacts with rat TNF. Dexamethasone, pentoxifylline and denbufylline inhibited TNF production with IC₅₀s of 6.0±2.0 nM, 20.6±8.00 M and 138.0 nM, respectively. When rats were dosed p.o. with dexamethasone or pentoxifylline or i.p. with denbufylline and 1.5h later TNF production was assessedex vivo by LPS-stimulated blood, a dose-related inhibition of TNF production occurred with ID₅₀s of approximately 0.08, 250.0 and 5.0 mg/kg, respectively. These results demonstrate that rat blood provides a useful test system for the detection andex vivo evaluation of inhibitors of TNF biosynthesis.     

Effect of defensin HNP-1 of human neutrophils on production of tumor necrosis factor α by human blood monocytes in vitro

Research Institute of Hematology and Blood Transfusion, Ministry of Health of Belarus', Minsk. Institute of Experimental Hematology and Biotechnology, Moscow. University of California, Los Angeles. (Presented by Academician of the Academy of Medical Sciences B. F. Semenov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 113, No. 5, pp. 524–527, May, 1992.     

Down-regulation of tumor necrosis factor α activity by acute ethanol treatment in human peripheral blood monocytes

As the most commonly used drug that can modulate both metabolic and immune pathways, ethanol is evaluated in this report as a regulator of tumor necrosis factor (TNF) production in human peripheral blood monocytes (M) in combination with a variety of stimuli. While acute ethanol treatment did not induce TNF in M, it was a potent down-regulator of M TNF production whether induced by the combination of interferon- plus muramyl dipeptide (MDP) (P<0.001), lipopolysaccharide (LPS) alone (P<0.01), or interferon- plus LPS. Down-regulation of M TNF by ethanol was dose dependent and statistically significant in the biologically relevant, 25–150 mM, ethanol concentration range. We also demonstrate that these ethanol concentrations did not affect M viability. TNF down-regulation by ethanol was most effective when ethanol was administered 4 hr prior to MDP stimulation; however, it was also effective—though to a lesser extent—if it was added at the time of MDP stimulation. Furthermore, ethanol also down-regulated TNF production of thein vivo preactivated M of trauma patients, which produce hyperelevated levels of TNF. We have previously shown that the majority of posttrauma elevated M TNF is produced by the M subpopulation expressing high-affinity type I Fc receptors (FcRI). When the FcRI cross-linking-stimulated M subpopulation was treated with acute ethanol, TNF production was suppressed again both inin vivo preactivated M of trauma patients and in M of normal controls. In experiments utilizing cyclooxygenase inhibitor, we also demonstrate that ethanol has a direct, prostaglandin E₂-independent, effect on M TNF production. These results demonstrate that acute ethanol exposure has the potential to down-regulate M production of TNF significantly regardless of the TNF-inducing stimulus. Decreased capacity of M to produce TNF might, therefore, contribute to the immunological and metabolic abnormalities described after ethanol uptake.     

Lipopolysaccharide inhibits macrophage phagocytosis of apoptotic neutrophils by regulating the production of tumour necrosis factor α and growth arrest-specific gene 6

Removal of apoptotic cells from inflammatory sites by macrophages is an important step in the resolution of inflammation. However, the effect of inflammatory modulators on phagocytic clearance of apoptotic cells remains to be clarified. In this paper, we demonstrate that lipopolysaccharide (LPS), a potent inflammatory agent, inhibits the phagocytosis of apoptotic neutrophils by mouse peritoneal macrophages. This inhibition can be attributed to both LPS-mediated induction of tumour necrosis factor (TNF-α) and suppression of growth arrest-specific gene 6 (Gas6) in macrophages. We found that LPS-induced TNF-α production inhibited phagocytic ability of macrophages in an autocrine manner. In contrast, Gas6 expression in macrophages was blocked by LPS, which also contributes to the inhibition of macrophage phagocytosis by LPS. Our data suggest that phagocytic clearance of apoptotic neutrophils by macrophages can be regulated by local pro- and anti-inflammatory factors in two opposite states.     

