Evidence of Reduced CBG Cleavage in Abdominal Obesity: A Potential Factor in Development of the Metabolic Syndrome.

Corticosteroid-binding globulin (CBG) is involved in the regulation of cortisol delivery. Neutrophil elastase-mediated cleavage of high to low affinity CBG (haCBG to laCBG) induces cortisol release at inflammatory sites. Past studies have shown reduced CBG in obesity, an inflammatory state, particularly in central adiposity/metabolic syndrome. We performed an observational, cross-sectional study of the effects of obesity, age and sex on ha/laCBG in 100 healthy volunteers. Total and haCBG levels were 11% higher in women but did not vary with age or menopausal status. Total CBG levels were lower with increased body weight and waist circumference; laCBG levels were lower with increased body weight, waist circumference, body mass index and body fat; higher haCBG levels were seen with increased body fat. The relation between CBG and adiposity appeared to be driven predominantly by the metabolic syndrome group. The results suggest reduced CBG cleavage in central obesity, possibly contributing to the characteristic inflammatory phenotype of the central obesity and metabolic syndrome. The mechanism of gender differences in CBG levels is unclear.

Corticosteroid-Binding Globulin: A Review of Basic and Clinical Advances.

Corticosteroid-binding globulin (CBG, transcortin) is the primary cortisol binding protein. It is a non-inhibitory serine protease inhibitor, capable of conformational change from a high cortisol-binding affinity form to a low affinity form upon cleavage of its reactive centre loop by various proteases, such as neutrophil elastase. The burgeoning inflammatory role of CBG applies to acute, severe inflammation where depletion is associated with mortality, and to chronic inflammation where defects in cortisol delivery may perpetuate inflammation. Naturally occurring human mutations influence a wide range of CBG properties and point toward a role in hitherto unexplained chronic musculoskeletal pain and fatigue disorders as well as potentially affecting fertility outcomes including offspring gender. In vitro and knock-out animal models of CBG propose a role for CBG in cortisol transport to the brain, providing a foundation for understanding the human observations in those with CBG mutations and sex differences in stress-related mood and behaviour. Finally, CBG measurement has a practical role in the estimation of free cortisol, useful in clinical circumstances where CBG levels or cortisol binding affinity is reduced. Taken together, novel data suggest a role for cortisol in targeted cortisol delivery, with implications in acute and chronic inflammation, as well as roles in metabolism and neurocognitive function, implying that CBG is a multifaceted component in the mechanisms of hypothalamic-pituitary-adrenal axis related homeostasis.

Depletion of high-affinity corticosteroid-binding globulin corresponds to illness severity in sepsis and septic shock; clinical implications.

OBJECTIVE: Corticosteroid-binding globulin (CBG) is cleaved by neutrophil elastase converting the high-affinity (haCBG) conformation of CBG to a low-affinity (laCBG) conformation with a ninefold reduced cortisol-binding affinity. These in vitro data suggest that cortisol release by CBG cleavage results in the targeted delivery of cortisol to areas of inflammation. Our objective was to determine whether CBG cleavage alters circulating levels of haCBG and laCBG in vivo in proportion to sepsis severity. DESIGN: Prospective, observational cohort study in an adult tertiary level Intensive Care Unit in Adelaide, Australia. PATIENTS: Thirty-three patients with sepsis or septic shock grouped by illness severity [sepsis, septic shock survivors, septic shock nonsurvivors and other shock]. MEASUREMENTS: Plasma levels of haCBG and laCBG were assessed using a recently developed in-house assay in patients. Plasma total and free cortisol levels were also measured. RESULTS: Plasma total CBG and haCBG levels fell significantly, in proportion to disease severity (P < 0·0001 for both). There was a nonsignificant increase in free and total cortisol as illness severity worsened (P = 0·19 and P = 0·39, respectively). Illness severity was better correlated with haCBG levels than either free or total cortisol levels. CONCLUSIONS: Increasing illness severity in sepsis and septic shock is associated with markedly reduced circulating haCBG concentrations in vivo. We propose that low levels of haCBG in chronic inflammation may limit the availability of cortisol to inflammatory sites, perpetuating the inflammatory process.

Assessing the Effectiveness of Selected Biomarkers in the Acute and Cumulative Physiological Stress Response in Professional Rugby Union through Non-invasive Assessment.

