Induction of TNF-alpha and MnSOD by endotoxin: role of membrane CD14 and Toll-like receptor-4.

Endotoxin (LPS) is a potent inducer of tumor necrosis factor-alpha (TNF-alpha) and manganese superoxide dismutase (MnSOD). Recent evidence suggests that LPS induction of TNF-alpha and MnSOD mRNAs is mediated through distinct intracellular signal transduction pathways. Membrane CD14 (mCD14) and Toll-like receptor-4 (TLR4) mediate LPS induction of TNF-alpha in macrophages. In the current study, we evaluated the role of mCD14 and TLR4 in LPS induction of MnSOD using peritoneal macrophages from CD14 knockout (CD14-KO) mice and mice with the Tlr4 gene point mutation (C3H/HeJ) or deletion (C57BL/10ScCr). We studied mCD14-dependent (1 and 10 ng/ml) and mCD14-independent (1,000 ng/ml) concentrations of LPS. Compared with control (BALB/c) macrophages, LPS at 1 and 10 ng/ml failed to induce TNF-alpha or MnSOD mRNA in CD14-KO macrophages. However, LPS at 1,000 ng/ml induced TNF-alpha and MnSOD mRNAs equally in macrophages from CD14-KO and control mice. LPS (1, 10, or 1,000 ng/ml) failed to induce TNF-alpha or MnSOD mRNA and failed to activate nuclear factor-kappaB in C3H/HeJ or C57BL/10ScCr macrophages. Measurements of TNF-alpha and MnSOD enzyme activity paralleled TNF-alpha and MnSOD mRNA levels. These data demonstrate that, like TNF-alpha, induction of MnSOD by LPS is mediated by mCD14 and TLR4 in murine macrophages.

Differential induction of TNF-alpha and MnSOD by endotoxin: role of reactive oxygen species and NADPH oxidase.

Endotoxin (lipopolysaccharide [LPS]) is known to induce the production of tumor necrosis factor (TNF)-alpha and the induction of manganese superoxide dismutase (MnSOD). We have recently demonstrated that induction of TNF-alpha and MnSOD by LPS is mediated through different signal transduction pathways. In the current study, we investigated the role of reactive oxygen species (ROS) in the induction of TNF-alpha and MnSOD messenger RNAs (mRNAs) in human monocytes. Hypoxia (1% O2) inhibited the production of superoxide (O2-) and the induction of MnSOD, but not TNF-alpha, mRNA. Diphenylene iodonium (DPI), a potent inhibitor of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, had no effect on LPS induction of MnSOD mRNA, whereas it markedly inhibited LPS-induced O2- production. Neither hypoxia nor DPI had any effect on LPS activation of nuclear factor (NF)-kappaB. These results suggest that (1) ROS is important in the induction of MnSOD, but not TNF-alpha, mRNA by LPS, (2) ROS from sources other than NADPH oxidase is involved in LPS induction of MnSOD mRNA, and (3) ROS-mediated LPS induction of MnSOD mRNA is independent of NF-kappaB activation.

Superoxide dismutase and pulmonary oxygen toxicity: lessons from transgenic and knockout mice (Review).

Superoxide (O2-) has been implicated in the pathogenesis of pulmonary O2 toxicity. The studies using transgenic and knockout mice of each of the three isoforms of superoxide dismutase (SOD) e.g. , CuZnSOD, MnSOD and extracellular SOD (EC-SOD), have demonstrated that O2- produced in the mitochondria from its electron transport system and extracellular O2- generated by infiltrating neutrophils, and possibly its derivatives e.g., hydroxyl radical and peroxynitrite, are important mediators of hyperoxia-induced pulmonary injury, while cytoplasmic O2- plays a limited, if any, role in the pathogenesis of pulmonary O2 toxicity. Distal airway epithelial cells including type II alveolar and non-ciliated bronchiolar epithelial cells, are important targets for O2 radicals under the hyperoxic condition. The accessibility of these distal airway epithelial cells to in vivo gene transfer through the tracheal route of administration, suggests the potential for in vivo transfer of MnSOD and EC-SOD genes as a future approach in the prevention of pulmonary O2 toxicity.

Resveratrol induces Fas signalling-independent apoptosis in THP-1 human monocytic leukaemia cells.

