Lactate induces tumour necrosis factor-alpha, interleukin-6 and interleukin-1beta release in microglial- and astroglial-enriched primary cultures

Hyperammonaemia has deleterious effects on the CNS in patients with liver dysfunction. Cellular mechanisms underlying the effects of hyperammonaemia are largely unknown, although astrocytes have been the main target of interest. This study investigated how treatment with NH4Cl and lactate, which increase in the brain as a consequence of hyperammonaemia, affects cells in primary rat cultures enriched in either astrocytes or microglia. Morphological changes were studied over time using light microscopy. Release of the proinflammatory cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-6 and IL-1beta was measured using ELISA. NH4Cl was found to induce vacuole formation in both culture systems. Lactate treatment altered astrocytic appearance, resulting in increased space between individual cells. Microglia adopted a round morphology with either NH4Cl or lactate treatment. Lactate, but not NH4Cl, induced release of TNF-alpha and IL-6 in both astroglial- and microglial-enriched cultures, while IL-1beta was released only in microglial cultures. Cytokine release was higher in the microglial- than in the astroglial-enriched cultures. Additionally, the astroglial-enriched cultures containing approximately 10% microglial cells released more cytokines than cultures containing about 5% microglial cells. Taken together, our data suggest that most TNF-alpha, IL-6 and IL-1beta release comes from microglia. Thus, microglia could play an important role in the pathological process of hyperammonaemia.     

Chronic cigarette smoking enhances spontaneous release of tumour necrosis factor-alpha from alveolar macrophages of rats

Some biological effects of chronic cigarette smoking (two cigarettes for 2 h, daily for 4 months) in rats were evaluated. During the smoking period, body weight of smoker rats was always significantly lower than that of control rats. Immediately after the last smoking session the carboxyhaemoglobin concentration in the blood was about 8.5% and the polymorphonuclear cells in the bronchoalveolar fluid increased significantly. At the same time, enzymatic analyses on the supernatants of bronchoalveolar fluid revealed a significant increase of beta-glucuronidase in the smoker group. Alveolar macrophages, collected 0, 8 and 24 h after the last smoking session, significantly increased the generation of superoxide anion and, after incubation for 24 h at 37( degrees ) C in a humidified atmosphere, released significantly high amounts of TNF-alpha. When challenged with lipopolysaccharide, alveolar macrophages of smoker rats released much more TNF-alpha but, in such a case, TNF-alpha release was about one half of that observed in the control group. Peritoneal macrophages of both control and smoker rats were unable either to generate high levels of superoxide anion or to release significant amounts of TNF-alpha. The results clearly demonstrated the activated state of alveolar macrophages and the resting state of peritoneal macrophages.     

Bradykinin stimulates tumor necrosis factor and interleukin-1 release from macrophages

Bradykinin and related kinins have been implicated in the initiation and maintenance of inflammation. Cytokines appear to be the primary mediators of many inflammatory diseases. The potential ability of bradykinin to stimulate release of tumor necrosis factor and interleukin-1 from macrophages was examined. Bradykinin stimulated release of both cytokines from P388-D1 and RAW264.7 murine macrophages. Studies with selective agonists and antagonists suggest that cytokine release is mediated by a B1 kinin receptor.     

Human fibroblasts release reactive oxygen species in response to interleukin-1 or tumour necrosis factor-alpha.

Human fibroblasts in primary culture released reactive oxygen species upon stimulation with cytokines such as interleukin-1 alpha (IL-1) or tumour necrosis factor-alpha (TNF). The primary radical produced was O2.- as determined by e.s.r. spin trapping and cytochrome c reduction. In contrast to the oxidative burst in granulocytes and monocytes, radical formation took place continuously for at least 4 h. Low-level chemiluminescence was increased by stimulation with IL-1 and TNF. Spectral characteristics and tests with azide led to the conclusion that the photoemissive species were excited carbonyls and not singlet oxygen. Further, there was a liberation of ethane from the cells. Radical production and light emission were not altered by either xanthine or allopurinol, nor by azide, cyanide or rotenone. O2.- production increased in the presence of NADH or NADPH, making an NAD(P)H oxidase a likely source.     

