James G. Lewis

Serotonin-Related Gene Polymorphisms and Central Nervous System Serotonin Function *

Central nervous system (CNS) serotonergic function affects a wide range of biological and behavioral functions affecting health and disease. Our objective in this study was to determine whether functional polymorphisms of the genes that encode for the serotonin transporter promoter (5HTTLPR) and monoamine oxidase A (MAOA-uVNTR) are associated with CNS serotonin turnover—indexed by cerebrospinal fluid levels of 5-hydroxyindoleacetic acid (5-HIAA)—in a community sample of healthy adults. Subjects were 165 community volunteers without current medical or psychiatric illness, stratified with respect to ethnicity, gender, and socioeconomic status who underwent inpatient evaluation in the General Clinical Research Center of a university medical center. A significant ethnicity×genotype interaction (P=0.008) indicated that, compared to the long/long and long/short genotypes, the 5HTTLPR short/short genotype was associated with higher CSF 5-HIAA levels in African Americans, but with lower levels in Caucasians. A gender×genotype interaction (P=0.04) indicated that 5HTTLPR short/short genotype was associated with higher 5-HIAA levels in women but with lower levels in men. MAOA-uVNTR 3.5 and 4 repeat alleles were associated with higher 5-HIAA (P=0.03) levels in men, but were unrelated to 5-HIAA levels in women. These findings suggest that effects of serotonin-related gene polymorphisms on CNS serotonergic function vary as a function of both ethnicity and gender. Further research will be required to determine the mechanism(s) underlying these differential effects. In the meanwhile, both ethnicity and gender should be taken into account in research evaluating effects of these and related polymorphisms on CNS serotonergic function, as well as the broad range of biological and behavioral functions that are regulated by CNS serotonergic function.

Enhanced expression of cytokines and chemokines by blood monocytes to in vitro lipopolysaccharide stimulation are associated with hostility and severity of depressive symptoms in healthy women.

The current study investigated the relation of hostility and severity of depressive symptoms, separately and jointly, to the capacity of blood monocytes to secrete an array of cytokines when stimulated by bacterial lipopolysaccharide (LPS). Subjects were 44 healthy, non-smoking, premenopausal women (aged 23-49 years) not currently taking oral contraceptives. Data were collected during the follicular phase of the menstrual cycle. The Cook-Medley Hostility (Ho) scale and the Beck Depression Inventory (BDI) were used to assess hostility and severity of depressive symptoms, respectively. Dual-color flow cytometry was used to measure the total expression of interleukin (IL)-1alpha, IL-1beta, IL-8, tumor necrosis factor (TNF)-alpha, monocyte chemotactic protein (MCP)-1 and monocyte inflammatory protein (MIP)-1alpha in blood monocytes following 4 h in vitro LPS stimulation of whole blood. In analyses adjusting for age, body mass index (BMI), fasting cholesterol, alcohol use, race and 17beta-estradiol (E(2)), higher Ho scores were associated with greater LPS-stimulated expression of IL-1alpha (beta = 0.033, p = 0.02), IL-8 (beta = 0.046, p = 0.01) and IL-1beta (beta = 0.024, p = 0.06). Higher BDI scores were associated with greater expression of TNF-alpha (beta = 0.042, p = 0.02) and IL-8 (beta = 0.045, p = 0.04). The linear combination of Ho and BDI scores was significantly associated with IL-1beta (beta = 0.18, p = 0.057), IL-8 (beta = 0.36, p = 0.01), TNF-alpha (beta = 0.25, p = 0.03), and IL-1alpha (beta = 0.18, p < 0.07). Thus, in healthy women, these psychological risk factors, alone and in combination, induce a proinflammatory phenotype in circulating monocytes characterized by the up-regulation of proinflammatory cytokines, supporting the hypothesis that inflammation may be a key pathway whereby hostility and depressive symptoms contribute to atherosclerosis and subsequent coronary heart disease (CHD).

Central nervous system serotonin function and cardiovascular responses to stress.

