Stephen B. Pruett

TLR4, Ethanol, and Lipid Rafts: A New Mechanism of Ethanol Action with Implications for other Receptor-Mediated Effects

Ethanol (EtOH) is the most widely abused substance in the United States, and it contributes to well-documented harmful (at high dosages) and beneficial (at low dosages) changes in inflammatory and immune responses. Lipid rafts have been implicated in the regulation and activation of several important receptor complexes in the immune system, including the TLR4 complex. Many questions remain about the precise mechanisms by which rafts regulate the assembly of these receptor complexes. Results summarized in this review indicate that EtOH acts by altering the LPS-induced redistribution of components of the TLR4 complex within the lipid raft and that this is related to changes in actin cytoskeleton rearrangement, receptor clustering, and subsequent signaling. EtOH provides an example of an immunomodulatory drug that acts at least in part by modifying lipid rafts, and it could represent a model to probe the relationships between rafts, receptor complexes, and signaling.

Evaluation of nitrite production by human monocyte-derived macrophages.

Reactive nitrogen intermediates are important in the anti-tumor and anti-microbial activities of rodent macrophages, but it is not known whether this is the case for human macrophages. In the present study, nitrite concentrations in vitro were used as an indicator of reactive nitrogen intermediate production by mouse, rat, and human macrophages. Human macrophages derived by culturing peripheral blood monocytes did not consistently produce detectable nitrite levels in response to any stimulus examined. Human macrophages were viable and metabolically active as indicated by the MTT assay, and their respiratory burst response to phorbol myristate acetate was increased following incubation with Interferon-gamma, as expected for typical macrophages. In contrast, rat or mouse peritoneal macrophages produced nitrite concentrations of approximately 20-100 microM in response to lipopolysaccharide, Interferon-gamma, or both. These results demonstrate substantial differences in the production of nitrites by rodent and human macrophages. Because of the heterogeneity among macrophage populations, these findings may not be applicable to all human macrophage populations, but they suggest a need for caution in extrapolating from rodent studies regarding the role of reactive nitrogen intermediates in anti-tumor or anti-microbial functions of human macrophages.

Quantifying the Relationship Between Multiple Immunological Parameters and Host Resistance: Probing the Limits of Reductionism

Although reductionist experimental designs are excellent for identifying cells, molecules, or functions involved in resistance to particular microbes or cancer cells, they do not provide an integrated, quantitative view of immune function. In the present study, mice were treated with either dexamethasone (DEX) or cyclosporin A (CyA), and immune function and host resistance were evaluated. Multivariate statistical methods were used to describe the relative importance of a broad range of immunological parameters for host resistance in mice treated with various dosages of DEX. Multiple regression and logistic regression analysis indicated that changes in 24 immunological parameters explained a substantial portion of the changes in resistance to B16F10 tumor cells or streptococcus group B. However, at least 40% of the change in host resistance remained unexplained. DEX at all dosages substantially suppressed numerous relevant immunological parameters, but significantly decreased resistance to Listeria monocytogenes only at the highest dosage. In contrast, CyA substantially decreased resistance to L. monocytogenes at dosages that caused relatively minor suppression of just a few immunological parameters (unfortunately, CyA data and host resistance data for L. monocytogenes were not suitable for multivariate analysis). These results illustrate that mathematical models can be used to explain changes in host resistance on the basis of changes in immune parameters, and that moderate changes in relevant immunological parameters may not produce the types of changes in host resistance expected on the basis of results from reductionist experimental designs.

Suppression of Innate Immunity by Acute Ethanol Administration: A Global Perspective and a New Mechanism Beginning with Inhibition of Signaling through TLR3

Excessive consumption of ethanol (EtOH) suppresses innate immunity, but the mechanisms have not been fully delineated. The present study was conducted to determine whether EtOH suppresses TLR signaling in vivo in mice and to characterize the downstream effects of such suppression. Degradation of IL-1R-associated kinase 1 induced by a TLR3 ligand in peritoneal cells (∼90% macrophages) was suppressed by EtOH. Phosphorylation of p38 kinase in peritoneal macrophages (F4/80+) was suppressed, as was nuclear translocation of p-c-Jun and p65 in peritoneal cells. EtOH decreased IL-6 and IL-12 (p40), but did not significantly affect IL-10 in peritoneal lavage fluid or in lysates of peritoneal cells. Changes in cytokine mRNAs (by RNase protection assay) in macrophages isolated by cell sorting or using Ficoll were generally consistent with changes in protein levels in cell lysates and peritoneal lavage fluid. Thus, suppression of TLR signaling and cytokine mRNA occurred in the same cells, and this suppression generally corresponded to changes in i.p. and intracellular cytokine concentrations. DNA microarray analysis revealed the suppression of an IFN-related amplification loop in peritoneal macrophages, associated with decreased expression of numerous innate immune effector genes (including cytokines and a chemokine also suppressed at the protein level). These results indicate that EtOH suppresses innate immunity at least in part by suppressing TLR3 signaling, suppressing an IFN-related amplification loop, and suppressing the induction of a wide range of innate effector molecules in addition to proinflammatory cytokines and chemokines.

