Aims: Microglia are involved in neurodegeneration, are prime targets for anti‐inflammatory therapy and are potential biomarkers of disease progression. For example, positron emission tomography imaging employing radioligands for the mitochondrial translocator protein of 18 kDa (TSPO, formerly known as the peripheral benzodiazepine receptor) is being scrutinized to detect neuroinflammation in various diseases. TSPO is presumably present in activated microglia, but may be present in other neural cells. Methods: We sought to elucidate the protein expression in normal human central nervous system, several neurological diseases (HIV encephalitis, Alzheimer's disease, multiple sclerosis and stroke) and simian immunodeficiency virus encephalitis by performing immunohistochemistry with two anti‐TSPO antibodies. Results: Although the overall parenchymal staining was minimal in normal brain, endothelial and smooth muscle cells, subpial glia, intravascular monocytes and ependymal cells were TSPO‐positive. In disease states, elevated TSPO was present in parenchymal microglia, macrophages and some hypertrophic astrocytes, but the distribution of TSPO varied depending on the disease, disease stage and proximity to the lesion or relation to infection. Staining with the two antibodies correlated well in white matter, but one antibody also stained cortical neurones. Quantitative analysis demonstrated a significant increase in TSPO in the white matter of HIV encephalitis compared with brains without encephalitis. TSPO expression was also increased in simian immunodeficiency virus encephalitis. Conclusions: This report provides the first comprehensive immunohistochemical analysis of the expression of TSPO. The results are useful for informing the usage of positron emission tomography as an imaging modality and have an impact on the potential use of TSPO as an anti‐inflammatory pharmacological target.

Nitric oxide generated by the inducible form of nitric oxide synthase (iNOS) may contribute to the pathogenesis of multiple sclerosis (MS). In this report, we studied postmortem tissues of MS patients for the expression of iNOS by in situ hybridization and immunocytochemistry. Immunocytochemistry for nitrotyrosine, a putative footprint for peroxynitrite formation was also performed. In acute MS lesions, intense reactivity for iNOS mRNA and protein was detected in reactive astrocytes throughout the lesion and in adjacent normal appearing white matter. Staining of macrophages, inflammatory cell infiltrates, and endothelial cells was variable from case to case, but generally detected only in acute lesions. In chronic MS lesions reactive astrocytes at the lesion edge were positive for iNOS whereas the lesion center was nonreactive. Normal appearing white matter demonstrated little reactivity, as did tissues from noninflamed control brains. Staining for nitrotyrosine was also detected in acute but not chronic MS lesions, and displayed a diffuse parenchymal, membranous, and perivascular pattern of immunoreactivity. These results support the conclusion that iNOS is induced in multiple cell types in MS lesions and that astrocyte-derived nitric oxide could be important in orchestrating inflammatory responses in MS , particularly at the blood-brain barrier. Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) that is thought to be mediated by an autoimmune attack directed against components of the myelin sheath. MS lesions are characterized by loss of myelin, oligodendrocytes, and axons associated with a mononuclear inflammatory infiltrate and a reactive gliosis. Although the mechanisms that lead to loss of function associated with these events remain poorly understood, the activation of T cells and macrophages that secrete freely diffusable factors has been widely implicated. Included in these factors are the proinflammatory cytokines interleukin (IL)-1, tumor necrosis factor-␣, IL-12, and interferon (IFN)-␥, and reactive oxygen and reactive nitrogen species. All of these factors have been shown to be elevated in active MS lesions, and animal models support a role for them in disease pathogenesis. 1,2 The anti-proliferative and/or cytotoxic effects of nitric oxide (NO) have been associated with the persistent production of high levels of NO that occurs after the activation of the inducible form of nitric oxide synthase (iNOS). 3 The expression of this enzyme in various cell types is known to be transcriptionally regulated and to be activated by a combination of pro-inflammatory signals such as ligands that activate toll-like receptors and/or cytokines such as IL-1, tumor necrosis factor-␣, and interferon-␥ (IFN-␥). 3 NO by itself demonstrates only weak toxic activity, but congeners formed by auto-oxidation such as NO 2⅐ , N 2 O 3 , and S-nitrosothiols enhance its cytotoxic potential. The toxicity of NO is also greatly enhanced when it combines with O 2Ϫ to generate peroxynitrite (ONOOϪ),...

We examined cytokine‐mediated neuronal death in neuron‐astrocyte cultures from second trimester human fetal cerebrum. In these cultures, high‐output inducible nitric oxide synthase (NOS) and tumor necrosis factor‐α (TNFα) are expressed in astrocytes after exposure to IL‐1β/IFNγ. Neuronal cell death was evident at ≥48 h following cytokine stimulation. Neutralizing anti‐TNFα antiserum inhibited (≈48%) neurotoxicity in IL‐1β/IFNγ‐treated cultures, demonstrating a role for endogenously produced TNFα. Interestingly, the degree of neuroprotection conferred by superoxide dismutase or N‐methyl D‐aspartate (NMDA) receptor antagonists in these cultures was smaller and variable. Similarly, the effect of the NOS inhibitor, NG‐monomethyl L‐arginine (NMMA) on IL‐1β/IFNγ‐induced neuronal death was variable, showing no statistically significant effect when results from more than 30 independent cultures were averaged. Neurons die by apoptosis in cytokine‐treated human fetal CNS cultures as shown by the characteristic nuclear morphology as well as positive labeling for TUNEL. Our results demonstrate a potent neurotoxicity mediated by the cytokine combination IL‐1β/IFNγ in primary human neuron‐astrocyte cultures and a crucial role for endogenous TNFα in mediating neurotoxicity in this system. These results firmly establish the neurotoxic potential of the inflammatory cytokines IL‐1β and TNFα in the human CNS. GLIA 28:114–127, 1999. © 1999 Wiley‐Liss, Inc.

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In the CNS, microglia are the primary targets of HIV infection. In this study, we investigated the effect of activation of the innate antiviral receptors TLR3 and TLR4 on HIV infection of primary human microglia, as well as microglial cell signaling and gene expression. Ligands for both TLR3 and TLR4 potently inhibited HIV replication in microglia through a pathway requiring IRF3. Surprisingly, a remarkably similar pattern of cell signaling and gene expression was observed in TLR3- and TLR4-activated microglia, suggesting a relatively minor role for MyD88 following TLR4 activation in these cells. HIV did not activate IRF3 but rather decreased IRF3 protein, indicating that HIV does not activate TLR3 or RIG-like helicases in microglia. Taken together, these results indicate that activation of TLR3 or TLR4 will elicit antiviral immunity, in addition to inducing proinflammatory responses. We suggest that a balanced expression between inflammatory and innate immune genes might be achieved by IRF3 overexpression.