Synergy between tumour necrosis factor α and granulocyte-macrophage colony-stimulating factor in neutrophil stimulation

We have examined the interaction between the cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor α (TNFα) on polymorphonuclear leukocyte (PMN) function and on PMN responses to further stimulation with formyl Met Leu Phe (fMLP). Incubation of PMN with TNFα (0.3 nM) and, to a lesser extent, with GM-CSF (1 nM) directly stimulated superoxide anion (O ⁻₂ ) generation and increased the response to subsequent stimulation of PMN by fMLP (100 nM). However, the combination of GM-CSF and TNFα did not result in increased O ⁻₂ generation and there was no synergistic effect of the combination of these cytokines on the priming of fMLP-induced O ⁻₂ generation. The combination of TNFα and GM-CSF did result in a striking synergism in the stimulation of PAF generation, and, whereas neither stimulus alone resulted in detectable PAF release, the combination elicited the release of significant levels of PAF. The observation of significant PAF release from PMN exposed to TNFα and GM-CSF indicates that overt neutrophil stimulation with phagocytic or soluble stimuli may not be required for expression of at least some of the PMN pro-inflammatory capacity.

     

Recombinant interleukin-1 and tumor necrosis factor induce neutrophil migration “in vivo” by indirect mechanisms

The and forms of recombinant interleukin-1 (IL-1 and IL-1) and of recombinant Tumor Necrosis Factor (TNF and TNF) induced dose-dependent neutrophil migration into rat peritoneal cavities. Migration induced by both IL-1s showed a bell-shaped dose-response curve and IL-1 was 3-fold more potent than IL-1. Pretreatment of the animals with dexamethasone or depletion of the peritoneal macrophage population, abolished the neutrophil migration induced by the four cytokines. In vitro stimulation of macrophage monolayers with IL-1 and the TNFs released a factor into the supernatant which, unlike these cytokines, induced neutrophil migration in dexamethasone pretreated animals. These results suggest that the neutrophil migration induced by IL-1, IL-1 and TNF is not due to a direct effect on neutrophils, but occurs via the release of a chemotactic factors(s) from resident macrophages.     

Acute ethanol consumption synergizes with trauma to increase monocyte tumor necrosis factor α production late postinjury

The hypothesis that acute ethanol uptake plus trauma can synergize to increase immunosuppression was tested. We found that, unlike non-alcohol-exposed patients, patients with acute alcohol use prior to trauma have a transient decrease in monocyte tumor necrosis factor (TNF) production during the very early postinjury (0–3 days) period. However, TNF production by these alcoholexposed patients' monocytes (MØ) became hyperelevated late postinjury (>9 days). Consequently, these massively elevated MØ TNF levels can contribute to posttrauma immunosuppression after acute alcohol use. We also demonstrate that normal monocyte activation with the superantigen,Staphylococcus enterotoxin B (SEB), results in a preferential induction of cellassociated MØ TNF production, described as characteristic of immunosuppressed trauma patients. Acutein vitro ethanol treatment down-regulated the elevated TNF production by trauma patients' MØ after either SEB, muramyl-dipeptide (MDP), interferon- plus MDP, or lipopolysaccharide (LPS) stimulation. Both SEB- and LPS-induced TNF mRNA induction was inhibited by acute alcohol treatment in normal MØ, indicating that ethanol can regulate cytokine gene expression. An additional immunosuppressive effect of acute ethanol's stimulation was suggested by its induction of elevated transforming growth factor production in trauma patients' activated MØ.     

Increased hippocampal uptake of tumor necrosis factor α and behavioral changes in mice

Brain trauma may alter the function of the blood-brain barrier (BBB) and affect psychomotor activity. We have shown that the transport system for tumor necrosis factor α (TNFα) at the BBB undergoes regulatory changes after spinal cord injury. In this study, we show in CD1 mice that mild trauma by weight-drop to the right temporal region specifically increases the uptake of blood-borne TNFα. This increase, measured by use of radiolabeled murine TNFα, occurred only in the right hippocampus 24 h after injury and returned to normal at 1 week. There was no increase in the uptake of the vascular marker albumin at 1 h, 24 h, or 1 week postinjury, indicating that the BBB remained relatively intact. Human interleukin-1β, which does not cross the BBB by saturable transport, showed no significant changes in brain uptake after trauma. Therefore, the selective entry of TNFα in the injured right hippocampus may be explained by enhanced transport across the BBB. To explore the functional relevance of this transport regulation, we measured mouse behavior by the staircase test. The number of rearings, mainly reflective of exploratory behavior, decreased at 1 h and 1 day after injury but increased at 1 week after a 30-g weight-drop injury. The number of stairs ascended, mainly indicative of locomotor activity, was unchanged at all times tested. We conclude that mild, blunt brain trauma involving the hippocampus causes specific upregulation of TNFα transport and a selective change in exploratory behavior. Although no causal relationship can be established at this time, the behavioral changes might be related to the increased TNFα transport after trauma. Electronic Publication     

