Role for neuronally derived fractalkine in mediating interactions between neurons and CX3CR1-expressing microglia

A recently identified chemokine, fractalkine, is a member of the chemokine gene family, which consists principally of secreted, proinflammatory molecules. Fractalkine is distinguished structurally by the presence of a CX3C motif as well as transmembrane spanning and mucin-like domains and shows atypical constitutive expression in a number of nonhematopoietic tissues, including brain. We undertook an extensive characterization of this chemokine and its receptor CX3CR1 in the brain to gain insights into use of chemokine-dependent systems in the central nervous system. Expression of fractalkine in rat brain was found to be widespread and localized principally to neurons. Recombinant rat CX3CR1, as expressed in Chinese hamster ovary cells, specifically bound fractalkine and signaled in the presence of either membrane-anchored or soluble forms of fractalkine protein. Fractalkine stimulated chemotaxis and elevated intracellular calcium levels of microglia; these responses were blocked by anti-CX3CR1 antibodies. After facial motor nerve axotomy, dramatic changes in the levels of CX3CR1 and fractalkine in the facial nucleus were evident. These included increases in the number and perineuronal location of CX3CR1-expressing microglia, decreased levels of motor neuron-expressed fractalkine mRNA, and an alteration in the forms of fractalkine protein expressed. These data describe mechanisms of cellular communication between neurons and microglia, involving fractalkine and CX3CR1, which occur in both normal and pathological states of the central nervous system.

Microglia at brain stab wounds express connexin 43 and in vitro form functional gap junctions after treatment with interferon-γ and tumor necrosis factor-α

Gap junctional communication between microglia was investigated at rat brain stab wounds and in primary cultures of rat and mouse cells. Under resting conditions, rat microglia (FITC-isolectin-B4-reactive cells) were sparsely distributed in the neocortex, and most (95%) were not immunoreactive for Cx43, a gap junction protein subunit. At brain stab wounds, microglia progressively accumulated over several days and formed aggregates that frequently showed Cx43 immunoreactivity at interfaces between cells. In primary culture, microglia showed low levels of Cx43 determined by Western blotting, diffuse intracellular Cx43 immunoreactivity, and a low incidence of dye coupling. Treatment with the immunostimulant bacterial lipopolysaccharide (LPS) or the cytokines interferon-γ (INF-γ) or tumor necrosis factor-α (TNF-α) one at a time did not increase the incidence of dye coupling. However, microglia treated with INF-γ plus LPS showed a dramatic increase in dye coupling that was prevented by coapplication of an anti-TNF-α antibody, suggesting the release and autocrine action of TNF-α. Treatment with INF-γ plus TNF-α also greatly increased the incidence of dye coupling and the Cx43 levels with translocation of Cx43 to cell–cell contacts. The cytokine-induced dye coupling was reversibly inhibited by 18α-glycyrrhetinic acid, a gap junction blocker. Cultured mouse microglia also expressed Cx43 and developed dye coupling upon treatment with cytokines, but microglia from homozygous Cx43-deficient mice did not develop significant dye coupling after treatment with either INF-γ plus LPS or INF-γ plus TNF-α. This report demonstrates that microglia can communicate with each other through gap junctions that are induced by inflammatory cytokines, a process that may be important in the elaboration of the inflammatory response.

Developmental plasticity of CNS microglia

Microglia arise from CD45+ bone marrow precursors that colonize the fetal brain and play a key role in central nervous system inflammatory conditions. We report that parenchymal microglia are uncommitted myeloid progenitors of immature dendritic cells and macrophages by several criteria, including surface expression of “empty” class II MHC protein and their cysteine protease (cathepsin) profile. Microglia express receptors for stem cell factor and can be skewed toward more dendritic cell or macrophage-like profiles in response to the lineage growth factors granulocyte/macrophage colony-stimulating factor or macrophage colony-stimulating factor. Thus, in contrast to other organs, where terminally differentiated populations of resident dendritic cells and/or macrophages outnumber colonizing precursors, the majority of microglia within the brain remain in an undifferentiated state.

kappa opioid receptors in human microglia downregulate human immunodeficiency virus 1 expression.

