Binding of bacillus Calmette-Guérin-activated macrophages to tumor targets. Selective inhibition by membrane preparations from homologous and heterologous neoplastic cells.

The binding of tumor cells by activated macrophages is an initial and necessary event in the cytolysis of these targets. The data here indicate that membrane preparations from RL sigma 1 leukemia targets, EL-4 lymphoma targets, and P815 mastocytoma targets each inhibited binding of its homologous target to bacillus Calmette-Guérin (BCG)-activated murine macrophages in a dose-dependent fashion. Similar amounts of membrane from lymphocytes did not alter binding of the three neoplastic target to BCG-macrophages. Membranes of the three targets also inhibited binding of the heterologous neoplastic targets. Inhibitory activity of membrane preparations from P815, EL-4, and RL sigma 1 targets could be adsorbed by incubation of limiting concentrations of the membrane preparations with BCG-activated macrophages but not with thioglycollate broth-elicited macrophages. Exposure of BCG macrophages to membrane preparations from RL sigma 1, FL-4, or P815 targets inhibited subsequent cytolysis of the three targets. Inhibitory activity was increased in preparations enriched for plasma membrane. The data suggest that binding of three murine, nonadherent neoplastic targets to BCG-activated murine macrophages is mediated, in part, by recognition structures present within the plasma membranes of the three targets.

Biochemical and functional responses stimulated by platelet-activating factor in murine peritoneal macrophages.

Platelet-activating factor (PAF) is a potent stimulant of leukocytes, including macrophages. To analyze the mechanisms of its effects upon macrophages, we determined whether macrophages bear specific surface receptors for PAF. By competitive radioactive binding assays, we determined two classes of specific receptors to be present on purified membranes derived from murine peritoneal macrophages (one having a Kd of approximately 1 X 10(-10) M and one a Kd of approximately 2 X 10(-9) M). When the macrophages were incubated with PAF, rapid formation of several inositol phosphates including inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate were observed. PAF also elevated intracellular levels of calcium to 290 +/- 27% of basal levels which were 82.7 +/- 12 nM. Increases in calcium were observed first in submembranous areas of the macrophages. PAF also led to increases of 1,2-diacylglycerol of approximately 200 pmol/10(7) cells. A characteristic pattern of enhanced protein phosphorylation, similar to that initiated by both phorbol 12,13-myristate and lipopolysaccharide, was observed and involved enhanced phosphorylation of proteins of 28, 33, 67, and 103 kD. The half-maximal dose of PAF for initiating all the above effects was approximately 5 X 10(-9) M. PAF also initiated significant chemotaxis of the cells; the half-maximal dose for this effect was approximately 1 X 10(-11) M. Taken together, these observations suggest that murine mononuclear phagocytes bear specific membrane receptors for PAF and that addition of PAF leads to generation of break-down products of polyphosphoinositides, subsequent changes in intracellular calcium and protein phosphorylation, and chemotaxis.

Retina: ultrastructural alterations produced by extremely low levels of coherent radiation.

The effect of low levels of coherent radiation on the eye is not fully established, but is generally presumed to be noninjurious. Irradiation of the retina with a Q-switched ruby laser emitting low amounts of energy (0.1 percent probability of creating damage) consistently produces ultrastructural alterations in rods and cones. Outer segments of these cells are broken and disorganized and their lamellae are in disarray 1 day after such irradiation.

Receptors for maleylated proteins regulate secretion of neutral proteases by murine macrophages.

Receptors for maleylated or acetylated proteins as well as for alpha-2-macroglobulin-protease complexes on macrophages serve as scavengers by mediating the uptake of macromolecules from the extracellular compartment. Described in this report is a novel function of these receptors on macrophages: regulation of neutral protease secretion. The binding of maleylated bovine serum albumin to macrophages triggered secretion of three neutral proteases: neutral caseinases, plasminogen activator, and cytolytic proteinase. Release of acid phosphatase, however, was not induced. An important biological consequence of protease secretion by macrophages, tumor-cytolysis, was also triggered by engagement of the receptor for maleylated bovine serum albumin. By contrast, the binding of alpha-2-macroglobulin-protease complexes to the macrophages suppressed secretion of all three proteases. Thus two receptors heretofore believed to serve principally as scavengers also regulate secretory functions of macrophages.