Resveratrol, a natural product present in wine, has recently been shown to inhibit the growth of a number of cancer cell lines in vitro. In the current study, we have demonstrated that resveratrol inhibits the growth of THP-1 human monocytic leukaemia cells in a dose-dependent manner with a median effective dose of 12 microM. It did not induce differentiation of THP-1 cells and had no toxic effect on THP-1 cells that had been induced to differentiate into monocytes/macrophages by phorbol myristate acetate. A significant fraction of resveratrol-treated cells underwent apoptosis as judged by flow cytometric analysis of DNA content, DNA fragmentation and caspase-specific cleavage of poly(ADP-ribosyl) polymerase. Resveratrol treatment had no effect on the expression of Fas receptor or Fas ligand (FasL) in THP-1 cells, nor did it induce clustering of Fas receptors. In addition, THP-1 cells were resistant to activating anti-Fas antibody, and neutralizing anti-Fas and/or anti-FasL antibodies had no protective effect against resveratrol-induced inhibition of THP-1 cell growth. The effect of resveratrol on THP-1 cells was reversible after its removal from the culture medium. These results suggest that (1) resveratrol inhibits the growth of THP-1 cells, at least in part, by inducing apoptosis, (2) resveratrol-induced apoptosis of THP-1 cells is independent of the Fas/FasL signalling pathway and (3) resveratrol does not induce differentation of THP-1 cells and has no toxic effect on differentiated THP-1 cells. Thus, resveratrol may be a potential chemotherapeutic agent for the control of acute monocytic leukaemia.

Differential induction of tumor necrosis factor alpha and manganese superoxide dismutase by endotoxin in human monocytes: role of protein tyrosine kinase, mitogen-activated protein kinase, and nuclear factor kappaB.

A mutant Escherichia coli lipopolysaccharide (LPS) lacking myristoyl fatty acid markedly stimulates the activity of manganese superoxide dismutase (MnSOD) without inducing tumor necrosis factor alpha (TNFalpha) production by human monocytes (Tian et al., 1998, Am J Physiol 275:C740.), suggesting that induction of MnSOD and TNFalpha by LPS are regulated through different signal transduction pathways. The protein tyrosine kinase (PTK)/mitogen-activated protein kinase (MAPK) pathway plays an important role in the LPS-induced TNFalpha production. In the current study, we determined the effects of PTK inhibitors, genistein and herbimycin A, on the induction of MnSOD and TNFalpha in human monocytes. Genistein (10 microg/ml) and herbimycin A (1 microg/ml) markedly inhibited LPS-induced protein tyrosine phosphorylation, phosphorylation and nuclear translocation of MAPK (p42 ERK, extracellular signal-regulated kinase), and increases in the steady state level of TNFalpha mRNA as well as TNFalpha production. In contrast, at similar concentrations, genistein and herbimycin A had no effect on the LPS-induced activation of nuclear factor kappaB (NFkappaB) and induction of MnSOD (mRNA and enzyme activity) in human monocytes. In addition, inhibition of NFkappaB activation by gliotoxin and pyrrodiline dithiocarbamate, inhibited LPS induction of TNFalpha and MnSOD mRNAs. These results suggest that (1) while PTK and MAPK are essential for the production of TNFalpha, they are not necessary for the induction of MnSOD by LPS, and (2) while activation of NFkappaB alone is insufficient for the induction of TNFalpha mRNA by LPS, it is necessary for the induction of TNFalpha as well as MnSOD mRNAs.

Induction of TNF-alpha and MnSOD by endotoxin: effect of E5531, a synthetic non-toxic lipid A analog.

E5531, a synthetic lipid A analog, has been shown to inhibit endotoxin (lipopolysaccharide, LPS)-induced tumor necrosis factor-alpha (TNF-alpha) production by human monocytes and murine macrophages. Whether it also inhibits LPS induction of manganese superoxide dismutase (MnSOD) is not clear. In the current study, we demonstrated that E5531, while having no effect on TNF-alpha and MnSOD mRNAs by itself, markedly inhibited LPS- and lipid A-, but not TNF-alpha-, induced increases in TNF-alpha and MnSOD mRNAs in human monocytes. In contrast, E5531 at concentrations and conditions that markedly inhibit LPS-induced increases in TNF-alpha and MnSOD mRNAs, and TNF-alpha production by human monocytes, had no effect on murine peritoneal macrophages. These results demonstrate that E5531 is a potent LPS antagonist in human monocytes. However, it does not show antagonist action against LPS in murine macrophages in the range of concentrations tested, suggesting that E5531 is a more potent antagonist in humans than in mice.