Tumor-stimulated release of tumor necrosis factor-alpha by human monocyte-derived macrophages.

Tumor necrosis factor-alpha (TNF) release by monocytes and macrophages may be an important determinant of the physiologic response of the host to neoplastic disease; however, the mechanisms which regulate TNF release by macrophages in hosts with neoplastic diseases are poorly understood. The purpose of this study was to determine if cell membranes and growth medium from human leukemia cell lines and solid tumor cell lines induced TNF release by cultured human blood monocyte-derived macrophages. The capacity for TNF release and direct tumor killing was highest in monocytes cultured for 7 to 11 days. Cell membranes and culture media from K562 erythroleukemia and several small cell lung carcinoma cell lines, including H82, induced the release of up to 1500 TNF units per 10(6) macrophages over 24 hr. By contrast, allogeneic peripheral blood lymphocytes, cell membranes from normal mixed donor peripheral blood leukocytes, or growth medium from normal embryonic lung fibroblasts induced the release of little or no TNF during culture up to 24 hr, suggesting that this macrophage response was specific for tumor cells. Release of TNF by tumor-stimulated macrophages was gradual, peaking 24 hr following the addition of stimuli. Induction of macrophage TNF release was concentration dependent, with half-maximal TNF levels induced by 12.5 and 25 micrograms/ml cell membranes prepared from K562 and H82, respectively. Pretreatment of tumor cell membranes with polymixin B, which inhibits many of the actions of endotoxin, failed to neutralize tumor induction of TNF, suggesting that endotoxin was not responsible for this activity. Depletion of macrophages by treatment with 3C10 monoclonal antibody and complement abrogated tumor-induced TNF release, indicating that macrophages were the source of the secreted TNF. HPLC analysis of H82 growth medium demonstrated a single peak of macrophage activating activity with approximate 40-kDa molecular weight. We have demonstrated that cell membranes and growth medium from some human leukemia and solid tumor cell lines, but not from normal human cells, induce human peripheral blood monocytes and monocyte-derived macrophages to release functionally active TNF. This process may contribute to the host response to some neoplastic diseases.     

Induction by mycoplasmas of tumor necrosis factor-alpha from macrophages

Several species of mycoplasmas including M. pneumoniae, the causative agent of human respiratory infection, were investigated for tumor necrosis factor-alpha (TNF-alpha) induction. The cytotoxic activity to Meth A cells of peritoneal macrophages purified from BALB/c mice was enhanced markedly when cultured with either viable or nonviable mycoplasmas. The supernatant of macrophage culture mixed with mycoplasmas, M. pneumoniae or A. laidlawii, showed a potent cytotoxic activity to TNF-alpha-sensitive but not to TNA-alpha-insensitive L cells. Addition of anti-TNA-alpha antiserum inhibited completely the cytotoxic activity of the supernatant, indicating that the cytotoxic activity is due mostly to TNF-alpha. These results strongly suggest that mycoplasmas possess an activity to induce TNF-alpha, which enhances the cytotoxic activity of macrophages and prevent infection with mycoplasmas in vivo.     

Mitogen-activated protein kinases regulate Mycobacterium avium-induced tumor necrosis factor-alpha release from macrophages

Tumor necrosis factor-alpha (TNF-alpha) is one of the key cytokines elicited by host macrophages upon challenge with pathogenic mycobacteria. Infection of human peripheral blood mononuclear cells or the murine macrophage cell line J774A-1 with Mycobacterium avium induced activation of the mitogen-activated protein kinases (MAPKs) ERK1/2, p38 and c-Jun N-terminal kinase. U0126, an MEK-specific inhibitor, abrogated M. avium-induced TNF-alpha secretion. Transfection of cells with dominant-negative MEK1 led to the suppression of TNF-alpha release in M. avium-challenged macrophages. M. avium activated p38 MAPK and use of the p38 MAPK inhibitor, SB203580, revealed that the p38 signaling pathway negatively regulates activation of ERK1/2 and release of TNF-alpha. Taken together, these results provide evidence that M. avium-induced TNF-alpha release from macrophages depends on an interplay between the ERK1/2 and the p38 MAPK signaling pathways.     

