A hypoxia-responsive element mediates a novel pathway of activation of the inducible nitric oxide synthase promoter.

Picolinic acid, a catabolite of L-tryptophan, activates the transcription of the inducible nitric oxide synthase gene (iNOS) in IFN-gamma-treated murine macrophages. We performed functional studies on the 5' flanking region of the iNOS gene linked to a CAT reporter gene to identify the cis-acting element(s) responsible for the activation of iNOS transcription by picolinic acid. Transient transfection assays showed that the full-length iNOS promoter in the murine macrophage cell line ANA-1 was activated by the synergistic interaction between IFN-gamma and picolinic acid. Deletion or mutation of the iNOS promoter region from -227 to -209, containing a sequence homology to a hypoxia-responsive enhancer (iNOS-HRE), decreased picolinic acid- but not LPS-induced CAT activity by more than 70%. Functional studies using a tk promoter-CAT reporter gene plasmid demonstrated that the iNOS-HRE was sufficient to confer inducibility by picolinic acid but not by IFN-gamma or LPS. Electrophoretic mobility shift assays confirmed that picolinic acid alone induced a specific binding activity to the iNOS-HRE. Furthermore, we found that the iNOS-HRE activity was inducible by hypoxia and that hypoxia in combination with IFN-gamma activated the iNOS promoter in transient transfection assays and induced iNOS transcription and mRNA expression. These data establish that the iNOS-HRE is a novel regulatory element of the iNOS promoter activity in murine macrophages and provide the first evidence that iNOS is a hypoxia-inducible gene.

Heterogeneity of hematopoietic cells immortalized by v-myc/v-raf recombinant retrovirus infection of bone marrow or fetal liver.

The J2 recombinant retrovirus expressing v-myc/v-raf (also known as MYC/RAF1) immortalized macrophages from the bone marrow of lipopolysaccharide-responsive mouse strains, producing the ANA-1 cell line from C57BL/6 mice and the INF-3A cell line from C3H/HeN mice. In contrast, J2 recombinant retrovirus infection of the fetal liver from C57BL/6-Ly-5a mice immortalized a cell line (GGD) that did not exhibit the characteristics of mature macrophages. The GGD cell line was classified as leukocytic on the basis of its expression of the Ly-6B.2, Fc gamma R, and Ly-5.2 antigens. Our results indicate that the J2 recombinant retrovirus selectively immortalizes macrophages from the bone marrow of C57BL/6 and C3H/HeN mice but immortalizes cells without definitive macrophage characteristics from murine fetal liver under the same culture conditions.

Bryostatin-1 and IFN-gamma synergize for the expression of the inducible nitric oxide synthase gene and for nitric oxide production in murine macrophages.

Bryostatin-1 (Bryo) is a nontumor-promoting protein kinase C modulator that has been shown to have both in vitro and in vivo activity against several murine and human tumors. In this study, we investigated the effects of Bryo on nitric oxide production, measured as accumulated nitrite (NO2-) in culture supernatant, and inducible nitric oxide synthase (iNOS) gene expression in the murine macrophage cell line ANA-1. ANA-1 macrophages did not produce NO2- or iNOS mRNA constitutively, and very little or no NO2- or iNOS mRNA were detectable upon exposure to IFN-gamma. Bryo, although ineffective alone, and IFN-gamma synergized to produce high levels of NO2- and iNOS mRNA. The activity of Bryo was evident at a concentration of 0.1 ng/ml and reached its maximum at 1 ng/ml. The effects of Bryo were time dependent because expression of iNOS mRNA was detectable as early as 6 h and increased through 24 h. Analyses of the molecular mechanisms involved indicate that Bryo and IFN-gamma mainly regulate iNOS gene expression posttranscriptionally through stabilization of iNOS mRNA. Experiments designed to investigate the role of tumor necrosis factor alpha (TNF-alpha) in NO2- production by Bryo- and IFN-gamma-activated macrophages revealed that ANA-1 macrophages expressed low levels of TNF-alpha mRNA constitutively that were not augmented in the presence of IFN-gamma. However, Bryo alone augmented the TNF-alpha mRNA expression, which was only slightly increased with the addition of IFN-gamma. A polyclonal antibody to TNF-alpha was able to completely neutralize TNF-alpha secreted in either medium or Bryo plus IFN-gamma-treated cultures. Neutralizing concentrations of anti-TNF-alpha antibody suppressed the Bryo plus IFN-gamma-induced NO2- production approximately by 50%, suggesting that NO2- produced by Bryo plus IFN-gamma-treated ANA-1 macrophages may involve both TNF-alpha-dependent and TNF-alpha-independent mechanisms. Overall, these findings provide the first evidence that Bryo and IFN-gamma can synergize for the induction of NO2- production as well as iNOS gene expression and show the involvement of posttranscriptional mechanisms in the induction of iNOS mRNA.