Role of Na+/H+ exchange by interferon-gamma in enhanced expression of JE and I-A beta genes.

The rapid transductional sequences initiated by interferon-gamma (IFN-gamma) on binding to its receptor regulate functional and genomic responses in many cells but are not well defined. Induction of macrophage activation is an example of such functional and genomic changes in response to IFN-gamma. Addition of IFN-gamma to murine macrophages, at activating concentrations, produced rapid (within 60 seconds) alkalinization of the cytosol and a concomitant, rapid influx of 22Na+. Amiloride inhibited the ion fluxes and the accumulation of specific messenger RNA for two genes induced by IFN-gamma (the early gene JE and the beta chain of the class II major histocompatibility complex gene I-A). The data indicate that IFN-gamma initiates rapid exchange of Na+ and H+ by means of the Na+/H+ antiporter and that these amiloride-sensitive ion fluxes are important to some of the genomic effects of IFN-gamma.

Cellular immunity in rats with primary brain tumors: inhibition of macrophage migration by soluble extracts of avian sarcoma virus-induced tumors.

Rats bearing primary tumors of the brain induced by avian sarcoma virus (ASV) were studied with the migration-inhibition factor (MIF) assay for the presence of cell-mediated immunity to tumor-associated antigens. Astrocytomas and sarcomas of the brain were induced in 34 neonatal F344 rats by the intracerebral inoculation of Bratislava-77 ASV. At weekly intervals from 4 to 9 weeks after the inoculation with virus, peritoneal exudate cells (PEC) from rats bearing brain tumors were tested an an MIF assay against soluble and KCl-treated extracts of a syngeneic, ASV-induced sarcoma. Incubation of the PEC with a soluble extract of syngeneic liver or with a KCl extract of a syngeneic, chemically induced tumor served as controls. Of 14 rats tested against the soluble tumor extract, 6 (43%) had statistically significant inhibition of migration (P less than or equal to 0.05). Of 23 animals tested against the KCl extract, 16 (70%) had significant inhibition. Immunity to the KCl extract was significant in most rats at each period. Ten rats were tested against a KCl extract of a hamster ASV-induced tumor; 7 gad significant inhibition of migration. None of 3 tested against a soluble extract of a syngeneic, chemically induced tumor had significant inhibition. Rats bearing ASV-induced brain tumors displayed cell-mediated immunity to tumor-associated antigen or antigens of ASV-induced tumors, which could be solubilized.

Induction of 5,6-ring-saturated thymine bases in NIH-3T3 cells by phorbol ester-stimulated macrophages: role of reactive oxygen intermediates.

Because oxygen intermediates secreted by inflammatory leukocytes are postulated to play a role in potentiating carcinogenesis, we investigated the ability of macrophages to induce oxidative DNA damage in eukaryotic cells. Murine macrophages, obtained from sites of inflammation and stimulated with 12-O-tetradecanoylphorbol-15-acetate, induced the formation of 5,6-ring-saturated thymine bases in the DNA of cocultured NIH-3T3 cells; macrophages or 12-O-tetradecanoylphorbol-15-acetate alone did not induce such alterations. Reagent H2O2, at concentrations produced by macrophages in the ambient medium (i.e., approximately 10(-5) M), induced saturated thymines in the target cells in a dose-dependent manner. The reaction between reagent H2O2 and cellular DNA was rapid, reaching maximum levels in 30 min, and similar amounts of saturated thymines were induced at 4 degrees or 37 degrees. The 3T3 targets were able to repair the saturated thymines rapidly (i.e., over 70% of the lesion was removed in 2 hr). Catalase completely inhibited macrophage-mediated induction of saturated thymines, although superoxide dismutase enhanced induction. Taken together, the data indicate that macrophages exposed to phorbol diesters can induce a specific, quantifiable lesion in the DNA of bystander eukaryotic cells and that reactive oxygen species from the macrophages participate in producing the lesion.

Enhanced release of hydrogen peroxide and metabolites of arachidonic acid by macrophages from SENCAR mice following stimulation with phorbol esters.

