Differential effects of albumin on microglia and macrophages; implications for neurodegeneration following blood–brain barrier damage

Microglial activation by blood-borne factors following blood–brain barrier damage may play a significant role in subsequent neuropathogenesis of several neurodegenerative diseases. Exposure of primary cultured rat brain microglia to pure, fatty acid- and lipid-deficient rat serum albumin or fraction V, (fatty acid and lipid-containing rat serum albumin), caused inducible nitric oxide synthase (iNOS) expression, glutamate release, tumour necrosis factor alpha (TNFα) and transforming growth factor-beta1 release. iNOS expression was attenuated by the MAPK/extracellular signal-regulated kinase pathway inhibitor U0126 and the phosphorylated forms of extracellular signal-regulated kinase 1 and 2 were detectable in microglia treated with albumin or fraction V. Glutamate release was prevented by l -α-aminoadipate and glutathione levels in microglia rose on exposure to albumin. Conditioned medium from microglia exposed to albumin or fraction V was neurotoxic. Peripheral macrophages were resistant to the effects of albumin but both microglia and macrophages responded to lipopolysaccharide, which induced interleukin-1 beta and tumour necrosis factor alpha release, cyclooxygenase-2 and iNOS expression in both cell types, indicating a discrete desensitised pathway in macrophages for albumin which was not desensitised in microglia. Thus, exposure of microglia in the brain to albumin may contribute to neuronal damage following blood–brain barrier breakdown and point to resident microglia rather than infiltrating macrophages as therapeutic targets.     

HIV receptors within the brain: A study of CD4 and MHC-II on human neurons,astrocytes and microglial cells

We have investigated the level of expression of CD4 and MHC-II antigens on CNS cells and compared it to that on monocytes. MHC-II antigens were expressed spontaneously on cultured astrocytes and monocytes, whereas they were detected only after IFNγ stimulation of microglial cells. In vitro, CD4 receptor was present on monocytes but not on neurons, astrocytes or microglial cells. In normal brain, CD4 antigen was expressed on perivascular microglial cells, a specialized microglia expressing monocytic markers, whereas in HIV1-infected brain, CD4⁺ cells were numerous and scattered throughout the whole parenchyma. These CD4⁺ macrophages may be HIV1-infected monocytes which have crossed the blood-brain barrier after infection, or perivascular microglial cells infected by HIV1-infected blood lymphocytes or free virions.     

Identification of a peptide sequence in albumin that potentiates superoxide production by microglia

Microglial activation has recently been recognized as a cause of damage in various neurodegenerative diseases. A possible mechanism underlying this damage is the activation of microglia by serum factors leaked through a disruption of the blood-brain barrier, which in turn trigger microglial cell proliferation and the release of various substances toxic to neurons, such as superoxide (O(2)(-)). We recently reported that serum albumin enhanced O(2)(-) production in cultured rat microglia stimulated by phorbol ester. In the present report, we identify the active site of this enhancement within the albumin molecule. We purified an active subfragment from trypsin-treated bovine serum albumin that was composed of 12-mer and 33-mer peptides connected by a disulfide bond. The chemically synthesized 12-mer peptide showed activity within a concentration range ( approximately 10(-7) M:) equivalent to that of albumin. The activities of a series of synthesized peptides conclusively indicated that the minimum active sequence was Leu-His-Thr-Leu. The present study may shed light on the mechanism of neuronal cell damage in various neurodegenerative diseases.     

Astrocytes in injury states rapidly produce anti-inflammatory factors and attenuate microglial inflammatory responses