Regulation of transforming growth factor beta1 by nitric oxide.

Many tumor cells or their secreted products suppress the function of tumor-infiltrating macrophages. Tumor cells often produce abundant transforming growth factor beta1 (TGF-beta1), which in addition to other immunosuppressive actions suppresses the inducible isoform of NO synthase. TGF-beta1 is secreted in a latent form, which consists of TGF-beta1 noncovalently associated with latency-associated peptide (LAP) and which can be activated efficiently by exposure to reactive oxygen species. Coculture of the human lung adenocarcinoma cell line A549 and ANA-1 macrophages activated with IFN-gamma plus lipopolysaccharide resulted in increased synthesis and activation of latent TGF-beta1 protein by both A549 and ANA-1 cells, whereas unstimulated cultures of either cell type alone expressed only latent TGF-beta1. We investigated whether exposure of tumor cells to NO influences the production, activation, or activity of TGF-beta1.A549 human lung adenocarcinoma cells exposed to the chemical NO donor diethylamine-NONOate showed increased immunoreactivity of cell-associated latent and active TGF-beta1 in a time- and dose-dependent fashion at 24-48 h after treatment. Exposure of latent TGF-beta1 to solution sources of NO neither led to recombinant latent TGF-beta1 activation nor modified recombinant TGF-beta1 activity. A novel mechanism was observed, however: treatment of recombinant LAP with NO resulted in its nitrosylation and interfered with its ability to neutralize active TGF-beta1. These results provide the first evidence that nitrosative stress influences the regulation of TGF-beta1 and raise the possibility that NO production may augment TGF-beta1 activity by modifying a naturally occurring neutralizing peptide.

Interleukin 12 primes macrophages for nitric oxide production in vivo and restores depressed nitric oxide production by macrophages from tumor-bearing mice: implications for the antitumor activity of interleukin 12 and/or interleukin 2.

Interleukin-12 (IL-12) is a recently described immunoregulatory cytokine with potent therapeutic activity in various preclinical models of infectious or malignant disease. As part of our ongoing evaluation of potential mechanisms accounting for the potent antitumor activity of IL-12, we have investigated the influence of IL-12 administration on total serum nitrate/nitrite (NO(x)(-)) levels and the production of nitric oxide (NO) by peritoneal macrophages from normal and tumor-bearing mice. We report here that IL-12 administration to either normal or tumor-bearing mice for periods of time ranging from 7-19 days induced progressive increases in serum NO(x)(-) levels and primed peritoneal macrophages for NO production on subsequent exposure to lipopolysaccharide or IL-2 ex vivo. Treatment of resident peritoneal macrophages or the macrophage cell line ANA-1 with IL-12 alone or IL-12 in combination with various other stimuli failed to induce NO production, suggesting that the effects of IL-12 occurred via an indirect mechanism. Furthermore, we have shown that not only was the production of NO by macrophages from untreated long-term, tumor- bearing mice suppressed compared with control mice treated with vehicle or IL-12, but also that IL-12 administration overcame this suppression and delayed tumor growth. Lastly, we have shown that administration of weekly pulses of IL-2 in combination with IL-12 additively enhanced the priming of macrophages for NO production ex vivo and delayed tumor growth far more effectively than either agent alone. These observations and reports in the literature regarding the potential influence of NO on development of the immune response and on the regulation of tumor growth and vascularization suggest that NO may play a significant role in the antitumor activity of IL-12 and IL-2.

Murine Th1 and Th2 cell clones differentially regulate macrophage nitric oxide production.

