John B. Hibbs

Evidence for cytokine-inducible nitric oxide synthesis from L-arginine in patients receiving interleukin-2 therapy.

An interferon-gamma, tumor necrosis factor, and interleukin-1-inducible, high-output pathway synthesizing nitric oxide (NO) from L-arginine was recently identified in rodents. High-dose interleukin-2 (IL-2) therapy is known to induce the same cytokines in patients with advanced cancer. Therefore, we examined renal cell carcinoma (RCC; n = 5) and malignant melanoma (MM; n = 7) patients for evidence of cytokine-inducible NO synthesis. Activity of this pathway was evaluated by measuring serum and urine nitrate (the stable degradation product of NO) during IL-2 therapy. IL-2 administration caused a striking increase in NO generation as reflected by serum nitrate levels (10- and 8-fold increase [P less than 0.001, P less than 0.003] for RCC and MM patients, respectively) and 24-h urinary nitrate excretion (6.5- and 9-fold increase [both P less than 0.001] for RCC and MM patients, respectively). IL-2-induced renal dysfunction made only a minor contribution to increased serum nitrate levels. Metabolic tracer studies using L-[guanidino-15N2]arginine demonstrated that the increased nitrate production was derived from a terminal guanidino nitrogen atom of L-arginine. Our results showing increased endogenous nitrate synthesis in patients receiving IL-2 demonstrate for the first time that a cytokine-inducible, high-output L-arginine/NO pathway exists in humans.

Metabolic fate of L-arginine in relation to microbiostatic capability of murine macrophages.

L-arginine is required for the fungistatic action of murine macrophages in vitro. To further investigate this requirement, L-arginine metabolism by macrophages was measured under conditions where fungistasis either succeeded or failed. Macrophage fungistasis correlated with metabolism of L-arginine to citrulline, nitrite, and nitrate. The metabolic rate was dependent on extracellular L-arginine concentration, reaching a maximum of 67 nmol nitrite/h per mg protein. It accounted for one-third of arginine consumed by fungistatic macrophages. Equimolar amounts of citrulline and total nitrite plus nitrate accumulated in medium. This was consistent with the hypothesis that one of the equivalent guanidino nitrogens of L-arginine was oxidized to both nitrite and nitrate leaving L-citrulline as the amino acid reaction product. The analogue, NG-mono-methyl-L-arginine, selectively inhibited nitrogen oxidation and it was shown previously that it inhibited fungistatic capability. Resident macrophages were not fungistatic and their nitrogen oxidation was low. Once macrophages began producing nitrite/nitrate, protein synthesis was not required during the next 8 h for either fungistasis or nitrogen oxidation. Two-thirds of L-arginine consumption was due to macrophage arginase yielding L-ornithine and urea, which accumulated in medium. This activity was dissociated from macrophage fungistasis. Nitrogen oxidation metabolism by macrophages is linked to a mechanism that inhibits proliferation of fungi. This may involve synthesis of an intermediate compound(s) that has antimicrobial properties.

Heterocytolysis by Macrophages Activated by Bacillus Calmette-Guérin: Lysosome Exocytosis into Tumor Cells

The cytotoxic activity of activated macrophages against tumorigenic target cells appears to be mediated by lysosomal enzymes of activated macrophage origin. Lysosomes of activated macrophages are secreted directly into the cytoplasm of susceptible target cells, which subsequently undergo heterolysis. This reaction can be inhibited by agents which prevent the exocytosis of macrophage lysosomes (hydrocortisone) or which interfere with the action of lysosomal enzymes (trypan blue).

Macrophage fibrinolytic activity: Identification of two pathways of plasmin formation by intact cells and of a plasminogen activator inhibitor

Endotoxin-stimulated macrophages hydrolyze fibrin by a plasmin-mediated process in the absence of detectable soluble plasminogen activator (PAs). The data show that macrophages also activate plasmin by a membrane-associated plasminogen activator (PAm). In the presence of endotoxin, PAm activity increases, and plasmin is formed only by PAm. In addition, endotoxin stimulates macrophages to secrete a proteinase inhibitor that blocks PAs activity but not PAm or plasmin activity. The increased PAm activity and the PA inhibitor secretion in response to endotoxin explains the ability of intact macrophages to hydrolyze fibrin in the absence of detectable PAs. Endotoxin, 100 ng/ml, induced an intracellular PA inhibitor in cultured macrophages, and this correlated with accumulation of inhibitor in medium over the cells. The intracellular PA inhibitor was found to be 50--60 kilodaltons by gel chromatography, to be of anionic charge at pH 7.4 and to inhibit urokinase esterolytic and proteolytic activity but not preformed plasmin. These results define two pathways of plasmin formation by intact macrophages and identify the macrophage cell surface as a site of PA activity relatively protected from soluble proteinase inhibitors.

Injury of neoplastic cells by murine macrophages leads to inhibition of mitochondrial respiration.

