Carol A. Colton

Production of superoxide anions by a CNS macrophage, the microglia.

Microglia have been implicated in both physiological and pathological processes of the brain. Their possible roles have been compared to those of macrophages and granulocytes. Here we demonstrate the specific ability of microglia to secrete the superoxide radical ion in response to a complement activated agent, opsonized zymosan, and to phorbol myristate acetate. As in other organs, this endogenously produced reactive oxygen intermediate could have both beneficial and deleterious effects.

The chemical biology of nitric oxide: implications in cellular signaling.

Nitric oxide (NO) has earned the reputation of being a signaling mediator with many diverse and often opposing biological activities. The diversity in response to this simple diatomic molecule comes from the enormous variety of chemical reactions and biological properties associated with it. In the past few years, the importance of steady-state NO concentrations has emerged as a key determinant of its biological function. Precise cellular responses are differentially regulated by specific NO concentration. We propose five basic distinct concentration levels of NO activity: cGMP-mediated processes ([NO]<1-30 nM), Akt phosphorylation ([NO] = 30-100 nM), stabilization of HIF-1alpha ([NO] = 100-300 nM), phosphorylation of p53 ([NO]>400 nM), and nitrosative stress (1 microM). In general, lower NO concentrations promote cell survival and proliferation, whereas higher levels favor cell cycle arrest, apoptosis, and senescence. Free radical interactions will also influence NO signaling. One of the consequences of reactive oxygen species generation is to reduce NO concentrations. This antagonizes the signaling of nitric oxide and in some cases results in converting a cell-cycle arrest profile to a cell survival profile. The resulting reactive nitrogen species that are generated from these reactions can also have biological effects and increase oxidative and nitrosative stress responses. A number of factors determine the formation of NO and its concentration, such as diffusion, consumption, and substrate availability, which are referred to as kinetic determinants for molecular target interactions. These are the chemical and biochemical parameters that shape cellular responses to NO. Herein we discuss signal transduction and the chemical biology of NO in terms of the direct and indirect reactions.

Heterogeneity of Microglial Activation in the Innate Immune Response in the Brain

The immune response in the brain has been widely investigated and while many studies have focused on the proinflammatory cytotoxic response, the brain’s innate immune system demonstrates significant heterogeneity. Microglia, like other tissue macrophages, participate in repair and resolution processes after infection or injury to restore normal tissue homeostasis. This review examines the mechanisms that lead to reduction of self-toxicity and to repair and restructuring of the damaged extracellular matrix in the brain. Part of the resolution process involves switching macrophage functional activation to include reduction of proinflammatory mediators, increased production and release of anti-inflammatory cytokines, and production of cytoactive factors involved in repair and reconstruction of the damaged brain. Two partially overlapping and complimentary functional macrophage states have been identified and are called alternative activation and acquired deactivation. The immunosuppressive and repair processes of each of these states and how alternative activation and acquired deactivation participate in chronic neuroinflammation in the brain are discussed.

Nitric oxide and redox mechanisms in the immune response

The role of redox molecules, such as NO and ROS, as key mediators of immunity has recently garnered renewed interest and appreciation. To regulate immune responses, these species trigger the eradication of pathogens on the one hand and modulate immunosuppression during tissue‐restoration and wound‐healing processes on the other. In the acidic environment of the phagosome, a variety of RNS and ROS is produced, thereby providing a cauldron of redox chemistry, which is the first line in fighting infection. Interestingly, fluctuations in the levels of these same reactive intermediates orchestrate other phases of the immune response. NO activates specific signal transduction pathways in tumor cells, endothelial cells, and monocytes in a concentration‐dependent manner. As ROS can react directly with NO‐forming RNS, NO bioavailability and therefore, NO response(s) are changed. The NO/ROS balance is also important during Th1 to Th2 transition. In this review, we discuss the chemistry of NO and ROS in the context of antipathogen activity and immune regulation and also discuss similarities and differences between murine and human production of these intermediates.

Mechanisms of the antioxidant effects of nitric oxide

The Janus face of nitric oxide (NO) has prompted a debate as to whether NO plays a deleterious or protective role in tissue injury. There are a number of reactive nitrogen oxide species, such as N2O3 and ONOO-, that can alter critical cellular components under high local concentrations of NO. However, NO can also abate the oxidation chemistry mediated by reactive oxygen species such as H2O2 and O2- that occurs at physiological levels of NO. In addition to the antioxidant chemistry, NO protects against cell death mediated by H2O2, alkylhydroperoxides, and xanthine oxidase. The attenuation of metal/peroxide oxidative chemistry, as well as lipid peroxidation, appears to be the major chemical mechanisms by which NO may limit oxidative injury to mammalian cells. In addition to these chemical and biochemical properties, NO can modulate cellular and physiological processes to limit oxidative injury, limiting processes such as leukocyte adhesion. This review will address these aspects of the chemical biology of this multifaceted free radical and explore the beneficial effect of NO against oxidative stress.

APOE genotype-specific differences in the innate immune response.