Rugby union is a sport involving high force and frequency impacts making the likelihood of injury a significant risk. The aim of this study was to measure and report the individual and group acute and cumulative physiological stress response during 3 professional rugby games through non-invasive sampling. 24 professional rugby players volunteered for the study. Urine and saliva samples were collected pre and post 3 matches. Myoglobin, salivary immunoglobulin A, cortisol, neopterin and total neopterin (neopterin+7,8-dihydroneopterin) were analysed by high performance liquid chromatography or enzyme linked immunosorbent assay. Significant increases in cortisol, myoglobin, neopterin and total neopterin when urine volume was corrected with specific gravity were observed (p<0.05). Significant decreases in salivary immunoglobulin A concentration were observed for games 1 and 2 while secretion rate decreased after games 2 and 3. Significant decreases were seen with the percent of 7,8-dihydroneopterin being converted to neopterin following games 2 and 3. The intensity of 3 professional rugby games was sufficient to elicit significant changes in the physiological markers selected for our study. Furthermore, results suggest the selected markers not only provide a means for analysing the stress encountered during a single game of rugby but also highlight the unique pattern of response for each individual player.

Characteristics of plasma NOx levels in severe sepsis: high interindividual variability and correlation with illness severity, but lack of correlation with cortisol levels.

Objectives Nitric oxide (NO) concentrations are elevated in sepsis and their vasodilatory action may contribute to the development of hyperdynamic circulatory failure. Hydrocortisone infusion has been reported to reduce nitric oxide metabolite (NOx) concentrations and facilitate vasopressor withdrawal in septic shock. Our aim was to determine whether NOx concentrations relate to (i) protocol-driven vasopressor initiation and withdrawal and (ii) plasma cortisol concentrations, from endogenous and exogenous sources. Demonstration of a relation between NOx, cortisol and vasopressor requirement may provide an impetus towards the study of hydrocortisone-mediated NOx suppression as a tool in sepsis management. Design A prospective study of 62 patients with severe sepsis admitted to the intensive care unit. Measurements Plasma NOx, total and free cortisol, and corticosteroid-binding globulin (CBG) concentrations were measured and related to protocol-driven vasopressor use for 7 days following admission. Results Patients who developed septic shock (n = 35) had higher plasma NOx, total and free cortisol, and lower CBG concentrations than the nonseptic shock group (n = 27). Cortisol, CBG and NOx concentrations correlated with illness severity. Free cortisol, and to a lesser extent total cortisol, but not NOx concentrations, predicted septic shock. NOx concentrations were higher in nonsurvivors, and the concentrations were characteristically stable within individuals but marked interindividual differences were only partly accounted for by illness severity or renal dysfunction. NOx concentrations did not correlate with cortisol, did not relate to vasopressor requirement and did not fall after standard dose hydrocortisone, given for clinical indications. Conclusions Nitric oxide production increased with sepsis severity but did not correlate with plasma cortisol or vasopressor requirement. NOx levels were not suppressed reproducibly by hydrocortisone. High interindividual variability of NOx levels suggests that absolute NOx levels may not be a suitable target for individualized hydrocortisone therapy.

Corticosteroid-binding globulin, cortisol, free cortisol, and sex hormone-binding globulin responses following oral glucose challenge in spinal cord-injured and able-bodied men.

Circulating cortisol, corticosteroid-binding globulin, and sex hormone-binding globulin were measured retrospectively in plasma samples following the oral glucose tolerance test in 20 spinal cord-injured men and 20 able-bodied controls. Plasma-free cortisol responses attenuated more rapidly in the able-bodied men, compared to spinal cord-injured subjects, due to significant rise in circulating corticosteroid-binding globulin whereas changes in total plasma cortisol were similar in both groups. The changes in plasma-free cortisol in both groups paralleled changes in insulin and glucose and show that spinal cord-injured men had heightened exposure to free cortisol during this dynamic test. This raises the possibility that the mechanism of abdominal obesity and the propensity towards insulin resistance in spinal cord-injured men could be subtly mediated by perturbations in free cortisol. There were no significant changes in plasma sex hormone-binding globulin in either group.

Plasma sex hormone-binding globulin, corticosteroid-binding globulin, cortisol, and free cortisol levels in outpatients attending a lipid disorders clinic: a cross-sectional study of 1137 subjects.