Inhibitory effects of bisbenzylisoquinolines on synthesis of the inflammatory cytokines interleukin-1 and tumour necrosis factor-alpha

Synthesis of IL-1beta and TNFalpha by human monocytesmacrophages was significantly inhibited by eleven bisbenzylisoquinolines and one half-molecule (benzylisoquinoline), with IC(50) values in the muM range. The results indicate that these compounds may have value in the therapy of human diseases where these inflammatory cytokines have a central role in pathogenesis.     

Activated macrophages promote Wnt signalling through tumour necrosis factor-alpha in gastric tumour cells

The activation of Wnt/beta-catenin signalling has an important function in gastrointestinal tumorigenesis. It has been suggested that the promotion of Wnt/beta-catenin activity beyond the threshold is important for carcinogenesis. We herein investigated the role of macrophages in the promotion of Wnt/beta-catenin activity in gastric tumorigenesis. We found beta-catenin nuclear accumulation in macrophage-infiltrated dysplastic mucosa of the K19-Wnt1 mouse stomach. Moreover, macrophage depletion in Apc(Delta716) mice resulted in the suppression of intestinal tumorigenesis. These results suggested the role of macrophages in the activation of Wnt/beta-catenin signalling, which thus leads to tumour development. Importantly, the conditioned medium of activated macrophages promoted Wnt/beta-catenin signalling in gastric cancer cells, which was suppressed by the inhibition of tumour necrosis factor (TNF)-alpha. Furthermore, treatment with TNF-alpha induced glycogen synthase kinase 3beta (GSK3beta) phosphorylation, which resulted in the stabilization of beta-catenin. We also found that Helicobacter infection in the K19-Wnt1 mouse stomach caused mucosal macrophage infiltration and nuclear beta-catenin accumulation. These results suggest that macrophage-derived TNF-alpha promotes Wnt/beta-catenin signalling through inhibition of GSK3beta, which may contribute to tumour development in the gastric mucosa.     

Trypsin induces tumour necrosis factor-alpha secretion from a human leukemic mast cell line

Trypsin activating both proteinase-activated receptor (PAR) 2 and PAR4 plays an important role in inflammation. We have investigated the potential of trypsin to induce TNF-alpha secretion from the human leukemic mast cell line (HMC-1). HMC-1 cells co-express both PAR2 and PAR4, and their agonist trypsin signals to HMC-1 cells. Trypsin (100 nm), SLIGKV-NH(2) (100 microm, corresponding to the PAR2 tethered ligand), or GYPGQV-NH(2) (100 microm, corresponding to the PAR4 tethered ligand) induced tumour necrosis factor (TNF)-alpha secretion from HMC-1 cells. TNF-alpha secretion by trypsin was significantly blocked by pretreatment with 50 microm PD098059, MEK-1 inhibitor. Furthermore, trypsin stimulated the activation of extracellular signal-regulated kinase (ERK) in HMC-1 cells without any detectable activation of c-Jun N-terminal kinase (JNK) and p38 MAP kinase homologue. These results show that trypsin may induce TNF-alpha secretion following activation of ERK via both PAR2 and PAR4 on HMC-1 cells.     

Cyclooxygenase-1-dependent prostaglandin synthesis modulates tumor necrosis factor-alpha secretion in lipopolysaccharide-challenged murine resident peritoneal macrophages

Comprehensive studies of prostaglandin (PG) synthesis in murine resident peritoneal macrophages (RPM) responding to bacterial lipopolysaccharide (LPS) revealed that the primary PGs produced by RPM were prostacyclin and PGE(2). Detectable increases in net PG formation occurred within the first hour, and maximal PG formation had occurred by 6-10 h after LPS addition. Free arachidonic acid levels rose and peaked at 1-2 h after LPS addition and then returned to baseline. Cyclooxygenase-2 (COX-2) and microsomal PGE synthase levels markedly increased upon exposure of RPM to LPS, with the most rapid increases in protein expression occurring 2-6 h after addition of the stimulus. RPM constitutively expressed high levels of COX-1. Studies using isoform-selective inhibitors and RPM from mice bearing targeted deletions of ptgs-1 and ptgs-2 demonstrated that COX-1 contributes significantly to PG synthesis in RPM, especially during the initial 1-2 h after LPS addition. Selective inhibition of either COX isoform resulted in increased secretion of tumor necrosis factor-alpha (TNF-alpha); however, this effect was much greater with the COX-1 than with the COX-2 inhibitor. These results demonstrate autocrine regulation of TNF-alpha secretion by endogenous PGs synthesized primarily by COX-1 in RPM and suggest that COX-1 may play a significant role in the regulation of the early response to endotoxemia.     