Apolipoprotein-E protein is an endogenous immunomodulatory agent that affects both the innate and the adaptive immune responses. Since individuals with the APOE4 gene demonstrate worsened pathology and poorer outcomes in many neurological disorders, we examined isoform-specific differences in the response of microglia, the primary cellular component of the brains innate immune response, in detail. Our data demonstrate that microglia derived from APOE4/4 targeted replacement mice demonstrate a pro-inflammatory phenotype that includes altered cell morphology, increased NO production associated with increased NOS2 mRNA levels, and higher pro-inflammatory cytokine production (TNFalpha, IL-6, IL12p40) compared to microglia derived from APOE3/3 targeted replacement mice. The effect is gene dose-dependent and increases with the number of APOE4 gene alleles. The APOE genotype-specific immune profile observed in the microglial immune response is also observed in the cortex of aged APOE3/3 and APOE4/4 mice treated with lipopolysacchride (LPS) and in peripheral (peritoneal) macrophages. To determine if APOE4s action resulted from an isoform-specific difference in effective levels of the apolipoproteins, we generated mice expressing only a single allele of APOE3. Immune-stimulated macrophages from APOE3/0 mice demonstrated an increased inflammatory response compared to APOE3/3 mice, but less than in APOE4/4 mice. These data suggest that inhibition of inflammation depends upon the dose of apoE3 protein available and that apoE4 protein may alter inflammation partly by dose effects and partly by being qualitatively different than apoE3. Overall, these data emphasize the important role of apolipoprotein E and of the APOE genotype on the immune responses that are evident in most, if not all, neurological disease.

Activated human microglia produce the excitotoxin quinolinic acid

WE aimed to determine the relative role of quinolinic acid synthesis in purified human microglia, monocytederived macrophages and astrocytes in the human brain following immune stimulation. Microglia and macrophages significantly increased quinolinic acid synthesis from tryptophan following activation by either lipopolysaccharide or interferon-γ. Quinolinic acid synthesis by individual microglia was heterogeneous, and its production by activated macrophages was approximately 32-fold greater than its microglial synthesis. Quinolinic acid synthesis by astrocytes was undetectable. Microglia may, therefore, be the primary endogenous cell type responsible for quinolinic acid synthesis in the brain parenchyma. However, under pathological conditions which precipitate blood–brain barrier compromise and/or leukocytic infiltration, intracerebral quinolinic acid may be derived chiefly from cells of the peripheral immune system such as activated macrophages.

Characterization of interleukin-1 production by microglia in culture.

The production of interleukin-1 (IL-1) by cultured neonatal rat microglia was studied using the D10 cell assay. The results show that IL-1 was secreted in response to lipopolysaccharide (LPS) in a dose- and time-dependent fashion. IL-1 production was specific to microglia and was not induced in astrocytes. Indomethacin, which is known to modulate the release of IL-1 from monocytes, had no effect on LPS-stimulated microglia. Aging of the microglia from two weeks to 4 weeks in culture, however, reduced the release of IL-1 in response to LPS. Our data indicate that microglia are a major source of IL-1 and that the release of IL-1 depends on the presence of inflammatory mediators such as LPS and the age of the culture.

Progression of Amyloid Pathology to Alzheimer's Disease Pathology in an Amyloid Precursor Protein Transgenic Mouse Model by Removal of Nitric Oxide Synthase 2

Alzheimers disease (AD) is characterized by three primary pathologies in the brain: amyloid plaques, neurofibrillary tangles, and neuron loss. Mouse models have been useful for studying components of AD but are limited in their ability to fully recapitulate all pathologies. We crossed the APPSwDI transgenic mouse, which develops amyloid β (Aβ)-protein deposits only, with a nitric oxide synthase 2 (NOS2) knock-out mouse, which develops no AD-like pathology. APPSwDI/NOS2−/− mice displayed impaired spatial memory compared with the APPSwDI mice, yet they have unaltered levels of Aβ. APPSwDI mice do not show tau pathology, whereas APPSwDI/NOS2−/− mice displayed extensive tau pathology associated with regions of dense microvascular amyloid deposition. Also, APPSwDI mice do not have any neuron loss, whereas the APPSwDI/NOS2−/− mice have significant neuron loss in the hippocampus and subiculum. Neuropeptide Y neurons have been shown to be particularly vulnerable in AD. These neurons appear to be particularly vulnerable in the APPSwDI/NOS2−/− mice as we observe a dramatic reduction in the number of NPY neurons in the hippocampus and subiculum. These data show that removal of NOS2 from an APP transgenic mouse results in development of a much greater spectrum of AD-like pathology and behavioral impairments.

Mitogenic effect of neuropeptide Y in rat vascular smooth muscle cells

Neuropeptide Y (NPY) is a vasoconstrictor released with norepinephrine from perivascular sympathetic nerves. Since sympathetic nerves appear to play a role in vascular smooth muscle cell (SMC) hypertrophy, we studied the effects of NPY on proliferation of cultured rat aorta- and vena cava-derived SMC. Both cell types displayed high-affinity NPY binding sites with displacement characteristics of [Pro34]NPY > NPY(13-36) > NPY(18-36) in aorta and [Pro34]NPY = NPY(13-36) = NPY(18-36) in the vena cava. Incubation with NPY (50-1000 nM) for 48 h increased by up to twofold cell number and [3H]-thymidine incorporation in both cell types (aortic more sensitive to NPY than venous). Following incubation with NPY, the disappearance of NPY immunoreactivity (-IR) from media was markedly delayed in the presence of SMC, and cell content of NPY-IR increased in a dose-dependent manner, indicating that SMC either diminish degradation of the peptide (possibly by internalization) or secrete endogenous NPY (or both). Structure-activity relationship studies with NPY(18-36) indicated involvement of Y1 receptors in mitogenesis. Thus, NPY has a mitogenic effect (probably mediated by Y1 receptors) and, therefore, may be a sympathetic trophic factor involved in vascular hypertrophy.