We measured plasma sex hormone-binding globulin (SHBG), corticosteroid-binding globulin (CBG), and total cortisol, and calculated free plasma cortisol in 1 137 subjects attending a hospital outpatient lipid disorders clinic to investigate whether or not these analytes correlated with the degree of insulin resistance and the presence of the metabolic syndrome. In both males and females, plasma SHBG correlated inversely with anthropometric measures and with fasting glucose, insulin, insulin resistance, and triglycerides, and positively with HDL-cholesterol. However, in males with the metabolic syndrome, unlike females, the relationship between SHBG, some anthropometric measures, fasting glucose, insulin, and HDL-cholesterol were lost, which suggests that in males SHBG may not co-cluster with other components of the metabolic syndrome. In males and males with the metabolic syndrome, total plasma cortisol and calculated plasma free cortisol correlated positively with fasting glucose. Corticosteroid-binding globulin correlated inversely with percentage body fat and positively with HDL-cholesterol in males with and without the metabolic syndrome. CBG correlated negatively with age in both sexes. Overall, the results confirm the finding that SHBG is a marker of insulin resistance in males and females and that SHBG is associated with fasting triglycerides in males with the metabolic syndrome. Importantly, SHBG could be considered a stronger component of the metabolic syndrome in females than in males. However, the aetiological role of CBG and cortisol in insulin resistance is uncertain, although in males, cortisol and CBG could be subtly related to the degree of insulin resistance.

Comparison of indices of insulin resistance with metabolic syndrome classifications to predict the development of impaired fasting glucose in overweight and obese subjects: a 3-year prospective study.

OBJECTIVE: To compare the ability of biochemical indices of insulin resistance (IR) with metabolic syndrome (MetS) classifications to predict changes in blood glucose control over a 3-year period in overweight and obese subjects. DESIGN: This was a longitudinal, prospective study, with data collected at baseline, 18 and 36 months. SUBJECTS AND METHODS: A total of 175 overweight (body mass index (BMI)>25 kg m(-2)) and obese (BMI>30 kg m(-2)) subjects were enrolled in the study. The IR indices assessed included fasting insulin concentration, the insulin/glucose-derived indices, homeostasis assessment model of insulin resistance (HOMA-IR) and quantitative insulin sensitivity check index (QUICKI), the insulin/triglyceride-derived McAuley index, plasma adiponectin concentration and the triglyceride (trig) and high-density lipoprotein (HDL)-cholesterol ratio (trig:HDL). The two MetS classifications were assessed according to the definitions of the National Cholesterol Education Program-Third Adult Treatment Panel (NCEP-ATPIII) and the International Diabetes Federation (IDF). The potential of the IR indices and MetS classifications at baseline to predict the development of impaired fasting glucose (IFG) was examined using receiver-operator characteristic (ROC) curve analysis and analysis of variance. RESULTS: Complete data were collected on 158 subjects. In all, 51 (32%) subjects developed IFG during the study. The analysis of variance showed significant differences between the IFG and normoglycaemic group in the baseline values of the McAuley index, trig:HDL, plasma adiponectin concentration and prevalence of the MetS. The ROC curve analysis confirmed this result and showed that the strongest predictors of IFG were baseline trig:HDL and IDF MetS classification, followed in order by the McAuley index, plasma adiponectin concentration and NCEP-ATPIII MetS classification. In contrast, the baseline values of fasting insulin, HOMA-IR and QUICKI did not predict IFG. DISCUSSION: This study showed that the IR indices, derived, in part, from plasma triglyceride concentration, were sensitive predictors for the development of IFG in normoglycaemic overweight and obese subjects. Indices derived from glucose and insulin did not identify this at-risk group. The study also showed that the presence of MetS and its abnormalities of an increased trig:HDL ratio and low plasma adiponectin concentration were all sensitive predictors of IFG.

Antisense gene inhibition by oligonucleotides containing C-5 propyne pyrimidines.

Phosphorothioate oligodeoxynucleotides containing the C-5 propyne analogs of uridine and cytidine bind RNA with high affinity and are potent antisense inhibitors of gene expression. In a cellular assay, gene-specific antisense inhibition occurred at nanomolar concentrations of oligonucleotide, was dose-dependent and exquisitely sensitive to sequence mismatches, and was correlated with the melting temperature and length of oligonucleotide. Activity was independent of RNA target site and cell type but was detectable only when the oligonucleotides were microinjected or delivered with cell-permeabilizing agents. These oligonucleotides may have important applications in therapy and in studies of gene function.

Plasma levels of sex hormone-binding globulin, corticosteroid-binding globulin and cortisol in overweight subjects who develop impaired fasting glucose: a 3-year prospective study.