Objective The objective of this study was to evaluate the impact of indices of central nervous system (CNS) serotonin function on cardiovascular reactivity to mental stress. Methods Lumbar puncture was performed on 54 healthy volunteers to obtain cerebrospinal fluid (CSF) for determination of 5-hydroxyindoleacetic acid (5HIAA) levels. Genotypes were determined with respect to a functional polymorphism of the serotonin transporter gene promoter region (5HTTLPR). Subjects then underwent mental stress testing. Results Persons with one or two long (l) 5HTTLPR alleles had CSF levels of the major serotonin metabolite, 5HIAA, that were 50% higher than those of persons with the s/s 5HTTLPR genotype. Persons with one or two l alleles or higher CSF 5HIAA levels also exhibited greater blood pressure and heart rate responses to a mental stress protocol. Conclusions These findings suggest the 5HTTLPR polymorphism affects CNS serotonin function, and they are consistent with the general hypothesis that CNS serotonin function is involved in the regulation of potentially health-damaging biobehavioral characteristics. In particular, the l allele could contribute, through its association with increased cardiovascular reactivity to stress, to increased risk of cardiovascular disease.

The relation of aggression, hostility, and anger to lipopolysaccharide-stimulated tumor necrosis factor (TNF)-α by blood monocytes from normal men

Aggression, hostility, and anger significantly predict morbidity and mortality from atherosclerotic cardiovascular disease (ACVD). ACVD is believed to be an inflammatory disease characterized by increased expression of a number of proinflammatory cytokines, such as tumor necrosis factor (TNF)-alpha. This study examined the relation of aggression, hostility, and anger to monocyte-associated TNF-alpha expression following lipopolysaccharide (LPS) stimulation. Participants were 62 healthy, non-smoking men (aged 18-45 years). Hostility, anger, verbal, and physical aggression were assessed using the Buss-Perry aggression questionnaire (BPAQ). LPS-stimulated TNF-alpha expression was determined using dual-color flow cytometry gating for CD14(+) cells. After controlling for age, race, education, and alcohol use, scores on the hostility (p=.013), physical aggression (p=.010), and verbal aggression (p=.034) subscales, and the total score (p=.007) on the BPAQ were positively associated with LPS-stimulated TNF-alpha expression. The results suggest that hostility and aggression are associated with an increased expression of TNF-alpha, a cytokine implicated in ACVD.

Venous thrombosis in a family with defective release of vascular plasminogen activator and elevated plasma factor VIII/von Willebrand's factor

A family is described in which venous thrombosis developed in five members as early as 14 years of age. Routine coagulation studies, plasma antithrombin III, factor V, plasminogen, beta-thromboglobulin, fibrinopeptide A, prothrombin fragment F1+2, and thrombin-antithrombin III complex were all within normal limits. However, defective release of vascular plasminogen activator was observed on several occasions in all five subjects as compared with a control population of 125 persons (0.04 Committee on Thrombolytic Agents [CTA] units/ml plasma as compared with 0.21 CTS units/ml). In addition, levels of factor VII/von Willebrands factor were significantly elevated above the normal range in this pedigree.

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‐α, IL‐1α, 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. J. Leukoc. Biol. 57: 427–433; 1995.