Apoptotic neutrophils containing Staphylococcus epidermidis stimulate macrophages to release the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-6.

Staphylococcus epidermidis infections are usually nosocomial and involve colonization of biomaterials. The immune defense system cannot efficiently control the bacteria during these infections, which often results in protracted chronic inflammation, in which a key event is disturbed removal of neutrophils by tissue macrophages. While ingesting uninfected apoptotic neutrophils, macrophages release anti-inflammatory cytokines that lead to resolution of inflammation. In clinical studies, we have previously found elevated levels of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 in synovial fluid from prostheses infected with coagulase negative staphylococci. We show that macrophages phagocytosing apoptotic neutrophils containing S. epidermidis released TNF-alpha and interleukin-6, whereas macrophages phagocytosing spontaneously apoptotic neutrophils did not. This difference was not due to dissimilar phagocytic capacities, because macrophages ingested both types of neutrophils to the same extent. The activation was induced mainly by the apoptotic neutrophils themselves, not by the few remaining extracellular bacteria. Macrophages were not activated by apoptotic neutrophils that contained paraformaldehyde-killed S. epidermidis. Proinflammatory reactions induced by clearance of apoptotic neutrophils containing S. epidermidis might represent an important mechanism to combat the infective agent. This activation of macrophages may contribute to the development of chronic inflammation instead of inflammation resolution.     

Increased elastase release by CF neutrophils is mediated by tumor necrosis factor-alpha and interleukin-8

Cystic fibrosis (CF) is a lethal, hereditary disorder characterized by a neutrophil-dominated inflammation of the lung. We sought to determine whether neutrophils from individuals with CF release more neutrophil elastase (NE) than neutrophils from normal subjects. Our results showed that peripheral blood neutrophils (PBNs) from normal subjects and individuals with CF contained similar amounts of NE, but after preincubation with CF bronchoalveolar lavage (BAL) fluid, significantly more NE was released by CF PBNs, a release that was amplified further by incubation with opsonized Escherichia coli. To determine which components of CF BAL fluid stimulated this excessive NE release from CF PBNs, we repeated the experiments after neutralization or immunoprecipitation of tumor necrosis factor (TNF)-alpha and interleukin (IL)-8 in CF BAL fluid. We found that subsequent NE release from CF PBNs was reduced significantly when TNF-alpha and IL-8 were removed from CF BAL fluid. When TNF-alpha and IL-8 were used as activating stimuli, CF PBNs released significantly greater amounts of NE compared with PBNs from control subjects and individuals with bronchiectasis. These results indicate that CF PBNs respond abnormally to TNF-alpha and IL-8 in CF BAL fluid and react to opsonized bacteria by releasing more NE. This may help explain the increased NE burden seen in this condition.     

LPS induces interleukin-6 and interleukin-8 but not tumor necrosis factor-alpha in human adipocytes