Chronic inflammation has long been associated with carcinogenesis. Phorbol esters which are potent promoters of tumors in mouse skin are also potent inflammatory agents in skin and cause inflammatory cells to release large quantities of reactive oxygen intermediates and oxidized lipid products. SENCAR mice have been bred for their sensitivity to the promotion of tumors by phorbol esters and C57BL/6 mice have been shown to be resistant. We quantified the release of H2O2 and metabolites of arachidonic acid by macrophages obtained from SENCAR and C57BL/6 mice, following exposure to phorbol esters and other stimulants. The basal level for secretion of H2O2 in resident peritoneal macrophages was negligible in cells from both strains. Conversely, inflammatory macrophages from SENCAR mice, elicited by the injection of sterile casein, secreted 4 times more H2O2 than the corresponding cells from C57BL/6 mice. Furthermore, cells from SENCAR mice required less than one-third the amount of phorbol ester to obtain 50% of the maximal response than that required by cells from C57BL/6 mice. This difference was less when zymosan was used as a stimulant. Both resident and inflammatory macrophages from SENCAR mice released more metabolites of arachidonic acid than cells from C57BL/6 mice when exposed to phorbol esters, but macrophages from C57BL/6 mice released more metabolites when stimulated with zymosan. Few differences in the pattern of released metabolites were noted between the strains of mice. There were large differences in the relative amounts of individual metabolites released when different stimulants were used. The enhanced response to phorbol esters of chronic inflammatory cells from SENCAR mice correlates with the enhanced sensitivity to the promotion of tumors by phorbol esters in these animals.

Role of oxygen radicals in induction of DNA damage by metabolites of benzene.

Benzene is strongly suspected of being an animal and human carcinogen, but the mechanisms by which benzene induces tumors of lymphoid and hematopoietic organs are unknown. Binding studies in vivo suggest a very low level of covalent binding to the DNA of bone marrow elements. Since several metabolites of benzene have the potential to undergo autooxidation and thereby generate reactive oxygen intermediates, we have tested the hypothesis that benzene metabolites can induce DNA damage through the generation of oxygen radicals. Hydroquinone (HQ), benzoquinone (BQ), catechol, and 1,2,4-benzenetriol (BT) were first tested for their ability to generate O2-. at a physiological pH. BT, and to a lesser extent HQ, were autooxidized and produced significant quantities of O2-.. No detectable O2-. was produced by catechol or BQ. Similarly, BT was very efficient at degrading DNA, and this degradation was inhibited by scavengers of O2-., H2O2 and .OH. HQ did not degrade DNA but did induce single- and double-strand breaks. In contrast to the action of BT, the breakage of DNA by HQ was not inhibited by scavengers of reactive oxygen intermediates. The metabolites which did not produce O2-. (catechol and BQ) did not induce significant breakage of DNA. Taken together, the data support the hypothesis that certain benzene metabolites can induce DNA damage through the production of oxygen radicals; they further suggest that other metabolites may act, via another mechanism, to damage DNA.

Effect of pyran copolymer on activation of murine macrophages: evidence for incomplete activation by use of functional markers.

The degree of activation of peritoneal macrophages elicited by pyran copolymer (MVE-2) was studied in C57BL/6J mice. When cytotoxicity was examined under endotoxin-free culture conditions, the pyran-elicited macrophages could not complete cytolysis of tumor target cells. The macrophages, however, completed cytolysis when pulsed with endotoxin. These results were obtained when either the interval between injection of the pyran copolymer and harvest of the macrophage or the dose of pyran was varied. The pyran-elicited macrophages expressed five markers considered to be typical of inflammatory macrophages, and bound tumor cells to an augmented degree. The pyran-elicited macrophages were capable of secreting a potent cytolytic proteinase when pulsed with endotoxin, but did not secrete cytolytic proteinase spontaneously. The pyran-elicited macrophages, in contrast to inflammatory macrophages, could effect cytostasis of tumor cells; their cytostatic potential was also augmented by addition of endotoxin. Taken together, the results indicated that pyran copolymer elicits primed but not fully activated murine macrophages.

Effects of TNF alpha on the expression of class II MHC molecules in macrophages induced by IFN gamma: evidence for suppression at the level of transcription.