Bovine immunodeficiency virus: incidence of infection in Mississippi dairy cattle

Bovine immunodeficiency virus (BIV), a lentivirus, was originally derived from a Holstein cow with persistent lymphocytosis and severe wasting. The virus is known to occur sporadically throughout the United States and perhaps across the globe, but epidemiological data concerning the incidence of BIV are meager and the virus was previously unreported in Mississippi animals. This study examined the seroepidemiology of BIV infection from two Mississippi dairy herds (Coastal Plains and MSU). Serology revealed a 38% incidence of BIV infection in Coastal Plains animals and a 58% incidence in MSU animals. A cumulative BIV seroprevalence of 50% was found in the Mississippi animals, and BIV seroprevalence increased with increasing age of the animals. Peripheral blood leukocytes of age matched BIV seropositive and seronegative animals were enumerated to assess any effect of BIV infection on leukocyte populations. No significant differences were found in total leukocyte populations or leukocyte subpopulations between BIV seropositive or seronegative animals. These data indicate that BIV infection is prevalent in Mississippi animals, but the role of BIV in bovine disease remains unclear.

Binge Ethanol and Liver: New Molecular Developments

Binge consumption of alcohol is an alarming global health problem. Binge (acute) ethanol (EtOH) is implicated in the pathophysiology of alcoholic liver disease (ALD). New studies from experimental animals and from humans indicate that binge EtOH has profound effects on immunological, signaling, and epigenetic parameters of the liver. This is in addition to the known metabolic effects of acute EtOH. Binge EtOH alters the levels of several cellular components and dramatically amplifies liver injury in chronically EtOH exposed liver. These studies highlight the importance of molecular investigations into binge effects of EtOH for a better understanding of ALD and also to develop therapeutic strategies to control it. This review summarizes these recent developments.

The role of chemical‐induced stress responses in immunosuppression: A review of quantitative associations and cause‐effect relationships between chemical‐induced stress responses and immunosuppression

Although there is an increasing awareness that drugs and chemicals can modulate the immune system by indirect mechanisms, few compounds have been thoroughly evaluated in this regard. Several environmentally relevant chemicals induce stresslike responses, as indicated by elevated glucocorticoid levels. Comparable glucocorticoid levels induced by physical or psychological stressors are consistently associated with suppression of one or more immunological parameters. Thus, it seems likely that stress-related neuroendocrine mechanisms are important in immunosuppression by some environmental chemicals. Distinguishing direct and indirect (stress-related) mechanisms of immunosuppression is generally possible, and this could be done as a routine part of immunotoxicity assessment. Although it is clear that glucocorticoids can contribute to such immunosuppression, it is also clear that several other neuroendocrine mediators associated with stress responses can be immunomodulatory. Thus, correlation between glucocorticoid levels and immunosuppression does not conclusively demonstrate a cause-effect relationship. Demonstrating such relationships has been difficult, but it has been done in a few cases of drug-induced thymic hypoplasia by monitoring several parameters known to be affected by glucocorticoids and by measuring the ability of a glucocorticoid antagonist (RU 486) or adrenalectomy to block changes in these parameters. A similar strategy might be useful for evaluation of the role of glucocorticoids in drug- or chemical-induced suppression of a variety of immune functions, but the effects of RU 486 on neuroendocrine feedback circuits and the possibility of consequent immunological changes must be considered when the data are interpreted. This approach could also be applied to evaluation of the roles in chemical-induced immunosuppression of other neuroendocrine mediators for which antagonists or agents that block the synthesis or release of the mediator are available. However, it is likely that a comprehensive (and perhaps predictive) understanding of the relationship between chemically induced stress responses and immunosuppression will require more detailed and quantitative elucidation of the mechanisms and regulation of neuroendocrine-immune interactions.