THP-1 monocytic leukemia cells express Fas ligand constitutively and kill Fas-positive Jurkat cells.

Normal human monocytes express Fas and are susceptible to Fas ligand (FasL)-induced apoptosis. Because the myeloid leukemia cell lines HL-60 and THP-1 can be differentiated into functional monocytes and macrophages, we studied their expression of Fas and Fas ligand (FasL) to determine whether there were differentiation-associated changes in these proteins. THP-1, HL-60 and HCW-2, both before and after treatment with PMA, expressed high levels of Fas ligand (FasL), but did not express Fas. The FasL expressed by THP-1 cells was functional as measured by their ability to kill Jurkat T-cells by apoptosis. The THP-1 Fas gene appears to be silent, because bacterial lipopolysaccharide (LPS) induced Fas expression in fully differentiated THP-1 cells. Our results suggest that FasL expression by leukemia cells may account in part for the pathophysiology of myeloid leukemia, and that PMA-differentiated THP-1 cells, while possessing many of the functional properties of normal macrophages, are abnormal with respect to a major apoptotic pathway.

Pertussis toxin-induced lung edema. Role of manganese superoxide dismutase and protein kinase C.

The mechanism by which pertussis toxin (Ptx) causes lung edema is not clear. We investigated the role of pulmonary manganese superoxide dismutase (MnSOD) and protein kinase C (PKC) in Ptx-induced lung edema. We demonstrated that intraperitoneal injection of Ptx at a concentration of 5 microg/100 g body weight caused a similar degree of lung edema in 2 d, as measured by lung wet weight/dry weight ratio, in heterozygous MnSOD gene (Sod2)-knockout mice (Sod2(+/-)) and in their wild-type littermates (Sod2(+/+)). The level of lung MnSOD activity in Sod2(+/-) mice was approximately half that of Sod2(+/-) mice. Ptx had no effect on levels of lung MnSOD messenger RNA, immunoreactive protein, or enzyme activity in either Sod2(+/+) or Sod2(+/-) mice. Ptx also had no effect on lung copper-zinc SOD, catalase, and glutathione peroxidase activities in these mice. On the other hand, Ptx caused the activation of lung PKC, for example, by translocation of a 72-kD PKC isoform from the cytosolic fraction to the membrane fraction. Pretreatment of mice with bisindolylmaleimide, a PKC inhibitor, prevented both the Ptx-induced activation of PKC and lung edema. These data suggest that Ptx-induced lung edema in mice is, at least in part, due to the activation of lung PKC.

Induction of Mn SOD in human monocytes without inflammatory cytokine production by a mutant endotoxin.

Endotoxin selectively induces monocyte Mn superoxide dismutase (SOD) without affecting levels of Cu,Zn SOD, catalase, or glutathione peroxidase. However, little is known about the structure-activity relationship and the mechanism by which endotoxin induces Mn SOD. In this study we demonstrated that a mutant Escherichia coli endotoxin lacking myristoyl fatty acid at the 3' R-3-hydroxymyristate position of the lipid A moiety retained its full capacity to coagulate Limulus amoebocyte lysate compared with the wild-type E. coli endotoxin and markedly stimulated the activation of human monocyte nuclear factor-kappaB and the induction of Mn SOD mRNA and enzyme activity. However, in contrast to the wild-type endotoxin, it failed to induce significant production of tumor necrosis factor-alpha and macrophage inflammatory protein-1alpha by monocytes and did not induce the phosphorylation and nuclear translocation of mitogen-activated protein kinase. These results suggest that 1) lipid A myristoyl fatty acid, although it is important for the induction of inflammatory cytokine production by human monocytes, is not necessary for the induction of Mn SOD, 2) endotoxin-mediated induction of Mn SOD and inflammatory cytokines are regulated, at least in part, through different signal transduction pathways, and 3) failure of the mutant endotoxin to induce tumor necrosis factor-alpha production is, at least in part, due to its inability to activate mitogen-activated protein kinase.