Previous studies have demonstrated that the combination of the T helper cell 1 (Th1)-derived cytokines interleukin (IL)-2 and interferon (IFN)-gamma induces nitric oxide (NO) production and tumor cytolysis by mouse peritoneal macrophages and the mouse macrophage cell line ANA-1 in vitro. Conversely, the Th2-derived cytokine IL-4 inhibits IL-2 and IFN-gamma-induced NO production and tumor cytolysis by ANA-1 macrophages. To examine the paracrine regulatory effects of Th1 and Th2 cells on macrophages, various mouse T cell clones were tested for their ability to regulate NO production by mouse peritoneal macrophages or ANA-1 macrophages. Antigen, superantigen, and mitogen stimulated Th1 cells but not Th2 cells induced NO production by macrophages. Supernatants from these activated Th1 clones also induced NO production by peritoneal macrophages and ANA-1 macrophages. Neutralization analysis using monoclonal anticytokine antibodies revealed that both IL-2 and IFN-gamma production by activated Th1 cells were required for the production of NO by macrophages. Co-culture studies using a panel of Th2 cell clones that share the same antigen specificity revealed that these cells suppressed Th1-mediated macrophage activation. The Th2-mediated impairment of Th1-induced NO production was primarily due to the secretion of IL-4. IL-4 appeared to have a direct effect on macrophage activation because neither mitogen-induced proliferation of Th1 cells nor cytokine production by Th1 cells were affected by IL-4. Overall, these results suggest that a potent paracrine regulatory network involving Th1 cells and Th2 cells may control the activation of macrophages for NO production and antitumor cytotoxicity.

Characterization of nitric oxide-stimulated ADP-ribosylation of various proteins from the mouse macrophage cell line ANA-1 using sodium nitroprusside and the novel nitric oxide-donating compound diethylamine dinitric oxide.

We examined the ability of nitric oxide (NO) to stimulate the ADP-ribosylation of proteins from the mouse macrophage cell line ANA-1. To demonstrate a specific effect of NO, we used a novel compound named diethylamine dinitric oxide (DEA/NO; 1,1-diethyl-2-hydroxy-2-nitrosohydrazine, sodium salt; [Et2NN(O)NO]Na), which releases NO in aqueous solution at neutral pH. DEA/NO stimulated the ADP-ribosylation of at least three cytosolic proteins (M(r) = 28,000, 33,000 and 39,000) from ANA-1 macrophages. The effect of DEA/NO on the ADP-ribosylation of the predominant target p39 was dose dependent (EC50 = 80 microM). Moreover, the effect of DEA/NO was attributed specifically to released NO rather than diethylamine or nitrite. Sodium nitroprusside (SNP) also stimulated the ADP-ribosylation of cytosolic proteins from ANA-1 mouse macrophages. However, SNP exhibited different time- and dose-dependent effects on the modification of p39. NO synthesized via the activity of interferon-gamma plus lipopolysaccharide-induced NO synthase also enhanced the ADP-ribosylation of p39, confirming that the effects of DEA/NO and SNP could be attributed to NO or reactive nitrogen oxide species. Neither pertussis toxin nor cholera toxin stimulated the ADP-ribosylation of p39; however, cholera toxin stimulated the ADP-ribosylation of proteins with approximate molecular weight of 28,000 and 33,000. These data suggest that the induced expression of NO synthase in tumoricidal macrophages may be associated with autocrine and paracrine effects of NO that include the ADP-ribosylation of various proteins. Moreover, these results indicate that DEA/NO and related compounds may be useful as pharmacologic tools for investigating the effects of NO and reactive nitrogen oxide species on macrophages.

LPS-inducible nuclear factor in human monocytes that binds the negative regulatory element of the HIV LTR.

We studied the constitutive and lipopolysaccharide (LPS)-induced expression of nuclear protein binding to the negative regulatory element (NRE) of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in fresh human monocytes. We demonstrated the existence of a constitutive factor binding to the NRE 73-bp HpaII/HpaII fragment (-216 to -143) whose expression is up-regulated by LPS treatment. Competition experiments with overlapping oligonucleotides covering the HpaII/HpaII fragment and with mutated oligonucleotides mapped the binding within the TTTCATCAC region (-171 to -163). This binding pattern is unique to human monocytes.