Effect of macrophage activation on killing of Listeria monocytogenes. Roles of reactive oxygen or nitrogen intermediates, rate of phagocytosis, and retention of bacteria in endosomes

The role of macrophage activation in the killing of L. monocytogenes is unclear. Some studies suggest that activation for enhanced production of reactive oxygen and nitrogen intermediates may not be of central importance. Recent data have indicated an important role for interferon‐gamma (IFN‐γ) induced retention of L. monocytogenes in endosomes. Data from the present study indicate that proteose peptone‐elicited macrophages from DBA2/J, CD‐1, and C3H/HeN mice are listericidal. Activation of these ceils in vitro or 20 h by IFN‐γ (20 or 500 U/ml) increased HM2O2 or nitrite production, but did not increase the number of L. monocytogenes killed during a subsequent 6‐h or 7‐h culture. Incubation of macrophages with IFN‐γ plus lipopolysaccharide (LPS) caused greater activation and increased the number of Listeria killed during a 6‐h or 7‐h culture. However, this seems primarily attributable to enhanced phagocytosis. Proteose peptone‐elicited macrophages were significantly more effective than resident macrophages in preventing the escape of L. monocytogenes from endosomes into the cytoplasm. This capability was not significantly enhanced by IFN‐γin vitro. but was enhanced by IFN‐γ plus LPS. This correlates well with the effects of these activation stimuli on killing of L. monocytogenes by proteose peptone‐elicited macrophages. These results indicate that enhanced retention of L. monocytogenes in endosomes is induced by proteose peptone elicitation and that further macrophage activation in vitro by IFN‐γ does not improve listericidal activity.

Immunotoxicological Characteristics of Sodium Methyldithiocarbamate

This study was conducted to assess immunotoxicological effects and selected general toxicological effects of sodium methyldithiocarbamate (SMD). Initially, the compound was administered orally to female B6C3F1 mice at 300 mg/kg/day for 3, 5, 10, or 14 days. Body, liver, kidney, spleen, and thymus weights were measured. Selected hematological and bone marrow parameters were examined. Flow cytometric analysis was used to assess changes in lymphocyte subpopulations in the thymus and spleen, and production of antibody-forming cells in vitro was measured. Major effects included decreased thymus weight at all time points; increased spleen weight after 10 or 14 days of exposure, increased bone marrow cellularity after 10 or 14 days of exposure, significant decreases in mature lymphocyte subpopulations which were greater in the thymus than in the spleen, relatively selective depletion of the major subpopulation of thymocytes (CD4+CD8+), and decreased body weight. Overall patterns of changes were consistent with the conclusion that SMD rapidly depletes most CD4+CD8+ thymocytes, more slowly depletes a smaller number of mature lymphocytes in the thymus and spleen, and induces compensatory and/or detoxication mechanisms after 10-14 days of exposure. Subsequent experiments were done to assess selected immune function parameters. SMD at 50-300 mg/kg/day for 7 days caused substantial, dose-dependent suppression of NK cell activity. No suppression of antibody production in vivo or splenocyte responses to mitogens or allogeneic lymphocytes in vitro was detected. NK cell activity, thymus weight, and CD4+CD8+ thymocyte numbers were suppressed by dermal administration of SMD.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative analysis of the neuroendocrine-immune axis: linear modeling of the effects of exogenous corticosterone and restraint stress on lymphocyte subpopulations in the spleen and thymus in female B6C3F1 mice.

The effects of exogenous corticosterone and restraint stress on the number and percentage of lymphocyte subpopulations in the spleen and thymus were evaluated. The data were used to generate linear models that describe the relationship between these parameters and the area under the corticosterone concentration vs time curve (AUC). Comparison of the models revealed that the number of nucleated cells in the spleen was decreased similarly by exogenous corticosterone and restraint (at equivalent corticosterone AUC values). However, exogenous corticosterone caused a greater decrease in cell number in the thymus than it did in the spleen. Corticosterone preferentially depleted CD4+CD8+ cells in the thymus, whereas the same corticosterone exposure produced by restraint stress did not. In the spleen, cell number for all major cell types was decreased by both treatments, but there were minor differences in the change in percentage of some subpopulations induced by exogenous corticosterone as compared to restraint. The models derived here provide quantitative data that indicate the magnitude of corticosterone and stress-induced effects on lymphocyte populations in the spleen and thymus. These results have mechanistic implications, and they may be useful in future efforts to extrapolate from mouse to human by completing a risk assessment parallelogram.