Susceptibility of heterozygous MnSOD gene-knockout mice to oxygen toxicity.

Recent studies have shown that homozygous Mn superoxide dismutase (Sod2) gene-knockout mice (Sod2(-/-)) die shortly after birth with extensive myocardial injury, whereas heterozygous mutants (Sod2(+/-)) are phenotypically normal in room air. In the current study, we showed that Sod2(+/-) mice with approximately 50% of normal pulmonary MnSOD activity and normal levels of lung CuZnSOD, catalase, and glutathione peroxidase activities were not substantially more susceptible to 100% O2 toxicity than their normal Sod2(+/+) littermates. The mean (+/- SD) survival of Sod2(+/-) mice in 100% O2 was 101.4 +/- 14.8 h (n = 20) versus 103.2 +/- 11.3 h (n = 20) for Sod2(+/+) littermates (P > 0.60). In addition, Sod2(+/-) mice with approximately 50% of normal heart MnSOD activity and Sod2(+/+) mice did not develop any ultrastructural abnormalities in the myocardium at 75 h or 90 h after 100% O2 exposure. These results suggest that in mice, only 50% of MnSOD activity may be sufficient for normal resistance to 100% O2 toxicity.

Transplantation of allogeneic islets of Langerhans in the rat liver: effects of macrophage depletion on graft survival and microenvironment activation.

Early impairment of islet function and graft loss limit the success of allogeneic islet transplantation. Nonspecific inflammatory events occurring at the transplant site immediately after grafting, involving the production of cytokines and free radicals and sinusoidal endothelial cell (SEC) activation, may contribute to islet cell damage. To evaluate whether Kupffer cell inactivation would result in prolonged allograft survival in a model system of intrahepatic islet transplantation in rats, we systemically administered either gadolinium chloride (GdCl3) or dichloromethylene diphosphonate (Cl2MDP) to assess the effects of macrophage inactivation on rejection and on the release of proinflammatory molecules, as well as to assess the functional profile of SEC. The results obtained were compared with those observed in untreated, sham-injected animals and in rats receiving intraportal infusions of microbeads. Transient macrophage inhibition, particularly in hepatic Kupffer cells, is associated with significant prolongation of graft survival after intraportal islet allotransplantation (ITx) in rats: 7.2 days in the control group versus 11.9 days in the GdCl3 group (P < 0.01) and 15.6 days in the Cl2MDP group (P < 0.0006), respectively. Although systemic release of inflammatory mediators was observed only when islet transplantations were performed and it could be inhibited by macrophage-targeting treatments, perturbation of the functional profile of endothelial cells was also observed when microembolization was induced by the use of microbeads and could not be prevented by macrophage inhibition. These experiments provide evidence to support the concept that macrophages play a key role in early inflammatory events known to adversely affect islet engraftment and suggest that manipulation of nonspecific immune activation by inhibition of macrophage function may facilitate hepatic engraftment of islet allografts. The mechanisms mediating this effect are likely to include prevention of release of tumor necrosis factor-alpha, interleukin-1beta, and NO and interference with the rate of immune response to the islets.

Chronic relapsing thrombotic thrombocytopenic purpura: role of therapy with cyclosporine.

Relapsing thrombotic thrombocytopenic purpura (TTP) is a rare disorder with most individuals experiencing 1 to 5 relapses. We report a patient with 18 episodes of thrombotic thrombocytopenic purpura (TTP), the highest number of relapses thus far described. The last 11 episodes were treated with regimens containing cyclosporine. The patient's medical record was reviewed for pertinent clinical, laboratory, and treatment data. We summarized various parameters for each episode and compared characteristics of relapses treated with vs. without cyclosporine. The initial episode of TTP was unusual in that it failed to respond to plasmapheresis, glucocorticoids, and fresh frozen plasma (FFP). It remitted only following splenectomy. Episodes 2-7 responded to FFP plus prednisone. Episode 8 failed to respond to prednisone plus FFP but remitted promptly with cyclosporine plus prednisone. Subsequently, 2 relapses responded to cyclosporine alone, 2 to cyclosporine plus FFP, 4 to cyclosporine plus prednisone +/- FFP, and 2 to cyclosporine, FFP, prednisone, and plasma exchange. There was no difference in remission duration, or in severity or duration of relapses treated with vs. without cyclosporine. Use of cyclosporine, however, significantly decreased the requirement for prednisone and the length of maintenance therapy; thus it is effective mainly as an adjunctive therapy for TTP.