LPS stimulation of TNF-receptor deficient macrophages: a differential role for TNF-alpha autocrine signaling in the induction of cytokine and nitric oxide production.

To evaluate the role of autocrine TNF-alpha signaling in macrophage activation, immortalized macrophages from normal mice (B6/J2) and from mice containing gene targeted disruptions of the type 1 and type 2 TNF-receptor genes (TRN) were stimulated under CD14-dependent or serum-free conditions. Although the B6/J2 and TRN clones mounted similar nitric oxide responses to LPS in the presence of serum, the TRN macrophages responded poorly when stimulated with LPS under serum free conditions. LPS stimulation of TRN and B6/J2 under serum-free conditions resulted in equivalent levels of IL-1beta, TNF-alpha, and iNOS gene expression. However, Western blot analysis revealed that iNOS protein production by TRN was 2-fold lower than that produced by B6/J2. These results indicate that autocrine TNF-alpha stimulation contributes to the signaling pathways initiated by ligation of LPS receptors in the absence of LBP and is involved in iNOS post-transcriptional regulation.

Comparative studies on functional and secretory properties of macrophage cell lines derived from different anatomical sites.

In the present study, we compared four macrophage (M phi) cell lines from different anatomical origins for functional and secretory activities against the two morphogenetic forms of the fungus Candida albicans. We show that all the cell lines actively phagocytize the yeast and exert antimicrobial activity against both forms of Candida, although M phi of microglial origin are the most effective. When assessed for secretory properties, microglial M phi exhibit a peculiar pattern with respect to other M phi populations under either basal or stimulated conditions. In particular, only microglial M phi fail to respond to the hyphal form of the fungus (H-Candida), which instead acts as a potent tumor necrosis factor inducer in the other M phi cell lines. When exposed to H-Candida, microglial M phi are indistinguishable from other M phi in their ability to modulate specific surface adhesion molecules. In addition to strengthening the knowledge on functional heterogeneity among M phi, our data provide evidence on the peculiar behavior of microglial M phi. To what extent M phi heterogeneity may be related to tissue homeostasis is discussed.

Soil translocation by pocket gophers in a Mima moundfield.

We measured soil translocation due to the tunneling of valley pocket gophers (Thomomys bottae) in a Mima moundfield at Miramar Mounds National Landmark, San Diego, California, from December, 1984 through December, 1985. We placed 1-l soil plugs containing 20 11-g iron pellets into pocket gopher tunnels at locations between mound tops and points about one mound radius beyond mound edges. After about 4-10 d, sites to which the marker-containing soil had been translocated were located with a metal detector and the horizontal and vertical displacements measured. Between 1 October and 15 May (the cooler, wetter portion of the year), pocket gophers removed an average of 63% of the experimental plugs and moved an average of 38% of the markers that we recovered. From 15 May through 1 October (the hotter, drier portion of the year), only 32% of plugs were cleared and 12% of the recovered markers were moved. On average, markers that were moved were displaced 41 cm moundward and 4.9 cm upward in elevation. The intensity of moundward translocation increased with distance from the mound center. At a distance of 0.5-1.0 mound radius beyond the edge of the mound, the moundward translocation tendency averaged 71 cm. The intensity of moundward translocation was also inversely related to maximum mound height. These observations provide strong support for the fossorial rodent hypothesis of Mima mound origin, and constitute a first step in development of a mathematical model of mound formation.

Interventional neuroradiology: a joint effort of the neuroradiologist and neurosurgeon in the treatment of vascular lesions of the head and neck.

Close cooperation between the neuroradiologist and neurosurgeon has made possible the treatment of certain lesions of the head and neck by embolization procedures. The experience at Emory University Hospital with embolizations of arteriovenous malformations, carotid-cavernous fistulae, dural-cavernous sinus fistulae and glomus jugulare tumors is discussed.

Outpatient skin grafting of extremity burn wounds with the use of Unna Boot compression dressings.