Synthesis of tumor necrosis factor α (TNF-α) by human monocytes in vitro

Research Institute of Hematology and Blood Transfusion, Ministry of Health of the Belorussian SSR, Minsk. Institute of Experimental Hematology and Biotechnology, All-Union Hematologic Scientific Center, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR E. A. Zotikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 112, No. 9, pp. 287–289, September, 1991.     

Multiple mechanisms for HSV-1 induction of interferon α production by peripheral blood mononuclear cells

Summary.  UV-inactivated, infectious, and other forms of herpes simplex virus 1 (HSV-1) induced interferon (IFN) production by different routes in myeloid origin mononuclear cells (MOMC) (consisting predominantly of monocytes). GM-CSF activated the MOMC (G-MOMC) to produce greater amounts of interferon while differentiation to DC, by the addition of granulocyte macrophage colony stimulating factor (GM-CSF) and calcium ionophore (GA-MOMC), reduced the levels of interferon production upon challenge with some HSV strains. UV-inactivated virus induced more interferon than infectious virus. L-fucose, an antagonist of the mannose receptor, inhibited the induction of IFN-α by UV-inactivated virus and gB⁻ virus (defective in penetration) in MOMC and GA-MOMC but not G-MOMC. L-fucose had little effect on interferon induction by infectious HSV-1. The insensitivity of the G-MOMC to fucose inhibition distinguishes these interferon producing cells from the pDC2 cells previously described as natural interferon producing cells. The mannose receptor appears to be involved in the response to non-infectious forms of HSV but infectious virus appears to use a different pathway. These studies suggest that non-infectious virions and HSV infected cell debris effectively stimulate monocytes and pre-dendritic cells to produce IFN-α to initiate host protection against HSV infection. Received April 1, 2002; accepted August 17, 2002     

Muramyldipeptide and granulocyte-macrophage colony-stimulating factor enhance interferon-γ-induced nitric oxide production by rat alveolar macrophages

Rat alveolar macrophages incubated with recombinant rat interferon- producel-arginine-dependent nitric oxide, which is rapidly decomposed into nitrite: this production by interferon- was markedly enhanced by granulocyte-macrophage colony-stimulating factor and muramyldipeptide, but not by other cytokines. The enhancement was dependent on the presence ofl-arginine in the incubation medium. It was based on a simple synergism between interferon- and muramyldipeptide and a priming effect of granulocyte-macrophage colony-stimulating factor for interferon--induced nitrite production. These data suggest that cytokine networks are important in the induction of nitric oxide in rat alveolar macrophages.     

Tumour necrosis factor α augments the inhibitory effects of CTLA-4-Ig on osteoclast generation from human monocytes via induction of CD80 expression

