Microglia as effector cells in brain damage and repair: focus on prostanoids and nitric oxide
Introduction
Microglial cells, despite a long and protracted debate on their developmental origin (Ling and Wong, 1993), are generally considered the resident macrophages of the CNS. Evidence from immunocytochemical studies using macrophage-specific markers have shown that monocytes enter the developing CNS and give rise to microglia (Perry, 1994). In the immature brain, the cells show a rounded and simple morphology and are often referred to as ameboid microglia. These cells are thought to be involved in phagocytosis and removal of degenerating cells during CNS development. During the first postnatal weeks, the ameboid morphology of microglia undergoes a progressive change until the cells acquire long, fine, branched processes. These “ramified” or resting microglia, found in normal adult brain, show a down-regulated immunophenotype adapted to the specialized microenvironment of the CNS, but become rapidly activated in response to pathological events. Activated microglia express, in a graded fashion, antigenic and functional properties comparable to those of peripheral macrophages and tend to become again ameboid, phagocytic and motile (Perry, 1994; Kreutzberg, 1996; Moore and Thanos, 1996).
Although microglia represents a substantial fraction of all glial cells (10–20%, Perry, 1994), their role in the normal adult brain remains largely unknown. Undoubtedly, however, they are important effector cells in most pathological conditions. Due to their reactivity to a wide range of stimuli, they play a crucial role in host defence and facilitate neuroprotection and repair processes. On the other hand, the type or intensity of the noxious stimulus and/or the concurrence of other local factors may make them instrumental for the establishment or amplification of tissue damage (Banati and Graeber, 1994; Mallat and Chamak, 1994). It is therefore important to understand what are the conditions regulating the balance between neuroprotective and neurotoxic activities of these cells.
A common approach to study microglial functional properties is the isolation and culture of purified populations (>99%) of microglia from neonatal rodent brains (Giulian and Baker, 1986; Levi et al., 1993) or from fetal human brains (Lee et al., 1993b). The cells in these cultures show at least some of the morphological and antigenic features of ameboid cells, suggesting that they are partially activated. This may be due to their origin from the immature brain and to the manipulations required to obtain purified cultures. Although these models are very useful for analyzing several microglial properties, they may not be suitable for studying the functional features of resting microglia nor the initial events leading to cell activation. For this reason, methods for the bulk purification and culture of microglia from the adult rodent or human brain have been developed (Williams et al., 1992; Becher and Antel, 1996; Slepko and Levi, 1996). Bulk-isolated adult microglia exhibits a phenotype similar to that of resting, ramified microglia in situ. After a short period in culture, however, the cells progressively express a set of markers and functional features characteristic of activated microglia in vivo and of neonatal microglia in vitro or in vivo, even in the absence of added inducing stimuli.
Microglia has been studied also in situ, both in the normal brain and during disease in humans and experimental animals. The in situ studies have provided invaluable information concerning the progressive antigenic changes that these cells undergo in the various phases of different types of diseases. They also clarified some functional features related to microglia activation such as proliferation, phagocytosis and expression of inflammatory cytokines (Moore and Thanos, 1996and references therein). The complexity of the in vivo system, however, is not suitable for approaching the analysis of several aspects of microglial functions at the cellular and molecular level.
A striking feature of microglial reactivity is the ability to synthesize and secrete a large number of substances, which, alone or in concert with factors derived from other brain or hematogenous cells, may have a crucial role in host defence or in the establishment or maintenance of brain damage. A list of secretory microglial products involved in inflammatory and/or repair processes is reported in Table 1. The list is rapidly expanding and includes growth factors, cytokines, coagulation and complement factors, lipid mediators, extracellular matrix components, enzymes, free radicals and neurotoxins. Nonetheless, the circumstances under which each of them is produced and the factors regulating their synthesis and release are still largely unknown.