Circulating sex hormone-binding globulin (SHBG), corticosteroid-binding globulin (CBG), and total and calculated free cortisol were measured in 206 overweight subjects to investigate whether or not they were markers of insulin resistance. Measurements were carried out on two occasions 36 months apart and subjects were grouped according to fasting plasma glucose. Fifty-one subjects, with a normal basal fasting glucose (<5.6 mmol/l) developed impaired fasting glucose 3 years later (> or = 5.6 mmol/l). Analysis either in toto or based on gender showed a highly significant increase in fasting insulin and insulin resistance, a modest increase in body mass index (BMI), but importantly no change in plasma SHBG, CBG, or cortisol concentrations. Subjects (n=101) with a normal fasting glucose both at baseline (<5.6 mmol/l) and at 36 months showed no significant change in fasting insulin, insulin resistance, SHBG, CBG, cortisol, or BMI. Cross-sectional analysis of the study population showed that plasma SHBG correlated negatively with insulin resistance both in men and women. Overall SHBG at baseline was not predictive of changes in fasting glucose. In females, plasma CBG correlated negatively with BMI. The major finding is that overweight subjects who developed impaired fasting glucose showed no significant change in plasma SHBG, CBG or cortisol, and therefore these indices are probably not early markers of insulin resistance in overweight subjects.

Septic shock and sepsis: a comparison of total and free plasma cortisol levels.

CONTEXT: Severe systemic infection leads to hypercortisolism. Reduced cortisol binding proteins may accentuate the free cortisol elevations seen in systemic infection. Recently, low total cortisol increments after tetracosactrin have been associated with increased mortality and hemodynamic responsiveness to exogenous hydrocortisone in septic shock (SS), a phenomenon termed by some investigators as relative adrenal insufficiency (RAI). HYPOTHESIS: Free plasma cortisol may correspond more closely to illness severity than total cortisol, comparing SS and sepsis (S). DESIGN: This was a prospective study. SETTING: This study took place in a tertiary teaching hospital. PATIENTS: Patients had SS (n = 45) or S (n = 19) or were healthy controls (HCs; n = 10). AIM: The aim of the study was to compare total with free cortisol, measured directly and estimated by Coolens' method, corticosteroid-binding globulin (CBG), and albumin in patients with SS (with and without RAI) and S during acute illness, recovery, and convalescence. RESULTS: Comparing SS, S, and HC subjects, free cortisol levels reflected illness severity more closely than total cortisol (basal free cortisol, SS, 186 vs. S, 29 vs. HC, 13 nmol/liter, P < 0.001 compared with basal total cortisol, SS, 880 vs. S, 417 vs. HC, 352 nmol/liter, P < 0.001). Stimulated free cortisol increments varied greatly with illness category (SS, 192 vs. S, 115 vs. HC, 59 nmol/liter, P = 0.004), whereas total cortisol increments did not (SS, 474 vs. S, 576 vs. HC, 524 nmol/liter, P = 0.013). The lack of increase in total cortisol with illness severity is due to lower CBG and albumin. One third of patients with SS (15 of 45) but no S patients met a recently described criterion for RAI (total cortisol increment after tetracosactrin < or = 248 nmol/liter). RAI patients had higher basal total cortisol (1157 vs. 756 nmol/liter; P = 0.028) and basal free cortisol (287 vs. 140 nmol/liter; P = 0.017) than non-RAI patients. Mean cortisol increments in RAI were lower (total, 99 vs. 648 nmol/liter, P < 0.001; free, 59 vs. 252 nmol/liter, P < 0.001). These differences were not due to altered CBG or albumin levels. Free cortisol levels normalized more promptly than total cortisol in convalescence. Calculated free cortisol by Coolens' method compared closely with measured free cortisol. CONCLUSIONS: Free cortisol is likely to be a better guide to cortisolemia in systemic infection because it corresponds more closely to illness severity. The attenuated cortisol increment after tetracosactrin in RAI is not due to low cortisol-binding proteins. Free cortisol levels can be determined reliably using total cortisol and CBG levels.

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.

Cell specificity in hepatocarcinogenesis: preferential accumulation of O6-methylguanine in target cell DNA during continuous exposure to rats to 1,2-dimethylhydrazine.