Microglia are known to be a primary inflammatory cell type in the brain. However, microglial inflammatory responses are attenuated in the injured brain compared to those in cultured pure microglia. In the present study, we found that astrocytes challenged by oxygen-glucose deprivation (OGD) or H(2) O(2) released soluble factor(s) and attenuated microglial inflammatory responses. Conditioned medium prepared from astrocytes treated with OGD (OGD-ACM) or H(2) O(2) (H(2) O(2) -ACM) significantly reduced the levels of interferon-γ (IFN-γ)-induced microglial inflammatory mediators, including inducible nitric oxide synthase, at both the mRNA and protein levels. The anti-inflammatory effect of astrocytes appeared very rapidly (within 5min), but was not closely correlated with the extent of astrocyte damage. Both OGD-ACM and H(2) O(2) -ACM inhibited STAT nuclear signaling, as evidenced by a reduction in both STAT-1/3 binding to the IFN-γ-activated site and IFN-γ-activated site promoter activity. However, both phosphorylation and nuclear translocation of STAT-1/3 was unchanged in IFN-γ-treated microglia. The active component(s) in OGD-ACM were smaller than 3kDa, and displayed anti-inflammatory effects independent of protein synthesis. These results suggest that, in the injured brain, astrocytes may act as a controller to rapidly suppress microglial activation.     

Microglia release activators of neuronal proliferation mediated by activation of mitogen-activated protein kinase, phosphatidylinositol-3-kinase/Akt and delta-Notch signalling cascades

Microglia, the resident macrophage of the brain, can release substances that aid neuronal development, differentiation and survival. We have investigated the effects of non-activated microglia on the survival of cultured rat cerebellar granule neurones. Microglial-conditioned medium, collected from primary rat microglial cultures, was used to treat 7-day-in-vitro neurones, and neuronal viability and proliferation was assessed following a further 1 or 7 days in culture. Microglial-conditioned medium enhanced neuronal survival by up to 50% compared with untreated neurones and this effect was completely abated by pretreatment of the microglia with l-leucine methyl ester. The expression of the proliferation marker Ki-67 increased in neuronal cultures treated with microglial-conditioned medium suggesting enhanced proliferation of precursor neurones. Microglial-induced neuronal proliferation could be attenuated by specific inhibition of mitogen-activated protein kinase or phosphatidylinositol-3-kinase/Akt signalling pathways, and by selective fractionation and immunodepletion of the microglial-conditioned medium. Activation of the Notch pathway was enhanced as antibody against the Notch ligand, delta-1, prevented the microglial-induced neuronal proliferation. These results show that microglia release stable neurotrophic factors that can promote neuronal precursor cell proliferation.     

Microglia: phagocyte and glia cell

Microglia are the resident immune cells of the brain, and are located within the brain parenchyme behind the blood-brain barrier. They originate from mesodermal hemapoietic precursors and are slowly turned over and replenished by proliferation in the adult central nervous system. In the healthy brain resting, ramified microglia function as supportive glia cells, and their activation status is regulated by neurons through soluble mediators and cell-cell contact. However, in response to brain pathology microglia become activated: acquisition of innate immune cell functions render microglia competent to react towards brain injury through tissue repair or induction of immune responses. In certain pathological conditions, however, microglia activation may sustain a chronic inflammation of the brain, leading to neuronal dysfunction and cell death. This might be mediated by the microglial release of extracellular toxic reactive oxygen and nitrogen species. Nevertheless, in the future microglia may potentially be harnessed for therapeutical purposes.     

Microglia proliferation is regulated by hydrogen peroxide from NADPH oxidase

Microglia are resident brain macrophages that become activated and proliferate following brain damage or stimulation by immune mediators, such as IL-1beta or TNF-alpha. We investigated the mechanisms by which microglial proliferation is regulated in primary cultures of rat glia. We found that basal proliferation of microglia was stimulated by proinflammatory cytokines IL-1beta or TNF-alpha, and this proliferation was completely inhibited by catalase, implicating hydrogen peroxide as a mediator of proliferation. In addition, inhibitors of NADPH oxidase (diphenylene iodonium or apocynin) also prevented microglia proliferation, suggesting that this may be the source of hydrogen peroxide. IL-1beta and TNF-alpha rapidly stimulated the rate of hydrogen peroxide produced by isolated microglia, and this was inhibited by diphenylene iodonium, implying that the cytokines were acting directly on microglia to stimulate the NADPH oxidase. Low concentrations of PMA or arachidonic acid (known activators of NADPH oxidase) or xanthine/xanthine oxidase or glucose oxidase (generating hydrogen peroxide) also increased microglia proliferation and this was blocked by catalase, showing that NADPH oxidase activation or hydrogen peroxide was sufficient to stimulate microglia proliferation. In contrast to microglia, the proliferation of astrocytes was unaffected by the presence of catalase. In conclusion, these findings indicate that microglial proliferation in response to IL-1beta or TNF-alpha is mediated by hydrogen peroxide from NADPH oxidase.     