Protection of rats against oxygen toxicity by tracheal administration of plasmid DNA: role of endogenous tumor necrosis factor.

Recombinant plasmid DNA alone or in conjunction with a gene delivery system has been used increasingly for in vivo gene transfer. However, little is known about the direct biological effects of plasmid DNA. In this study, we demonstrated that tracheal administration of a number of plasmid DNA protected rats against oxygen toxicity. This protection required the presence of intact plasmid DNA, was not due to endotoxin contamination, and was not shared by salmon testis DNA. The plasmid DNA-induced protection against oxygen toxicity was associated with the production of tumor necrosis factor (TNF) and the enhancement of pulmonary Mn-superoxide dismutase (MnSOD), CuZnSOD, and glutathione peroxidase activities. Coadministration of plasmid DNA and anti-TNF antibody (but not nonspecific IgG) partially abolished the protective effect and reduced the pulmonary MnSOD activity, suggesting that the plasmid DNA-induced oxygen tolerance was in part mediated by the endogenous TNF and MnSOD. In view of these observations and the known immunostimulatory effects of bacterial DNA, caution should be exercised in interpreting the results of in vivo gene transfer using recombinant plasmid DNA.

Surfactant inhibits cationic liposome-mediated gene transfer.

We have demonstrated that tracheal insufflation of recombinant plasmid DNA results in transfection of rat lungs to the same extent as insufflation of plasmid-cationic liposome complex. To understand this observation better, we investigated the in vitro gene transfer of plasmid DNA in the presence and absence of cationic liposome and the effect of surfactant on gene transfer. The chloramphenicol acetyltransferase (CAT) expression plasmids pBL-CAT and pSV-CAT were studied in three cell types: rat fetal lung fibroblast (RFL-6), calf pulmonary artery endothelial cell (CPAE), and rat type II alveolar epithelial cell (type II AE). Three cationic liposomes were tested: DDAB (dimethyl-dioctadecyl ammonium bromide)-liposome, DOTAP (dioleoyltrimethyl ammonium propane)-liposome, and lipofectin. The results revealed that (i) plasmid DNA alone caused a dose-dependent, low-level transfection, most efficiently in RFL-6 followed by CPAE and type II AE, (ii) DDAB-liposome markedly enhanced gene transfer, most efficiently in RFL-6 followed by CPAE and type II AE, (iii) Survanta, a naturally derived surfactant preparation, and Exosurf, a synthetic surfactant, while having no effect on in vitro gene transfer by plasmid DNA alone, markedly inhibited cationic liposome-mediated gene transfer, (iv) dipalmitoyl phosphatidylcholine was responsible for the inhibitory effect of Exosurf, and (v) inhibition of cationic liposome-mediated gene transfer by Exosurf was not due to inhibition of plasmid DNA-cationic liposome complex uptake or interference with the promoter and enhancer. The observed inhibition of cationic liposome-mediated gene transfer by surfactant may in part explain our previous observation that tracheal insufflation of plasmid DNA and plasmid-cationic liposome complex results in equal lung gene transfer.

Superoxide dismutase and pulmonary oxygen toxicity.

The production of superoxide (O2-) under hyperoxic conditions is markedly accentuated leading to the generation of potent oxidants such as hydrogen peroxide (H2O2), hydroxyl radical (HO.), and peroxynitrite (ONOO-). Superoxide dismutase (SOD), by rapidly removing O2-, reduces the tissue concentration of O2- and prevents the production of HO. and ONOO-. Three forms of SOD exist in the lung: CuZnSOD, MnSOD, and extracellular SOD. Considerable supportive, though not all conclusive, evidence suggests that all three forms of SOD are essential for the pulmonary defense against oxygen toxicity, and that enhancement of pulmonary SOD has the potential of protecting against oxygen toxicity.

Pulmonary response to plasmid DNA and immunohistochemical localization of transgene expression.