Toxic effects of benzene and benzene metabolites on mononuclear phagocytes

Benzene is a potent bone marrow toxin in animals and man. Animal studies have shown that exposure to benzene can alter T lymphocyte functions and decrease the resistance of animals to Listeria monocytogenes and transplanted tumor cells. Mononuclear phagocytes participate in host resistance to Listeria and tumor cells. The purpose of the studies presented here was to determine the effects of benzene and benzene metabolites on macrophage functions and the ability of macrophages to be activated for functions which are important in host defense. Benzene had no effects on macrophage function or activation for any of the functions tested. Conversely, metabolites of benzene, catechol (CAT), hydroquinone (HQ), benzquinone (BQ), and 1,2,4-benzenetriol (BT) had potent and varied effects on macrophage function and activation. BQ inhibited the broadest range of functions including release of H2O2, Fc receptor-mediated phagocytosis, interferon gamma priming for tumor cell cytolysis, and bacterial lipopolysaccharide (LPS) triggering of cytolysis. BQ was also the most potent metabolite causing inhibition at lower concentrations than the other metabolites. HQ inhibited H2O2 release and priming for cytolysis and BT inhibited phagocytosis and priming for cytolysis. CAT only inhibited the release of H2O2. None of the compounds tested inhibited the induction of class II histocompatibility antigens on the cell surface. All of the effects measured occurred using concentrations of compounds which did not disrupt the cell integrity or inhibit general functions such as protein synthesis. Taken together these data suggest that benzene metabolites alter macrophage function through several mechanisms including inhibition of output enzymes and disruption of signal transduction systems.

Stress-induced changes in the expression of monocytic β2-integrins: The impact of arousal of negative affect and adrenergic responses to the Anger Recall Interview

Adhesion of circulating monocytes to the vascular endothelium is one of the earliest steps in the development of atherosclerosis. This leukocyte-to-endothelium interaction is mediated in part by beta2-integrins, a group of cell adhesion molecules that bind to endothelial ligands. Given the significance of this interaction to atherogenesis, we examined the effects of stress, operationalized as the arousal of negative affect (NA) and cardiovascular and catecholamine responses to the Anger Recall Interview (ARI), on the expression of LFA-1 (CD11a), Mac-1 (CD11b) and p150/95 (CD11c) on circulating monocytes (CD14+). Subjects were 173 healthy, nonsmoking men and women (60% men, 40% minorities, aged 18-49 year). Arousal of NA, cardiovascular responses (heart rate [HR], systolic blood pressure [SBP], diastolic blood pressure [DBP]), circulating catecholamines (epinephrine [Epi], norepinephrine [Ne]) and beta2-integrin (CD11/CD18) expression were determined prior to and following the ARI. The principal findings were that the ARI, on average, induced a decrease in monocyte expression of beta2-integrins. However, after adjusting for age, sex, body mass index, exercise status, and baseline level of beta2-integrin expression, those individuals who showed the largest increases in NA, Ne and DBP during the ARI showed an increase in monocyte beta2-integrin expression. Thus, heightened psychological and physiological stress responses induced phenotypic changes in monocytic expression of beta2-integrins that are consistent with the role of monocytes/macrophages in vascular inflammation and increased risk of atherosclerotic cardiovascular disease.

Fibrinolytic response and oral contraceptive associated thromboembolism

A case-control study of fibrinolytic activity was conducted comparing 12 women with a recent history of thromboembolism while taking oral contraceptives and 28 matched female controls without a history of thromboembolism. All subjects had stopped using oral contraceptives at least 12 months prior to study. A new assay, recently developed in this laboratory, was used to evaluate the mean fibrinolytic response to venous occlusion in both cases and controls. The fibrinolytic response of all subjects was stratified into quartiles as previously described. While the controls segregated as expected, all 12 cases occurred in subjects whose fibrinolytic response fell in the first two quartiles with 8 of the 12 subjects having first quartile responses. The mean fibrinolytic response for all controls was 12.3 units while cases showed a mean response of only 3.9 units. The above data supports and extends our recent suggestion that low fibrinolytic response may signal a natural predisposition to venous thromboembolism which could be triggered by use of synthetic estrogens. However, these data do not support the use of this assay as a screening test for oral contraceptive related risk to thromboembolism since at least 50% of the control population also segregate in the first two quarters.

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 macro phage 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, deter mined 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 in duced 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 in hibition of over 80% of RNA synthesis in L929 cells or lymphocytes induccd no measurable cytotoxicity. Macro phages 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 cyclohcximide and found that macro phages were also extremely sensitive to killing by these compounds, and there was a very close association be tween 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 syn thesis 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 synthe sis of certain proteins to avoid apoptosis. J. Leukoc. Biol. 57: 635–642; 1995.