ATP mediates calcium signaling between astrocytes and microglial cells: modulation by IFN-gamma

Calcium-mediated intercellular communication is a mechanism by which astrocytes communicate with each other and modulate the activity of adjacent cells, including neurons and oligodendrocytes. We have investigated whether microglia, the immune effector cells involved in several diseases of the CNS, are actively involved in this communication network. To address this issue, we analyzed calcium dynamics in fura-2-loaded cocultures of astrocytes and microglia under physiological conditions and in the presence of the inflammatory cytokine IFN-gamma. The intracellular calcium increases in astrocytes, occurring spontaneously or as a result of mechanical or bradykinin stimulation, induced the release of ATP, which, in turn, was responsible for triggering a delayed calcium response in microglial cells. Repeated stimulations of microglial cells by astrocyte-released ATP activated P2X(7) purinergic receptor on microglial cells and greatly increased membrane permeability, eventually leading to microglial apoptosis. IFN-gamma increased ATP release and potentiated the P2X(7)-mediated cytolytic effect. This is the first study showing that ATP mediates a form of calcium signaling between astrocytes and microglia. This mechanism of intercellular communication may be involved in controlling the number and function of microglial cells under pathophysiologic CNS conditions.     

Astrocyte-derived interleukin 3 as a growth factor for microglia cells and peritoneal macrophages

Astrocytes have been shown to release an interleukin 3 (IL 3)-like factor that induces the expression of 20-alpha-hydroxysteroid-dehydrogenase (20-alpha SDH) in nu/nu spleen cells, and the proliferation of the IL 3-dependent cell line 32DCL. We have investigated whether astrocyte-derived IL 3 supports growth of macrophages and their representatives in the brain, the microglia cells. Evidence for intercellular communication between murine astrocytes and macrophages became already detectable in co-culture experiments: astrocytes activated with endotoxin resulted in an increased growth of peritoneal macrophages on the astrocyte monolayer. Biochemical analysis of supernatants of activated astrocytes revealed that the IL 3-like factor that stimulated 32DCL cells and the expression of 20 alpha SDH also served as a growth factor for cultured peritoneal macrophages. The same results were obtained by using microglia cells isolated from primary brain cell cultures of newborn mice, which are characterized by their positive reaction for macrophage markers such as Mac-1 and nonspecific esterase. If secreted by reactive astrocytes in vivo, the IL 3-like factor may contribute to the accumulation of macrophages and microglia cells detected in brain lesions of patients with multiple sclerosis.     

Hydrogen peroxide and ADP-ribose induce TRPM2-mediated calcium influx and cation currents in microglia

Microglial cells are the host macrophages in the central nervous system and respond to brain injury and various neurological diseases. In this process, microglial cells undergo multiple morphological and functional changes from the resting cell toward a fully activated, phagocyting tissue macrophage. In culture, bacterial lipopolysaccharide (LPS) is a frequently used tool to induce this activation. By using calcium-imaging and patch-clamp techniques, we investigated the effect of hydrogen peroxide (H₂O₂), which is released by macrophagic cells themselves, on the intracellular calcium concentration and ion currents in cultured rat microglia. Application of 0.1–5 mM H₂O₂ for several minutes induced small responses in untreated cells but a large calcium influx and cation current in LPS-treated cells. In both untreated and LPS-treated microglia, internal perfusion of ADP-ribose (ADPR) via the patch pipette elicited large cation currents. Both stimuli, H₂O₂ and ADPR, have been reported to activate the recently cloned nonselective cation channel TRPM2. RT-PCR analysis from cultured rat glial and neuronal cells confirmed a strong expression of TRPM2 in rat microglia but not in astrocytes and cerebellar granule cells. In situ hybridizations from mouse brain showed a distribution of TRPM2, which is compatible with the expression in microglial cells. In conclusion, we describe here a novel calcium influx pathway in microglia coupled to hydrogen peroxide and ADPR and provide evidence that this pathway involves TRPM2. The increased sensitivity to H₂O₂ in LPS-stimulated cells suggests a role for TRPM2 in the calcium signaling of activated microglia. nonselective cation channel; transient receptor potential channel; H₂O₂; activated microglia     