Tracheal insufflation of chloramphenicol acetyltransferase (CAT) expression plasmid (pBL-CAT) DNA without any gene delivery system results in transfection of rat lungs (Am J Physiol. 1995;268:L1052-L1056). This study investigated the pulmonary response to tracheal insufflation of plasmid DNA and the immunohistochemical localization of transgene expression in the lungs. Insufflation of 300 micrograms pBL-CAT DNA resulted in lung transfection without causing pulmonary injury as judged from the protein content of alveolar lavage fluid, pleural effusion, and lung ultrastructure. There was, however, an acute alveolar inflammatory response at 6 h after insufflation due to contaminating endotoxin present in the plasmid DNA preparation. Reducing the amount of endotoxin from 0.022 to 0.0015 microgram per 300 micrograms pBL-CAT completely abolished the acute alveolar inflammatory response without affecting the lung transfecting efficiency of pBL-CAT. Immunohistochemistry revealed that insufflation of pBL-CAT transfected predominantly small airway epithelial cells.

Pulmonary O2 toxicity: role of endogenous tumor necrosis factor.

The role of endogenous tumor necrosis factor (TNF) in the pathogenesis of pulmonary O2 toxicity was investigated. Intratracheal insufflation of anti-TNF antibodies prolonged the survival of rats exposed to 100% O2. No TNF bioactivity or immunoreactive protein was detectable in the alveolar lavage fluid or lung homogenate of rats exposed to normoxia or hyperoxia. However, levels of pulmonary TNF mRNA were markedly enhanced in rats exposed to hyperoxia. These results suggest that hyperoxia may cause the production of low level TNF, which in turn enhances O2 toxicity.

Lung-specific direct in vivo gene transfer with recombinant plasmid DNA.

A number of gene delivery methods have been developed to facilitate gene transfer into mammalian somatic cells in vivo. In this study, we demonstrated that tracheal insufflation of two recombinant plasmids containing a bacterial gene, chloramphenicol acetyltransferase (CAT), driven by the cytomegalovirus (CMV) immediate early promoter/enhancer, either alone or complexed to cationic liposomes, resulted in efficient and selective transfection of the lungs in rats. When the simian virus 40 (SV40) promoter/enhancer was used, there was no detectable transfection. Insufflation of plasmid DNA was as efficient as plasmid-liposome complexes in transfecting the lungs. Expression of the CAT gene in the lungs was noted as early as 1 day after insufflation of plasmid DNA alone or plasmid-liposome complexes, and lasted for > 21 days. In contrast, intravenous injection of plasmid alone or plasmid-liposome complexes did not result in transfection of the lungs. Because of its simplicity, without the potential adverse effect of any gene delivery systems, intratracheal delivery of recombinant plasmid DNA may have potential implication for lung-specific gene therapy.

Role of endogenous cytokines in endotoxin- and interleukin-1-induced pulmonary inflammatory response and oxygen tolerance.

Endotoxin lipopolysaccharide and the cytokines, tumor necrosis factor (TNF) and interleukin-1 (IL-1), are known to protect adult rats against O2 toxicity. However, whether the effect of endotoxin is mediated by these cytokines is not clear. We have previously demonstrated that depletion of 84% rat alveolar macrophages (AM), which reduced lipopolysaccharide (LPS)-induced release of TNF by 86%, had no effect on LPS-induced O2 tolerance. In this study, we demonstrated that coinsufflation of LPS with anti-TNF antibody and IL-1 receptor antagonist (IL-1ra), which completely inhibited LPS-induced TNF and IL-1 activities, had no effect on LPS-induced alveolar inflammatory response and O2 tolerance. Likewise, coinsufflation of IL-1 and anti-TNF antibody, which completely neutralized IL-1-induced TNF activity, had no effect on IL-1-induced alveolar inflammatory response and O2 tolerance. In contrast, IL-1ra completely abolished IL-1-induced inflammatory response and markedly inhibited IL-1-induced O2 tolerance. These results suggest that LPS-induced alveolar inflammatory response and O2 tolerance are not mediated by endogenous TNF and IL-1. Similarly, endogenous TNF does not mediate IL-1-induced alveolar inflammatory response and O2 tolerance.