Cytotoxic T lymphocyte antigen-4-immunoglobulin (CTLA-4-Ig) exerts anti-rheumatic action via negative regulation of the co-stimulation process between antigen-presenting cells and T cells. CTLA-4-Ig also binds to CD80/CD86 on monocytes of osteoclast precursors. However, little is known about the effect of CTLA-4-Ig on osteoclastogenesis in rheumatoid arthritis (RA). In this study we evaluated the effects of CTLA-4-Ig on osteoclast generation from human blood monocytes (PBM) and rheumatoid synovial fluid monocytes (RSFM). Highly purified monocytes were cultured with receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) in the presence of CTLA-4-Ig. CTLA-4-Ig inhibited RANKL-induced osteoclast generation in PBM and RSFM, as determined by tartrate-resistant acid phosphatase (TRAP) staining and bone resorption assay using osteo assay surface plates. In addition, CTLA-4-Ig reduced the gene and protein expressions of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) and cathepsin K during osteoclastogenesis. Furthermore, CTLA-4-Ig significantly inhibited cell proliferation during osteoclastogenesis. Interestingly, the gene expression of indoleamine 2,3-dioxygenase-1, an inducer of apoptosis, was enhanced by CTLA-4-Ig. We next examined the effect of tumour necrosis factor (TNF)-α, a major inflammatory cytokine in rheumatoid synovium, on the expression of CD80 and CD86 by flow cytometric analysis. TNF-α potently induced the surface expression of CD80, which is known to have much higher affinity to CTLA-4-Ig than CD86, and this induction was observed at mRNA levels. Interestingly, freshly prepared rheumatoid synovial monocytes also expressed CD80 as much as TNF-α-treated PBM. Furthermore, TNF-α enhanced CTLA-4-Ig-induced inhibition of osteoclastogenesis and cell proliferation. Taken together, the TNF-α-induced CD80 may augment CTLA-4-Ig-induced inhibition of osteoclastogenesis, suggesting that CTLA-4-Ig potently inhibits osteoclast differentiation and protects bone destruction in rheumatoid inflamed joints.     

Tumour necrosis factor-α drives Alport glomerulosclerosis in mice by promoting podocyte apoptosis

Chronic renal failure involves the progressive loss of renal parenchymal cells. For example, Alport syndrome develops from mutated type IV collagen that fosters the digestion of glomerular basement membranes and podocyte loss, followed by progressive glomerulosclerosis, ie Alport nephropathy. Here we show that autosomal recessive Alport nephropathy in collagen 4a3-deficient mice is associated with increased intrarenal expression of the pro-apoptotic cytokine tumour necrosis factor-alpha (TNF-α) in glomerular cells including podocytes as well as in infiltrating leukocytes. We therefore hypothesized that TNF-α contributes to Alport glomerulosclerosis by inducing podocyte apoptosis. To address this issue, we treated 4-week-old collagen 4a3-deficient mice with either vehicle or the TNF-α antagonist etanercept for a period of 5 weeks. Etanercept treatment prolonged mean survival from 68 to 81 days as compared to vehicle-treated mice. The beneficial effect of etanercept on survival was associated with a significant improvement of the glomerulosclerosis score, proteinuria, and the glomerular filtration rate at 9 weeks of age. Etanercept treatment specifically reduced the numbers of apoptotic podocytes, increased total podocyte counts, and increased the renal mRNA expression of nephrin and podocin without affecting markers of renal inflammation. TNF-α-induced podocyte loss is a previously unrecognized pathological mechanism of Alport glomerulosclerosis, and TNF-α blockade might be a therapeutic option to delay the progression of Alport nephropathy and potentially of other forms of glomerulosclerosis.     

Effects of tumour necrosis factor α and interleukin 1 β on the proliferation of cultured glomerular epithelial cells

Rat glomerular epithelial cells were cultured with human monocyte supernatant or with recombinant cytokines. A primary glomerular culture and a glomerular epithelial cell culture were made; supernatant from monocyte cultures derived from healthy humans, and recombinant tumour necrosis factor (TNF ) or recombinant interleukin 1 (IL-1 ) were added. Cell proliferation rates were assayed by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. In serum-free media, consistent proliferation of glomerular epithelial cells (GEC) was observed throughout the 3 week culture period. Significant growth-stimulatory effects were induced by lipopolysaccharide-treated monocyte conditioned medium and by 1–50 ng/ml of TNF , growth being up to 400% more than in the control culture. The effect of TNF depended mainly on its interaction with epidermal growth factor (EGF). In contrast to TNF , IL-1 inhibited GEC proliferation; this was due to the early appearance and proliferation of mesangial cells, despite the culture being serum-free. This study showed that activated monocytes secrete growth factors for GEC in vitro, and that interaction between both TNF and IL-1 and between TNF and EGF can modulate GEC proliferation. These findings suggest that, under pathological conditions, monocytes or macrophages affect GEC proliferation, probably being involved in crescent formation.     