Microglial cells, the resident macrophages of the brain, play an important role in the neuropathogenesis of human immunodeficiency virus type 1 (HIV-1), and recent studies suggest that opioid peptides regulate the function of macrophages from somatic tissues. We report herein the presence of kappa opioid receptors (KORs) in human fetal microglia and inhibition of HIV-1 expression in acutely infected microglial cell cultures treated with KOR ligands. Using reverse transcriptase-polymerase chain reaction and sequencing analyses, we found that mRNA for the KOR was constitutively expressed in microglia and determined that the nucleotide sequence of the open reading frame was identical to that of the human brain KOR gene. The expression of KOR in microglial cells was confirmed by membrane binding of [3H]U69,593, a kappa-selective ligand, and by indirect immunofluorescence. Treatment of microglial cell cultures with U50,488 or U69,593 resulted in a dose-dependent inhibition of expression of the monocytotropic HIV-1 SF162 strain. This antiviral effect of the kappa ligands was blocked by the specific KOR antagonist, nor-binaltrophimine. These findings suggest that kappa opioid agonists have immunomodulatory activity in the brain, and that these compounds could have potential in the treatment of HIV-1-associated encephalopathy.

A tetracycline derivative, minocycline, reduces inflammation and protects against focal cerebral ischemia with a wide therapeutic window

The only treatment of patients with acute ischemic stroke is thrombolytic therapy, which benefits only a fraction of stroke patients. Both human and experimental studies indicate that ischemic stroke involves secondary inflammation that significantly contributes to the outcome after ischemic insult. Minocycline is a semisynthetic second-generation tetracycline that exerts antiinflammatory effects that are completely separate from its antimicrobial action. Because tetracycline treatment is clinically well tolerated, we investigated whether minocycline protects against focal brain ischemia with a wide therapeutic window. Using a rat model of transient middle cerebral artery occlusion, we show that daily treatment with minocycline reduces cortical infarction volume by 76 ± 22% when the treatment is started 12 h before ischemia and by 63 ± 35% when started even 4 h after the onset of ischemia. The treatment inhibits morphological activation of microglia in the area adjacent to the infarction, inhibits induction of IL-1β-converting enzyme, and reduces cyclooxygenase-2 expression and prostaglandin E2 production. Minocycline had no effect on astrogliosis or spreading depression, a wave of ionic transients thought to contribute to enlargement of cortical infarction. Treatment with minocycline may act directly on brain cells, because cultured primary neurons were also salvaged from glutamate toxicity. Minocycline may represent a prototype of an antiinflammatory compound that provides protection against ischemic stroke and has a clinically relevant therapeutic window.

Amyloid-β peptide–Receptor for Advanced Glycation Endproduct interaction elicits neuronal expression of macrophage-colony stimulating factor: A proinflammatory pathway in Alzheimer disease

In Alzheimer disease (AD), neurons are thought to be subjected to the deleterious cytotoxic effects of activated microglia. We demonstrate that binding of amyloid-beta peptide (Aβ) to neuronal Receptor for Advanced Glycation Endproduct (RAGE), a cell surface receptor for Aβ, induces macrophage-colony stimulating factor (M-CSF) by an oxidant sensitive, nuclear factor κB-dependent pathway. AD brain shows increased neuronal expression of M-CSF in proximity to Aβ deposits, and in cerebrospinal fluid from AD patients there was ≈5-fold increased M-CSF antigen (P < 0.01), compared with age-matched controls. M-CSF released by Aβ-stimulated neurons interacts with its cognate receptor, c-fms, on microglia, thereby triggering chemotaxis, cell proliferation, increased expression of the macrophage scavenger receptor and apolipoprotein E, and enhanced survival of microglia exposed to Aβ, consistent with pathologic findings in AD. These data delineate an inflammatory pathway triggered by engagement of Aβ on neuronal RAGE. We suggest that M-CSF, thus generated, contributes to the pathogenesis of AD, and that M-CSF in cerebrospinal fluid might provide a means for monitoring neuronal perturbation at an early stage in AD.