In the last few years, a number of comprehensive review articles (Banati and Graeber, 1994; Mallat and Chamak, 1994; Perry et al., 1994; McGeer and McGeer, 1995; Kreutzberg, 1996; Moore and Thanos, 1996) have discussed the role of these cells in brain inflammation, degeneration and ischemia. In the present article, we will restrict our discussion to two classes of compounds, prostanoids and nitric oxide (NO), which may be critical factors in determining the final outcome of microglial reaction to pathological stimuli.
Prostanoids, the arachidonic acid (AA) metabolites of the cyclooxygenase (COX) pathway, and NO, whose formation is catalyzed by nitric oxide synthase (NOS), are potent local mediators and play major roles in regulating inflammation, immune functions, vascular tone and neurotransmission (Appleton et al., 1996). While low levels of these molecules may participate to protective responses leading to enhanced disease resistance, their excessive production may be a cause of cytotoxicity.
Interactions between prostanoids and NO are of particular interest at inflammation sites, where their synthesis is often elicited in inflammatory cells by the same stimuli, and where they may have synergistic as well as antagonistic actions on common targets. To date, most of the information available on these compounds was obtained from in vitro or in vivo models of acute and chronic inflammation of peripheral organs. In spite of the close relationship between microglia and peripheral macrophages, however, it may not be appropriate to extend to the CNS the information acquired with peripheral cells. Indeed, inflammatory and immune responses in the CNS are peculiar (Perry and Gordon, 1991), probably due to the adaptation of the macrophagic phenotype of microglia to the unique cerebral environment.
In the next sections, we shall first provide a schematic description of prostanoid and NO metabolic pathways. Then we shall illustrate their regulation and reciprocal interactions in microglial cells, underlining the similarities and differences with peripheral macrophages.
Section snippets
Biosynthesis of prostanoids and other arachidonic acid metabolites
The first event in the prostanoid cascade is the liberation of AA (5-8-11-14-eicosatetraenoic acid), a polyunsaturated 20-carbon fatty acid, from membrane glycerophospholipids by the enzyme phospholipase. Several forms of phospholipases, acting on different substrates, can induce AA release. However, in most inflammatory cells, where the bulk of AA resides in the sn-2 position of phospholipids, phospholipase A2 (PLA2) seems to have a prominent role. Two major subtypes of PLA2 have been
Nitric oxide synthesis
Nitric oxide is an inorganic free radical gas, generated from l-arginine through a complex enzymatic reaction catalyzed by NOS (E.C.1.14.13.39). The reaction requires O2 and reduced nicotinamide adenine dinucleotide phosphate (NADPH) as co-substrates and tetrahydrobiopterin, flavin adenin nucleotide and flavin mononucleotide as cofactors.
As in the case of COX, constitutive and inducible isoforms of NOS have been identified. The constitutive NOS (cNOS or type I NOS) isoforms are typically
Prostanoid and nitric oxide reciprocal interactions
Prostanoids and NO have several common targets in both physiological and pathological conditions. Their normally low concentrations can increase during inflammation, trauma, ischemia, viral infection, etc., largely due to the expression of two inducible rate-limiting enzymes, COX-2 and iNOS. In several, but not all cases, the stimuli capable of inducing the expression of these enzymes are the same. In the brain, two populations of effector cells, astrocytes and microglia, appear to express
Conclusions
Microglial cells have been generally considered as aggressive cells, capable of inducing neuronal and oligodendroglial damage through their secretory products (nitrogen and oxygen radicals, inflammatory cytokines, glutamate, other excitotoxins, etc.) and of promoting autoimmune processes through their ability to present antigens and stimulate T-cell responses. It has recently become clear, however, that this is just one of the facets of microglial pathophysiology. As mentioned at the beginning
Acknowledgements
The original work presented in this article was supported by the following grants: Project on AIDS of the Italian Ministry of Health; Project on Multiple Sclerosis of the Istituto Superiore di Sanità; Project on Ageing, subproject on Gerontobiology of the Italian National Research Council. The secretarial help of Ms. Estella Sansonetti is gratefully acknowledged.
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