Thirty-one patients underwent split-thickness skin grafting for burn injuries of an extremity, after which Unna Boot compression dressings were applied for fixation of the graft. Three patients required hospitalization of 2 to 4 days, and 28 patients were treated strictly on an outpatient basis. The lower extremity was involved in 25 patients, and the upper extremity was involved in six. Average wound size was 284 cm2. Eighteen patients were treated with sheet grafts, and 13 received meshed grafts. Nine wounds extended across a joint. Patients were allowed immediate ambulation after surgery. All grafts resulted in 95% to 100% wound coverage, and no regrafting was required. Application of Unna boot compression dressings to extremity skin grafts provides excellent protection of both meshed and nonmeshed grafts and allows immediate ambulation and range of motion. Many patients with burn injuries may be treated on an outpatient basis with the use of this technique.

Assay for macrophage-mediated anti-tumor cytotoxicity.

This unit describes a procedure for determining ability of mouse macrophages to lyse tumor cells in vitro. The Basic Protocol outlines a three-stage assay that includes: (1) culture of macrophages (freshly explanted from mice or grown from a cell line) with suspected activating reagents; (2) extensive washing of the cultured macrophages to remove residual reagents, followed by incubation with [(111)In]-labeled tumor cells to allow lysis to occur; and (3) collection of cell-free culture supernatants and measurement of cytolytic activity as a function of (111)In released from tumor cells destroyed by activated macrophages. The Support Protocol outlines a method for radiolabeling tumor cells with [(111)In]oxine.

Limitations of computerized tomography scanning in acoustic neurinomas.

Computerized tomography (CT) of the head has certain limitations in detecting small lesions and subtle erosions of bone. The CT scan can give valuable information in the large cerebellopontine angle tumors; however, it should not replace polytomography of the petrous ridges in examining patients for small acoustic neurinomas.

8-Methoxypsoralen inhibits lymphocyte proliferation in vitro in the absence of ultraviolet radiation.

8-Methoxypsoralen was assessed for its effects on in vitro lymphocyte proliferation in the absence of ultraviolet radiation. 8-Methoxypsoralen inhibited both the phytohemagglutinin and concanavalin A-induced proliferation of normal human peripheral blood lymphocytes in a time and dose-dependent manner. This inhibition did not require the photoactivation of 8-methoxypsoralen in these cells. Suppressed lymphocyte proliferation in the presence of 8-methoxypsoralen could not be overcome by addition of exogenous interleukin-2. The observed decrease in lymphocyte proliferation hyporesponsiveness to interleukin-2 correlated with the ability of 8-methoxypsoralen to induce a dose-dependent decrease in interleukin-2 receptor expression on phytohemagglutinin-stimulated lymphocytes. Since interleukin-2 receptors play a central role in lymphocyte proliferation and immune reactivity, their decrease may explain the mechanism by which 8-methoxypsoralen impairs lymphocyte function.

Picolinic acid, a catabolite of L-tryptophan, is a costimulus for the induction of reactive nitrogen intermediate production in murine macrophages.

In this study we investigated the effects of picolinic acid, a catabolite of L-tryptophan, on the production of L-arginine-derived reactive nitrogen intermediates in the murine macrophage cell line ANA-1. ANA-1 macrophages did not produce nitrite (NO2-) constitutively, but accumulated detectable levels of NO2- on exposure to IFN-gamma. Picolinic acid, although ineffective by itself, augmented IFN-gamma-induced NO2- production. The activity of picolinic acid was evident at 1 mM and reached its maximum at 4 mM. Picolinic acid also augmented the IFN-gamma-dependent expression of TNF-alpha mRNA, but did not appreciably affect the secretion of the TNF-alpha protein. Neutralizing concentrations of anti-TNF mAb completely abrogated IFN-gamma- and IFN-gamma plus rTNF-alpha-induced NO2- production in ANA-1 macrophages, but only decreased by approximately 50% the synergistic interaction between IFN-gamma and picolinic acid. Although IL-4 inhibited the expression of IFN-gamma plus picolinic acid-induced TNF-alpha mRNA and protein, it only partially suppressed picolinic acid-dependent NO2- production. Therefore, picolinic acid may affect NO2- production via both TNF-alpha-dependent and TNF-alpha-independent pathways. Overall, this study suggests that amino acid catabolites may be important for the activation and the expression of effector functions by murine macrophages, and provides the first evidence of a possible connection between tryptophan and arginine metabolism.