Innate immune responses in central nervous system inflammation

In autoimmune diseases of the central nervous system (CNS), innate glial cell responses play a key role in determining the outcome of leukocyte infiltration. Access of leukocytes is controlled via complex interactions with glial components of the blood-brain barrier that include angiotensin II receptors on astrocytes and immunoregulatory mediators such as Type I interferons which regulate cellular traffic. Myeloid cells at the blood-brain barrier present antigen to T cells and influence cytokine effector function. Myelin-specific T cells interact with microglia and promote differentiation of oligodendrocyte precursor cells in response to axonal injury. These innate responses offer potential targets for immunomodulatory therapy.     

Cholinergic modulation of microglial activation by alpha 7 nicotinic receptors

Almost all degenerative diseases of the CNS are associated with chronic inflammation. A central step in this process is the activation of brain mononuclear phagocyte cells, called microglia. While it is recognized that healthy neurons and astrocytes regulate the magnitude of microglia-mediated innate immune responses and limit excessive CNS inflammation, the endogenous signals governing this process are not fully understood. In the peripheral nervous system, recent studies suggest that an endogenous 'cholinergic anti-inflammatory pathway' regulates systemic inflammatory responses via alpha 7 nicotinic acetylcholinergic receptors (nAChR) found on blood-borne macrophages. These data led us to investigate whether a similar cholinergic pathway exists in the brain that could regulate microglial activation. Here we report for the first time that cultured microglial cells express alpha 7 nAChR subunit as determined by RT-PCR, western blot, immunofluorescent, and immunohistochemistry analyses. Acetylcholine and nicotine pre-treatment inhibit lipopolysaccharide (LPS)-induced TNF-alpha release in murine-derived microglial cells, an effect attenuated by alpha 7 selective nicotinic antagonist, alpha-bungarotoxin. Furthermore, this inhibition appears to be mediated by a reduction in phosphorylation of p44/42 and p38 mitogen-activated protein kinase (MAPK). Though preliminary, our findings suggest the existence of a brain cholinergic pathway that regulates microglial activation through alpha 7 nicotinic receptors. Negative regulation of microglia activation may also represent additional mechanism underlying nicotine's reported neuroprotective properties.     

Down regulation of CD4 expression in cultured microglia by immunosuppressants and lipopolysaccharide.

Microglia, brain macrophages, are thought to be the primary target of HIV-1 infection in the brain, because they exclusively express the CD4 antigen which is effectively used for viral entry. The expression of CD4 mRNA in cultured microglia could be detected by the reverse-PCR method. Using this and immunohistochemical staining, we found that the immunosuppressants cyclosporin A and FK506 decreased CD4 expression in cultured murine microglia without causing any significant decrease in cell viability. FK506 was more potent than cyclosporin A. Lipopolysaccharide also decreased CD4 mRNA expression in microglia. The effects of immunosuppressants and lipopolysaccharide seemed to be specific for microglia since these chemicals did not alter the CD4 expression in lymphocytes or peritoneal macrophages. These agents, if modified to pass through the blood-brain barrier, may prevent viral spread of HIV-1 infection in the central nervous system and the AIDS-dementia complex.     

Proliferation of microglial cells induced by 1-methyl-4-phenylpyridinium in mesencephalic cultures results from an astrocyte-dependent mechanism: role of granulocyte macrophage colony-stimulating factor