Adipose tissue-derived cytokines are presumably involved in obesity-associated pathologies including type 2 diabetes and atherosclerosis. Here we studied the lipopolysaccharide (LPS)-induced expression dynamics of tumor necrosis factor-alpha (TNFalpha), interleukin-6 (IL-6), IL-8 and IL-10 in human adipose tissue biopsies, in preadipocyte-derived adipocytes, and in mesenchymal stem cell (MSC)-derived adipocytes. TNFalpha, IL-6, IL-8 and IL-10 secretions by adipose tissue explants were increased 5.5-, 19.5-, 3.5- and 12.5-fold, respectively, by LPS (1 microg/mL) administration. Concordantly, IL-6 and IL-8 release was dose-dependently induced in MSC-derived adipocytes by LPS (>10 pg/mL). In contrast, TNFalpha and IL-10 remained undetectable even at the highest LPS dose (1 microg/mL) after 24h. In MSC- and preadipocyte-derived adipocytes, respectively, exposure to LPS evoked a weak and transient induction of TNFalpha mRNA whereas induction of IL-6 and IL-8 mRNA were pronounced and sustained for at least 24h. Basal glucose uptake, lipolysis and IL-6 mRNA were induced by exogenous TNFalpha (10 ng/mL) but not by IL-6 (10 ng/mL), IL-8 (100 ng/mL) and IL-10 (20 ng/mL). In this adipocyte model TNFalpha induces well known metabolic effects, but together with previous reports these data suggest that inflammation-induced TNFalpha may derive from non-adipocyte sources in adipose tissue, likely to be macrophages.     

Tumor necrosis factor-alpha induces vitamin D-1-hydroxylase activity in normal human alveolar macrophages.

The induction of 1-hydroxylase in alveolar macrophages by tumor necrosis factor-alpha (TNF) was examined in view of recent evidence suggesting that local production of 1,25-(OH)2D3 may play a role in the regulation of immune functions. Incubation of pulmonary alveolar macrophages from normal human subjects with recombinant TNF caused a 2- to 10-fold increase in 25-hydroxyvitamin D3-1-hydroxylase activity. The dose-response curve was linear over the range 0.05-5.0 IU/ml, and no further increase was seen at higher concentrations. The increase in 1-hydroxylase activity was present after 12 h and reached a maximum after 3 days. The effect of TNF was inhibited in a dose-dependent manner by the presence of 1,25(OH)2D3 (10(-10)-10(-8) M) in the incubation media for 5 days but was unaffected by 10(-9) M 1,25(OH)2D3 after 12 h. The enhancement of macrophage 1-hydroxylase activity by TNF was comparable to that induced by gamma interferon (IFN) but the effects of maximal doses of both agents were not additive. The presence of antibody to TNF resulted in a 76% inhibition of TNF-induced 1-hydroxylase but had no significant effect on IFN-induced 1-hydroxylase activity.     

Induction by Staphylococcus aureus L-form of tumor necrosis factor-alpha from macrophages

Induction of tumor necrosis factor-alpha (TNF-alpha) by Staphylococcus aureus L-form was investigated. The supernatant of a macrophage culture mixed with S. aureus L-form showed a potent cytotoxic activity to L cells. Addition of anti TNF-alpha antibody inhibited completely the cytotoxic activity of the supernatant, indicating that the activity might be due mostly to TNF-alpha. To investigate localization of TNF-alpha production, the membranes of hypotonicity treated L-form were layered on a step-gradient composed of an upper and lower layers of 35% and 50% sucrose, respectively. The membranes were banded at the interface of 35% and 50% of sucrose. The activity of TNF-alpha production of the membrane fraction was 10-times higher than that of the soluble fraction.     

Prostaglandin E(2) and tumour necrosis factor-alpha release by monocytes are modulated by phospholipids

The regulation of pro- and anti-mediator release from cells within the alveolar space would represent a desirable mechanism serving to protect this delicate gas-exchanging region of the lung. This study investigates the effect of alveolar surfactant lipids on the regulation of tumour necrosis factor alpha (TNF-alpha), a potent inflammatory cytokine, and prostaglandin E(2)(PGE(2)), a lipid mediator with anti-inflammatory properties. The results of this investigation reveal a marked effect on the release of these two important mediators from a monocytic cell line, MonoMac 6 (MM6), by phosphatidylcholine (PC), phosphatidylethanolamine (PE), cholesterol (Chol) and sphingomyelin (SM). PC, PE and Chol demonstrated marked downregulation of TNF-alpha production at lipid concentrations of 125 and 250 microg/ml. Interestingly, SM significantly up regulated the release of TNF-alpha at these concentrations. However, the release of PGE(2)in MM6 cells incubated with the same lipids was significantly increased with PC and Chol, and significantly decreased in cells pre-treated with SM. This indicates a role for these lipids in alveolar immunoregulation.