Tumor necrosis factor-alpha (TNF-alpha) induces surface expression of class II major histocompatibility (MHC) molecules (la molecules) in many cells, including macrophage-like cell lines. When we tested the effects of this cytokine on murine peritoneal macrophages, TNF alpha had little effect on surface expression of la. The strong expression of such molecules induced by interferon-gamma (IFN gamma) was, however, suppressed moderately by TNF alpha. These effects were reflected at the level of specific messenger RNA (mRNA) as detected by Northern blot analysis. Furthermore, the locus of control appears to be transcriptional; in nuclear run-on assays, TNF alpha suppressed the IFN gamma-induced enhancement of transcription for the murine beta-chain of I-A (I-A beta.). Taken together the data suggest that TNF alpha has little effect on class II MHC genes and surface expression in murine peritoneal macrophages, that TNF alpha is a modest suppressant of such molecules when their levels are raised by IFN gamma, and that these suppressive effects are mediated at the level of transcription.

Selective sensitivity of macrophages to cytotoxicity by inhibitors of macromolecular synthesis: induction of apoptosis.

Initial studies designed to measure the effect of inhibiting RNA synthesis by dactinomycin on macrophage functions revealed that the cells were uniformly killed at concentrations that have been routinely used to inhibit RNA synthesis in other cell types. We, thus, determined the dose curve for the cytotoxicity of dactinomycin for macrophages and two other cell types, L929 cells and splenic lymphocytes. Macrophages were extremely sensitive to the cytotoxicity of dactinomycin compared to the other cell types. Submicromolar concentrations that induced 100% cytotoxicity in macrophages caused little death in L929 cells or lymphocytes. Concentrations of dactinomycin that inhibited RNA synthesis by 40% in macrophages induced almost complete cell death but inhibition of over 80% of RNA synthesis in L929 cells or lymphocytes induced no measurable cytotoxicity. Macrophages did take up more dactinomycin than other cells but the amount was not sufficient to account for the large differences in cytotoxicity. We next tested the effects of doxorubicin and cycloheximide and found that macrophages were also extremely sensitive to killing by these compounds, and there was a very close association between the amount of inhibition of protein synthesis and the amount of toxicity. The morphology of macrophages exposed to these agents was consistent with death by apoptosis. This was further supported by assays measuring membrane integrity and DNA fragmentation. These data demonstrate that inhibition of macromolecular synthesis in macrophages, by different mechanisms, causes macrophages to undergo apoptosis. They further suggest that, in contrast to other cell types that require protein synthesis for apoptosis, macrophages require the synthesis of certain proteins to avoid apoptosis.

Gene regulation in macrophage activation: differential regulation of genes encoding for tumor necrosis factor, interleukin-1, JE, and KC by interferon-gamma and lipopolysaccharide.

Although macrophage activation is induced in a complex manner by signals such as interferon-gamma (IFN gamma) and bacterial lipopolysaccharide (LPS) and depends on alterations in levels of specific proteins due to differences in gene expression, the complexity of gene regulation during macrophage activation in regard to multiple signals is not fully appreciated. To probe this question, we selected four model genes encoding for tumor necrosis factor (TNF), interleukin-1 (IL-1), and the immediate early genes JE and KC. After analyses of Northern blots for specific mRNA, LPS was found to enhance levels of mRNA for IL-1, TNF, JE, and KC. IFN gamma initiated heightened mRNA levels for JE but did not alter specific mRNA for IL-1, TNF, or KC. When IFN gamma and LPS were combined, additive effects on levels of specific mRNA for JE, enhancement of mRNA for TNF, suppressed mRNA for KC, and no effect on mRNA for IL-1 were observed. When transcription of these genes was assessed by nuclear "run on" experiments, LPS increased transcription of KC and TNF but not of IL-1 or JE, implying that the increased levels of mRNA for JE and IL-1 were attributable to increased stability of mRNA. Likewise, IFN gamma did not initiate transcription of JE. When IFN gamma and LPS were given together, IFN gamma enhanced the LPS-induced transcription of TNF and KC, suggesting decreased stability of mRNA for KC. A distinct pattern of regulation for each of the four genes was thus observed. Taken together, the data suggest that gene regulation in macrophage activation represents a complex response of enhanced and suppressed transcription and mRNA stability, the precise pattern of which depends on the stimuli given to the macrophages and the gene examined.