Phenotype of arylsulfatase A-deficient mice: Relationship to human metachromatic leukodystrophy

Metachromatic leukodystrophy is a lysosomal sphingolipid storage disorder caused by the deficiency of arylsulfatase A. The disease is characterized by progressive demyelination, causing various neurologic symptoms. Since no naturally occurring animal model of the disease is available, we have generated arylsulfatase A-deficient mice. Deficient animals store the sphingolipid cerebroside-3-sulfate in various neuronal and nonneuronal tissues. The storage pattern is comparable to that of affected humans, but gross defects of white matter were not observed up to the age of 2 years. A reduction of axonal cross-sectional area and an astrogliosis were observed in 1-year-old mice; activation of microglia started at 1 year and was generalized at 2 years. Purkinje cell dendrites show an altered morphology. In the acoustic ganglion numbers of neurons and myelinated fibers are severely decreased, which is accompanied by a loss of brainstem auditory-evoked potentials. Neurologic examination reveals significant impairment of neuromotor coordination.

Neonatal gene transfer leads to widespread correction of pathology in a murine model of lysosomal storage disease

For many inborn errors of metabolism, early treatment is critical to prevent long-term developmental sequelae. We have used a gene-therapy approach to demonstrate this concept in a murine model of mucopolysaccharidosis type VII (MPS VII). Newborn MPS VII mice received a single intravenous injection with 5.4 × 106 infectious units of recombinant adeno-associated virus encoding the human β-glucuronidase (GUSB) cDNA. Therapeutic levels of GUSB expression were achieved by 1 week of age in liver, heart, lung, spleen, kidney, brain, and retina. GUSB expression persisted in most organs for the 16-week duration of the study at levels sufficient to either reduce or prevent completely lysosomal storage. Of particular significance, neurons, microglia, and meninges of the central nervous system were virtually cleared of disease. In addition, neonatal treatment of MPS VII mice provided access to the central nervous system via an intravenous route, avoiding a more invasive procedure later in life. These data suggest that gene transfer mediated by adeno-associated virus can achieve therapeutically relevant levels of enzyme very early in life and that the rapid growth and differentiation of tissues does not limit long-term expression.

Microglial activation precedes acute neurodegeneration in Sandhoff disease and is suppressed by bone marrow transplantation

Sandhoff disease is a lysosomal storage disorder characterized by the absence of β-hexosaminidase and storage of GM2 ganglioside and related glycolipids in the central nervous system. The glycolipid storage causes severe neurodegeneration through a poorly understood pathogenic mechanism. In symptomatic Sandhoff disease mice, apoptotic neuronal cell death was prominent in the caudal regions of the brain. cDNA microarray analysis to monitor gene expression during neuronal cell death revealed an upregulation of genes related to an inflammatory process dominated by activated microglia. Activated microglial expansion, based on gene expression and histologic analysis, was found to precede massive neuronal death. Extensive microglia activation also was detected in a human case of Sandhoff disease. Bone marrow transplantation of Sandhoff disease mice suppressed both the explosive expansion of activated microglia and the neuronal cell death without detectable decreases in neuronal GM2 ganglioside storage. These results suggest a mechanism of neurodegeneration that includes a vigorous inflammatory response as an important component. Thus, this lysosomal storage disease has parallels to other neurodegenerative disorders, such as Alzheimer's and prion diseases, where inflammatory processes are believed to participate directly in neuronal cell death.