Concentrations of the major methylated DNA purines were determined in two liver cell cell populations of Fischer 344 rats during administration of 30 ppm 1,2-dimethylhydrazine (SDMH) in the drinking water for periods of up to 4 weeks. Quantitation of 7-methylguanine and O6-methylguanine (O6MG) was achieved by highly sensitive optical methods following separation of DNA bases by high-performance liquid chromatography. Overall alkylation as indicated by the concentration of 7-methylguanine was near maximum in both hepatocytes and liver nonparenchymal cells by the third day of SDMH administration. Similar amounts of 7-methylguanine were present in the two liver cell populations at seven of nine time points during 4 weeks of exposure. In contrast, dramatic differences in the cumulative concentrations of O6MG were seen in the two cell populations. Nonparenchymal cells accumulated O6MG during the first 8 days of exposure and had up to a 30-fold greater concentration of this product than did the corresponding hepatocytes. In the hepatocytes, a rapid decline in O6MG concentration was observed between 1 and 3 days of exposure to SDMH. Thereafter, only low levels of this promutagenic lesion were present in hepatocytes. Exposure of rats to the same regimen of SDMH for up to 10 months caused angiosarcomas in the livers of over 90% of the animals, while hepatocellular carcinomas occurred in only 40%. Thus, a strong correlation exists between the inability to repair O6MG and cell specificity for carcinogenesis.

Circulating levels of isoflavones and markers of 5alpha-reductase activity are higher in Japanese compared with New Zealand males: what is the role of circulating steroids in prostate disease?

Epidemiological evidence implicates dietary isoflavone intake as protective against prostate disease. A putative mechanism is attenuated circulating androgen levels in male populations consuming an isoflavone rich diet. We investigated this hypothesis by collecting plasma from 60 Japanese and 60 New Zealand males aged between 21 and 31 years each consuming their traditional diets. We measured plasma testosterone, dihydrotestosterone (DHT), androstenedione, dehydroepiandrosterone sulfate (DHEAS), the combined levels of androsterone sulfate and epiandrosterone sulfate (AoS/epiAoS), sex hormone-binding globulin, and cortisol and corticosteroid-binding globulin as well as the isoflavones genistein and equol. Plasma genistein and equol levels were several times higher in Japanese males as would be expected from an isoflavone rich diet. However, androstenedione, DHEAS, calculated free testosterone and paradoxically markers of 5alpha-reductase, DHT and AoS/epiAoS were all also significantly higher in Japanese rather than the New Zealand male counterparts. All other comparisons were not significant. Plasma DHT and DHEAS correlated positively with plasma equol and plasma AoS/epiAoS correlated positively with genistein levels. Taken together the results suggest that, rather than reduced levels of steroidogenesis, Japanese males may have increased 5alpha-reductase activity and possibly altered 17beta OH steroid dehydrogenase activity. Significantly the positive association between isoflavones levels and 5alpha-steroids is counter-intuitive to isoflavone intake offering prostate protection, unless this is postulated to occur through other mechanisms.

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.

Exposure of cultured murine peritoneal macrophages to low concentrations of beryllium induces increases in intracellular calcium concentrations and stimulates DNA synthesis.

Exposure of humans to beryllium dusts can induce a specific form of chronic pneumonitis that consists mainly of noncaseating granulomas in the lungs. Multiple studies have documented both genetic and immune components of chronic berylliosis. Much work has focused on T cells and their reactivity in berylliosis, but less work has focused on the end effector cells in granulomatous inflammation, macrophages. Because macrophages must become activated to form granulomas, and they become activated by responding to numerous immunomodulatory signals, we investigated the effects of beryllium (BeCl2) on a central signal transduction pathway in macrophages, increases in intracellular calcium ([Ca2+]i). Exposure of cultured murine peritoneal macrophages to low, nontoxic concentrations induced successive spikes or oscillations in [Ca2+]i. Concentrations as low as 5 nM induced significant increases in [Ca2+]i. The source of the increased [Ca2+]i was exclusively extracellular in that increases in [Ca2+]i could be completely blocked by chelating extracellular Ca2+, were inhibited by the Ca2+ channel blocker verapamil, and exposure of macrophages to BeCl2 had no effect on IP3 concentrations. DNA synthesis, a Ca2+-sensitive function, was enhanced in dividing 1LN cells and induced de novo in quiescent macrophages. Furthermore, BeCl2 enhanced DNA synthesis in the absence of coexposure to the protein kinase C activator phorbol myristate acetate. These data support the hypothesis that beryllium toxicity is in part the result of altered Ca2+ metabolism in mononuclear phagocytes consequent to reversible opening of plasma membrane channels.

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.