Effects of oxidized LDL on mononuclear phagocytes: inhibition of induction of four inflammatory cytokine gene RNAs, release of NO, and cytolysis of tumor cells.

A critical step in development of atherosclerosis is the interaction of oxidized low-density lipoprotein (LDL) with mononuclear phagocytes. Oxidized LDL, as well as acetyl-LDL, is rapidly taken up into macrophages via a family of scavenger receptors. We report that macrophages treated with oxidized LDL have markedly lower levels of mRNA specific for the genes MCP-1, TNF-alpha, IL-1 alpha, and KC as measured by Northern blot analyses of lipopolysaccharide (LPS)-stimulated macrophages. By contrast, acetyl-LDL does not inhibit these genes at the doses at which oxidized-LDL is effective. Similar effects are observed whether the LDL is oxidized in the presence of Cu2+ or of Fe2+. Such inhibition also occurs when maleylated bovine serum albumin (BSA), which also clears by one or more scavenger receptors on macrophages, is used as the stimulant. Fe2+ or Cu2+ oxidized LDL inhibits release of nitric oxide when triggered by LPS and direct cytolysis of tumor cells when triggered by maleylated BSA or LPS. Taken together, the data presented indicate that oxidized LDL inhibits induction of several important gene RNAs as well as functional markers that characterize the development of inflammatory and fully activated macrophages.

Maleylated-BSA induces hydrolysis of PIP2, fluxes of Ca2+, NF-kappaB binding, and transcription of the TNF-alpha gene in murine macrophages.

The interaction of altered lipids or proteins with the several scavenger receptors (SR) on macrophages can lead to disparate results in both gene expression and cell function. However, the molecular bases of signaling induced by SR ligation have remained obscure. Here we report that maleylated-bovine serum albumin (maleyl-BSA) binds a low-affinity SR, initiating PIP2 hydrolysis, [Ca2+]i spikes, phospholipase A2 (PLA2) activation, nuclear factor-kappa(B) (NF-kappa(B)) binding to its cognate nucleotide and tumor necrosis factor alpha (TNF-alpha) gene transcription. We recently reported that oxidized low-density lipoprotein (ox-LDL), which binds another macrophage SR, induced pertussis-toxin-sensitive hydrolysis of PIP2 and elevations in [Ca2+]i [J. Biol. Chem. 270, 3475-3478, 1995]. By contrast, maleyl-BSA-initiated events were not pertussis toxin-sensitive and produced less [Ca2+]i spiking than ox-LDL. Furthermore, maleyl-BSA led to binding of NF-kappa(B) to its cognate nucleotide and TNF-alpha gene transcription, whereas ox-LDL suppressed these events. Collectively, this data suggests that maleyl-BSA and ox-LDL bind to distinct SR on murine macrophages, initiate distinct signal transduction pathways, and produce different functional effects.

A simple, sensitive assay for determining DNA in mononuclear phagocytes and other leukocytes.

An assay for determining the DNA content of mononuclear phagocytes is described. The assay is efficient and very sensitive, measuring as little as 1 microgram of DNA. Content of DNA is a linear function of the number of mononuclear phagocytes in the sample. One million murine macrophages contain 10.1 +/- 0.36 microgram of DNA. Consequently, samples of as few as 100,000 macrophages can be accurately quantified to be related to other biochemical analyses.

Incidence and nature of primary granulomatous inflammation in surgically removed material.

A large number of cases (303) of primary granulomatous inflammation, present in surgically removed specimens from a broad range of locations, were reviewed and examined in detail. The granulomas were characterized as to morphology, location, and etiology. Epithelioid granulomas without necrosis were most frequently due to sarcoidosis and mycobacterial infection, while epithelioid granulomas with necrosis were most commonly due to mycobacterial infection, fungal infection, rheumatoid arthritis and sarcoidosis. Mature granulomas, most of which did not contain necrosis, were generally due to foreign bodies. The responsible etiologic agents were generally identified in granulomas due to fungi, bacteria, or foreign bodies (92%). However, mycobacteria were infrequently identified in granulomas, even when the lesions were examined by the auramine-O technique (31%). Overall, the majority of the granulomas (76%), were due to five causes: sarcoidosis, mycobacterial infection, particulate inclusions, fungal infection, and rheumatoid arthritis. The frequency of a given etiology, however, varied widely depending on the location of the lesion. By classifying granulomas morphologically and knowing their location, useful predictive information concerning the etiology of a given granuloma, beyond that obtained by histochemical stains, could be derived.