Tetracyclines inhibit microglial activation and are neuroprotective in global brain ischemia

Ischemic stroke is the most common life-threatening neurological disease and has limited therapeutic options. One component of ischemic neuronal death is inflammation. Here we show that doxycycline and minocycline, which are broad-spectrum antibiotics and have antiinflammatory effects independent of their antimicrobial activity, protect hippocampal neurons against global ischemia in gerbils. Minocycline increased the survival of CA1 pyramidal neurons from 10.5% to 77% when the treatment was started 12 h before ischemia and to 71% when the treatment was started 30 min after ischemia. The survival with corresponding pre- and posttreatment with doxycycline was 57% and 47%, respectively. Minocycline prevented completely the ischemia-induced activation of microglia and the appearance of NADPH-diaphorase reactive cells, but did not affect induction of glial acidic fibrillary protein, a marker of astrogliosis. Minocycline treatment for 4 days resulted in a 70% reduction in mRNA induction of interleukin-1β-converting enzyme, a caspase that is induced in microglia after ischemia. Likewise, expression of inducible nitric oxide synthase mRNA was attenuated by 30% in minocycline-treated animals. Our results suggest that lipid-soluble tetracyclines, doxycycline and minocycline, inhibit inflammation and are neuroprotective against ischemic stroke, even when administered after the insult. Tetracycline derivatives may have a potential use also as antiischemic compounds in humans.

Implications of immune-to-brain communication for sickness and pain

This review presents a view of hyperalgesia and allodynia not typical of the field as a whole. That is, exaggerated pain is presented as one of many natural consequences of peripheral infection and injury. The constellation of changes that results from such immune challenges is called the sickness response. This sickness response results from immune-to-brain communication initiated by proinflammatory cytokines released by activated immune cells. In response to signals it receives from the immune system, the brain orchestrates the broad array of physiological, behavioral, and hormonal changes that comprise the sickness response. The neurocircuitry and neurochemistry of sickness-induced hyperalgesia are described. One focus of this discussion is on the evidence that spinal cord microglia and astrocytes are key mediators of sickness-induced hyperalgesia. Last, evidence is presented that hyperalgesia and allodynia also result from direct immune activation, rather than neural activation, of these same spinal cord glia. Such glial activation is induced by viruses such as HIV-1 that are known to invade the central nervous system. Implications of exaggerated pain states created by peripheral and central immune activation are discussed.

Human immunodeficiency virus type 1 infection alters chemokine beta peptide expression in human monocytes: implications for recruitment of leukocytes into brain and lymph nodes.

Two chemokine (chemoattractant cytokines) beta peptides, macrophage inflammatory proteins 1 alpha and 1 beta (MIP-1 alpha and MIP-1 beta), were induced in human monocyte cultures following infection with the human immunodeficiency virus type 1 (HIV-1). Induction depended on productive viral infection: not only did the kinetics of MIP-1 peptide induction closely follow those of viral replication, but monocyte cultures inoculated with heat-inactivated virus or infected in the presence of AZT failed to produce these chemokine beta peptides. In addition, HIV infection markedly altered the pattern of beta chemokine expression elicited by tumor necrosis factor (TNF), itself a potent proinflammatory cytokine upregulated during the development of AIDS. Reverse transcription (RT)-PCR and RT-in situ PCR studies on brain tissue from patients with AIDS dementia demonstrated elevated MIP-1 alpha and MIP-1 beta mRNA expression relative to comparable samples from HIV-1-infected patients without dementia. Cells expressing chemokines in HIV-1-infected brains were identified morphologically as microglia and astrocytes. As MIP-1 alpha and MIP-1 beta are potent chemoattractants for both monocytes and specific subpopulations of lymphocytes, this dysregulation of beta chemokine expression may influence the trafficking of leukocytes during HIV infection. These data, taken together, suggest a mechanism by which HIV-1-infected monocytes might recruit uninfected T cells and monocytes to sites of active viral replication or inflammation, notably the brain and lymph nodes.