Delta-9-tetrahydrocannabinol suppresses tumor necrosis factor α maturation and secretion but not its transcription in mouse macrophages

Various in vitro studies have shown that delta-9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, has a variety of inhibitory effects on immune functions including effects on macrophages. The present studies have examined the mechanism of THC's effects on tumor necrosis factor α (TNF-α), a major macrophage-produced cytokine and an important mediator involved in cytokine networks and in host defense mechanisms. Exposure of macrophages to medium containing THC has resulted in low levels of soluble TNF-α protein and reduced TNF-α bioactivity in the culture supernatant. However, THC did not inhibit the levels of LPS-induced TNF-α mRNA and intracellular TNF-α precursor protein, had only a weak effect on expression of membrane-bound TNF-α, but suppressed TNF-α maturation/secretion by macrophages. The higher the THC concentration in the medium during TNF-α induction, the greater the amount of intracellular TNF-α precursors that accumulated in the activated macrophages and the less mature TNF-α was released from the cells. Data suggest that TNF-α production by macrophages was altered greatly by exposure to THC at the levels of TNF-α precursor maturation and secretion.     

Expression of bone-morphogenetic protein 2 and tumor necrosis factor α correlates with bone metastases in bladder urothelial carcinoma

We have investigated the role of bone-morphogenetic protein (BMP) 2 and tumor necrosis factor α (TNF-α) in 33 patients with bladder cancer (BCa) with bone metastasis. Thirty nonmetastatic BCas were included as controls. Immunohistochemical staining with BMP-2 and TNF-α was performed. Expressions of the factors were quantified and studied statistically. As a result, a trend showing higher expression of BMP-2 and TNF-α was associated with advanced disease. Expressions of BMP-2 and TNF-α were significantly higher in BCa with bone metastases (P = .0002 and P = .0172, respectively). The expression of BMP-2 and TNF-α showed a direct correlation in metastatic and muscle-invasive cases (P = .0202 and P = .0004, respectively) but not in nonmetastatic or noninvasive BCa (P = .1834 and P = .9215, respectively). It is postulated that BMP-2 can be responsible for the mechanism involved in triggering bone metastasis in BCa. The correlation with TNF-α indicates that the interaction of the 2 factors may promote local invasion and distant metastasis, especially to bone.     

Salmeterol inhibits interferon-γ and interleukin-4 production by human peripheral blood mononuclear cells

T-lymphocytes play an important role in allergic asthma. In the present study, the effect of β₂ adrenoceptor agonists was examined on proliferation, interleukin-4 (IL-4) and interferon-γ (IFN-γ) production by human peripheral blood mononuclear cells (PBMC). The proliferation after 24 h phytohaemagglutinin (PHA) activation was significantly inhibited at high concentrations of salmeterol, isoprenaline and salbutamol (≥10⁻⁶M). A U-shaped concentration response curve was observed for the effect of all agonists on IL-4 production 24 h after PHA activation. Maximal inhibition occurred at 10⁻⁹M and amounted to 71% (P<0.02), 38% (P<0.01) and 49% (P<0.01) for salmeterol, isoprenaline and salbutamol, respectively. In contrast, no significant effect of salmeterol (10⁻¹¹-10⁻⁵ M) on IL-4 production could be detected after 96 h. A biphasic concentration response curve was observed for the inhibitory activity of all β-adrenoceptor agonists on IFN-γ production by PBMC 24 h after PHA activation. The first phase reached a plateau at 10⁻⁹ M and the inhibition amounted to 50% (P<0.05), 33% (P<0.01) and 44% (P<0.05) for salmeterol, isoprenaline and salbutamol, respectively. At higher concentrations of the three β-adrenoceptor agonists the inhibition was increased up to 80% (P<0.05), 60% (P<0.05) and 58% (P<0.01), respectively. Similar to the results obtained after 24 h, IFN-γ production after 96 h was biphasically inhibited by salmeterol, and this inhibition (60%) was significant at 10⁻⁵ M. Together, the present data provide clear evidence for concentration-dependent effects of β-adrenoceptor agonists on the IL-4 and IFN-γ production by human PBMC. These results suggest that β-agonists, at low concentrations, predominantly inhibit IL-4 production and may therefore act as anti-inflammatory drugs in allergic asthma.