Inflammation, oxidative DNA damage, and carcinogenesis.

Inflammation has long been associated with carcinogenesis, especially in the promotion phase. The mechanism of action of the potent inflammatory agent and skin promoter 12-tetradecanoyl phorbol-13-acetate (TPA) is unknown. It is thought that TPA selectively enhances the growth of initiated cells, and during this process, initiated cells progress to the preneoplastic state and eventually to the malignant phenotype. Many studies support the multistep nature of carcinogenesis, and a significant amount of evidence indicates that more than one genetic event is necessary for neoplastic transformation. Selective growth stimulation of initiated cells by TPA does not explain how further genetic events may occur by chronic exposure to this nongenotoxic agent. We and others have proposed that TPA may work, in part, by inciting inflammation and stimulating inflammatory cells to release powerful oxidants which then induce DNA damage in epidermal cells. Macrophages cocultured with target cells and TPA induce oxidized thymine bases in the target cells. This process is inhibited by both catalase and inhibitors of lipoxygenases, suggesting the involvement of both H2O2 and oxidized lipid products. Furthermore, macrophage populations that release both H2O2 and metabolites of arachidonic acid (AA) are more efficient at inducing oxidative DNA damage in surrounding cells than populations which only release H2O2 or metabolites of AA. In vivo studies demonstrated that SENCAR mice, which are sensitive to promotion by TPA, have a more intense inflammatory reaction in skin than C57LB/6 mice, which are resistant to promotion by TPA. In addition, macrophages from SENCAR mice release more H2O2 and metabolites of AA, and induce more oxidative DNA damage in cocultured cells than macrophages from C57LB/6 mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Simple and sensitive assay employing stable reagents for quantification of plasminogen activator.

A simple and sensitive indirect assay for quantifying plasminogen activator using [3H]-casein as substrate is described. The assay has been used to measure plasminogen activators from various sources including bacteria, cultured cells, and human plasma. The assay is linear with respect to concentration of plasminogen activator and time of incubation and is independent of concentration of plasminogen. The assay can routinely be used to quantify as few as 8 X 10(-3) international units ml-1 of streptokinase. Results obtained with the [3H]-casein assay correlate well with those obtained by the fibrin plate method (R = 0.83 by Spearman's ranked co-efficient of correlation). The reagents are extremely stable and easily stored. The ease with which the assay can be performed gives the clinical laboratory the potential to test large numbers of patients upon short notice and within a short period of time.

Divalent cation requirements for mounting a respiratory burst in response to phorbol diesters by macrophages from SENCAR and C57BL/6 mice.

Oxygen radicals are thought to play an important role in the promotion phase of carcinogenesis and the action of phorbol esters. Inflammatory cells are an abundant source of reactive oxygen intermediates (ROI) in the body and release large quantities of ROI when exposed to phorbol esters. Both protein kinase C (PKC), the receptor for phorbol esters, and the NADPH oxidase which generates ROI are Ca2+- and Mg2+-dependent. We investigated the requirements for Ca2+ and Mg2+ of macrophages from strains of mice sensitive and resistant to the promotion of tumors by phorbol esters. Macrophages from SENCAR mice, which are sensitive to phorbol ester promotion, required much lower levels of Ca2+ or Mg2+ to mount a full respiratory burst, as measured by the release of H2O2 in response to phorbol ester stimulation, than macrophages from C57BL/6 mice, which are resistant to promotion by phorbol esters. Conversely, when the particulate stimulus zymosan was used, there was little difference between macrophages from the two strains in requirements for Ca2+ and Mg2+ to release H2O2. Lowering the concentration of either cation in the absence of the other was more inhibitory than in the presence of the other cation. The studies demonstrate that differences in sensitivity to divalent cations by macrophages from these two strains is selective for phorbol ester stimulation and that lower requirements for Ca2+ and Mg2+ for ROI release correlates with sensitivity to the promotion of tumors by phorbol esters.