Cytotoxic activated macrophages (CM) inhibited the growth of neoplastic L1210 cells in vitro but L1210 cell death was minimal to nonexistent. L1210 cells injured by CM were separated from macrophages and studied in an isolated system. CM-injured L1210 cells had an absolute requirement for glucose or another glycolyzable hexose (mannose or fructose) for at least 40 h after removal from macrophages. If the culture medium lacked sufficient concentration of one of these sugars, CM-injured L1210 cells died within 4 h. Uninjured L1210 cells cultured alone or with peptone-stimulated macrophages had no such requirement and maintained complete viability in hexoseless medium. The hexose requirement of CM-injured L1210 cells could not be fulfilled by other naturally occurring monosaccharides, glucose or mannose derivatives, or substrates that can be oxidized by mitochondria. The concentration requirements for glucose, mannose, and fructose by CM-injured L1210 cells correlated with the concentrations required to support maximal glycolysis of these sugars by other murine ascites cells. A concentration of 2-deoxy-D-glucose which completely inhibited L1210 cell glycolysis also complete prevented the ability of glucose or mannose to maintain viability of CM-injured L1210 cells. Interaction with CM led to inhibition of L1210 cell mitochondrial oxidative phosphorylation. This was supported by the findings that: (a) CM-injured L1210 cells had no Pasteur effect; their rate of aerobic glycolysis was the same as the rate of anaerobic glycolysis of uninjured L1210 cells, (b) Endogenous respiration of CM-injured L1210 cells was 15% of normal. Maximal inhibition of uninjured L1210 cell respiration by a specific mitochondrial poison (oligomycin) was nearly the same (13% of normal). It followed that CM-injured L1210 cells required hexose for chemical energy production via the glycolytic pathway. CM-induced mitochondrial injury occurred in five other neoplastic cell lines tested.

Macrophage Nonimmunologic Recognition: Target Cell Factors Related to Contact Inhibition

Activated mouse macrophages were not cytotoxic to contact-inhibited nontumorigenic 3T3 fibroblasts, but caused marked destruction to non-contact-inhibited, tumorigenic 3T12 and simian virus 40-transformed fibroblasts. Nonimmunologic recognition and destruction of target cells by activated macrophages is independent of altered morphology, abnormal karyotype, and ability for continuous multiplication in vitro—all characteristics of 3T3 fibroblasts. A modification e f the target cell surface that results in a high in vitro saturation density, agglutinability by plant lectins, and tumorigenicity appears to evoke a cytotoxic response by activated macrophages.

Cytokines induce an L-arginine-dependent effector system in nonmacrophage cells

Treatment of EMT‐6 mammary adenocarcinoma cells with gamma interferon (rMuIFNγ) plus tumor necrosis factor (rMuTNFα) and/or interleukin‐1 (rHuIL‐1α) causes release of iron‐55 label, inhibition of DNA replication, and inhibition of aconitase activity. In addition, the same combinations of cytokines induce EMT‐6 cells to synthesize L‐citrulline, nitrite, and nitrate directly from L‐arginine. Lipopolysaccharide (LPS) can act as a cofactor in the induction of these metabolic effects when added to EMT‐6 cells in the presence of rMuIFNγ. The results show that increased levels of cytokines in the microenvironment can induce a novel effector pathway in somatic cells not specialized for host defense, resulting in specific metabolic effects as well as the inhibition of cellular proliferation.

Assembly and regulation of NADPH oxidase and nitric oxide synthase

Research over the past year has revealed several interesting advances in the biosynthesis of the superoxide anion and nitric oxide. Highlights include the demonstration that the G protein Rac 2 is required for NADPH oxidase activation, the finding that nitric oxide is a feedback inhibitor of nitric oxide synthase isoforms, and the discovery that the continuous catalytic activity of the immune/inflammatory nitric oxide synthase is due to strong calmodulin binding, which is independent of elevated calcium levels. Interferon-gamma primes neutrophils and macrophages for both O2- and nitric oxide synthesis. However, NADPH oxidase and immune/inflammatory nitric oxide synthase are differentially regulated such that their activities are not simultaneously induced.

Infection and Nitric Oxide

The author describes how his experience as an infectious disease fellow at Stanford University with Jack Remington from 1969 to 1971 influenced the direction of his subsequent laboratory investigation. The author reviews a series of studies that were intended to provide a mechanistic understanding of an in vitro cytotoxicity assay developed while he was a fellow with Jack Remington. These investigations resulted in the 1987 discovery of the synthesis of nitric oxide from L-arginine by cytokine-activated macrophages. This work provided the components (the precursor, products, and an inhibitor) of the enzymatic synthesis of nitric oxide by all three later-identified nitric oxide synthase isoforms. The significance of cytokine-induced nitric oxide synthesis during innate resistance and cell-mediated immune reactions is discussed briefly.

Activated macrophage conditioned medium: identification of the soluble factors inducing cytotoxicity and the L-arginine dependent effector mechanism.

Conditioned medium (CM) from cultures of cytotoxic activated macrophages causes inhibition of mitochondrial respiration, DNA synthesis, and aconitase activity in murine EMT‐6 mammary adenocarcinoma cells by an l‐arginine dependent effector mechanism. CM induces cytotoxicity and nitrite synthesis in EMT‐6 cells in a dose dependent manner. We have identified the soluble factors in CM that induce cytotoxicity and synthesis of inorganic nitrogen oxides from l‐arginine by EMT‐6 cells. Using functional inhibition experiments, the activity of lipopolysaccharide (LPS), tumor necrosis factor alpha (TNFα), and interferon gamma (IFNγ) in CM was investigated. The LPS inhibitor polymyxin B and TNFα antibody produced a modest decrease in nitrite production, while IFNγ antibody markedly inhibited both nitrite production and cytostasis. Simultaneous treatment with polymyxin B, TNFα antibody, and IFNγ antibody reduced EMT‐6 cell nitrite production by 81%, and cytostasis by 74%. By Western blot, IFNγ and TNFα were shown to be present in CM. When CM was subjected to hydrophobic interaction chromatography, a single peak of activity was eluted, and Western blot showed that the active fractions contained IFNγ. Furthermore, IFNγ antibody neutralized the activity in these chromatographic fractions. We conclude that induction of inorganic nitrogen oxide synthesis from l‐arginine by the synergistic combination of IFNγ, TNFα, and LPS accounts for most of the biologic activity of CM, and that IFNγ is the major priming factor.