There is evidence that an inflammatory microglial reaction participates in the pathophysiology of dopaminergic neuronal death in Parkinson's disease and in animal models of the disease. However, this phenomenon remains incompletely characterized. Using an in vitro model of neuronal/glial mesencephalic cultures, we show that the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+) stimulates the proliferation of microglial cells at concentrations that selectively reduce the survival of DA neurones. The mitogenic action of MPP+ was not the mere consequence of neuronal cell demise as the toxin produced the same effect in a model system of neuronal/glial cortical cultures, where target DA neurones are absent. Consistent with this observation, the proliferative effect of MPP+ was also detectable in neurone-free microglial/astroglial cultures. It disappeared, however, when MPP+ was added to pure microglial cell cultures suggesting that astrocytes played a key role in the mitogenic mechanism. Accordingly, the proliferation of microglial cells in response to MPP+ treatment was mimicked by granulocyte macrophage colony-stimulating factor (GM-CSF), a proinflammatory cytokine produced by astrocytes and was blocked by a neutralizing antibody to GM-CSF. Thus, we conclude that the microglial reaction observed following MPP+ exposure depends on astrocytic factors, e.g. GM-CSF, a finding that may have therapeutic implications.     

Xenobiotic-mediated production of superoxide by primary cultures of rat cerebral endothelial cells, astrocytes, and neurones

Previous works of our group demonstrated that xenobiotic metabolism by brain microsomes or cultured cerebral cells may promote the formation of reactive oxygen species. In order to characterise the risk of oxidative stress to both the central nervous system and the blood-brain barrier, we measured in the present work the release of superoxide in the culture medium of rat cerebrovascular endothelial cells during the metabolism of menadione, anthraquinone, diquat or nitrofurazone. Assays were run in the same experimental conditions on primary cultures of rat neurones and astrocytes. Quinone metabolism efficiently produced superoxide, but the production of radicals during the metabolism of diquat or nitrofurazone was very low, as a probable result of their reduced transport inside the cells. In all cell types assayed, superoxide production was time- and concentration-dependent, and cultured astrocytes always produced the highest amounts of radicals. Superoxide formation by microsomes prepared from the cultured cells was decreased by immunoinhibition of NADPH-cytochrome P450 reductase or by its irreversible inhibition by diphenyliodonium chloride, suggesting the involvement of this flavoprotein in radical production. Cerebrovascular endothelial cells cultured on collagen-coated filters produced equivalent amounts of superoxide both at their luminal side and through the artificial basement membrane, suggesting that in vivo, endothelial superoxide production may endanger adjacent astrocytes and neurones.     

Macrophages/microglial cells in patients with cerebral malaria

Cerebral malaria is a life threatening sequel of Plasmodium falciparum infection and contributes significantly to malaria mortality, especially among children. Accumulation of macrophages and proliferation of microglial cells play key roles in cerebral malaria and are thought to contribute to the pathophysiological alterations observed in these patients, which include enhanced adherence of infected erythrocytes to the cerebral vasculature by expression and secretion of proinflammatory molecules, disruption of the blood-brain barrier, recruitment of other inflammatory cells to the lesion site. In this review, recent advances in the understanding of the involvement of macrophages/microglial cells in the development of cerebral malaria are summarized.     

Debris of “dark” (compacted) neurones are removed from an otherwise undamaged environment mainly by astrocytes via blood vessels

By means of a condenser-discharge electric shock paradigm, “dark” granule neurones were momentarily produced in a sporadic distribution among normal ones in the otherwise undamaged (non-necrotic, non-excitotoxic, non-inflammatory or non-contused) hippocampal dentate gyri of the rat brain. In the electron microscope, the ultrastructural elements of the affected neurones remained undamaged but turned markedly electron-dense and the distances between them became strikingly reduced (compaction). A proportion of such neurones recovered in 1 day while others died. During the first week of survival, the dead “dark” granule neurones retained the compacted and electron-dense ultrastructure, but underwent cytoplasmic convolution and fragmentation. The fragments were enclosed by membranes and separated from each other and from the intact neuropil by astrocytic processes containing an excess of glycogen particles. Neither proliferation of microglial cells nor infiltration of haematogenous macrophages was observed. A few fragments were taken over by resting microglial cells, while the majority was engulfed by astrocytes. The latter transported the engulfed fragments, either unchanged or digested to various degrees, to capillaries, arterioles and venules. Thereafter, the astrocyte-engulfed neuronal fragments, as well as their partly or completely digested remnants, were either transferred to phagocytotic pericytes or discharged into vascular lumina.