Macrophages and microglia in the nervous system

13 Armstrong, C. M. and Lopez-Barneo, J. (1987) Science 236, 712-714 14 Tsien, R. W., Hess, P., McCleskey, E. W. and Rosenberg, R. L. (1987) Annu. Rev. Biophys. Chem. 16, 265-290 15 Begenisich, T. and Smith, C. (1984) in Current Topics in Membranes and Transport (Vol. 22) (Baker, P. F., ed.), pp. 353-369, Academic Press 16 Begenisich,T. and Lynch, C. (1974) J. Gen. Physiol. 63,675689 17 Cotman, C. W. and Iversen, L. L. (1987) Trends Neurosci. 10, 263-265 18 MacDermott, A. and Dale, N. (1987) Trends Neurosci. 10, 280-283 19 Ascher, P. and Nowak, L. (1987) Trends Neurosci. 10, 284288 20 Peters, S., Koh, J. and Choi, D. W. (1987) Science 236, 589593

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Macrophagesand microglia in the nervoussystem V. H u g h P e r r y a n d S i a m o n G o r d o n

Why might macr@hages be of interest to neurobiologists? Recent evidence shows that macrophages play a role in tissue homeostasis as well as in defence and repair of tissues. We will review here the possible functions of resident and recruited macrophages in the developing and adult nervous system and examine what contribution these cells might make to repair mechanisms in the central and peripheral nervous systems. There is increasing evidence that macrophages form an important component of the non-neuronal cell population in the nervous system and the tools are becoming available that allow us to study these cells in situ.

between central stromal macrophages and haematopoietic ceilsa. The study of macrophages in vitro, often a population of macrophages isolated from the peritoneum, has shown that these cells possess a remarkable repertoire of receptors 4 and secretory products 5 in addition to their well-known phagocytic and degradative capacities. As methods become available for the isolation and characterization of macrophages from different tissues, it is evident that the phenotype varies greatly according to the microenvironment.

V. Hugh Perryisat the Departmentof Expe#mental Psychologyand 5iamonGordonis at the 5it WilliamDunn Schoolof Pathology, Universityof Oxford, South ParksRoad, Oxford, UK.

Ontogeny of microglia Macrophages in tissues

Microgiia were first described by del Rio Hortega6 Macrophages differ from other haematopoietic cells in silver-stained preparations at the light microscope in that they form a resident population in many tissues level. We restrict our use of the term microglia to of the body 1. They are generated in the bone marrow those cells described by del Rio Hortega in the adult and circulate in the blood as monocytes before being CNS; they are a morphologically distinct type of cell delivered to their target tissues. Once they have with long, branched and crenellated processes. On entered the tissue they are known as macrophages, the basis of studies in which cells have been examined or in some tissues by more specialized names such as in the developing CNS using silver stains or electron the Kupffer cells of the liver. The macrophage microscopy, the microglial cell has at various times population is not static but there is variable turnover been described as mesodermal, monocytic or neuroof these cells in different tissues as senescent ones ectodermal in origin (reviewed in Refs 7, 8). If are replaced by cells from the blood and by local microglia were of monocytic origin it might have been proliferation, as in the lung. The number of macropha- expected that they would share cell surface antigens ges can be dramatically altered in response to injury with monocytes and this would allow their morpholoor infection by rapid recruitment from blood to the site gical differentiation to be followed. However, a of a local injury. These newly recruited cells, in number of immunocytochemical studies failed to contrast to the resident cells, have potent demonstrate such shared antigens. It has now been respiratory-burst activity (the capacity to generate shown in the mouse, using a monoclonal antibody highly reactive cytocidal oxygen metabolites) and are specific for mouse macrophages (F4/80), and sensiimportant for defence against infection 2. The activa- tive immunocytochemistry9, that during the tion of recruited macrophages by ~,,-interferon derived embryonic development of the retina and brain 1°'11, from stimulated T lymphocytes, represents an monocytes enter these structures and can be followed imrnunologically specific amplification mechanism to through a series of morphological transitions as they combat infection. Although some resident macropha- differentiate into microglia (Fig. 1). The number of ges may be relatively refractory to lymphokines 1, invading macrophages in the mouse CNS is greatest they are well placed at portals of entry to respond to during the late embryonic and early postnatal period. local injury or infection as a first line of defence. It is An important point to note is that the blood-brain becoming increasingly clear that the resident macro- barrier is intact at this stage 12 and not only must the phage plays a role in normal tissue homeostasis. In putative chemotactic signal be transmitted to the the bone marrow, for example, there is evidence for circulating monocytes but also these cells must cross non-phagocytic trophic and regulatory interactions the intact barrier. These results, taken in conjunction TINS, Vol. 11, No. 6, 1988

© 1988,ElsevierPublications,Cambridge 0378-5912/88/$02.00

273

NEWBORNWHITE MATTER

5 DAY O L D WHITE MATTER

5 DAY OLDGREY MATTER

..

Fig. 1. Changes in morphology as monocytes invade the developing nervous system and pass through a series of transitional forms as they differentiate into microglia in the mature CNS. The location of the cells is shown, but it should be noted that microglia in the adult are similar in form independent of whether they lie in the grey or white matter. Taken from sections in which the cells were labelled immunohistochemically with the monoclonal antibody F/80, specific for mouse macrophages. Bar = 25 Izm.

,

~t..

ADULT

with earlier studies 7 and the ever-increasing list of other cell surface antigens shared by macrophages and microglia 11'13'14, including molecules of known function, confirm that microglia are the resident macrophages of the brain (Table I). It should be noted that some of these cell surface markers are useful reagents for the study of microglia. Apart from the microglia there are macrophages with a different phenotype associated with the brain but lying outside the parenchyma. Macrophages are prominent in the leptomeninges and choroid plexus (Fig. 2). These cells have relatively short simple processes and express some antigens not found on mature microglia 11'14. The differences in form and phenotype of macrophages in the leptomeninges and choroid plexus when compared with the microglia suggest a different function. Small numbers of macrophages are found in the cerebrospinal fluid, which indicates that there may be traffic of macrophages through the choroid plexus to the cerebrospinal fluid in normal brain.

of the normal adult brain since macrophages in the developing brain have a different morphology (Fig. 1) and express different surface antigens when compared with microglia in the normal adult t4. In addition, the phagocytosis observed in the developing brain, and other receptor-mediated endocytic stimuli can influence the nature of products secreted by macrophages. The newly recruited cells in the developing brain may be more akin to those involved in an inflammatory response than to microglia. The number of macrophages in the CNS rapidly increases when there is considerable naturally occurring cell death in the retina as well as many CNS structures, and macrophages can be found phagocytosing dying cells l°'H. We believe that the dying cells in the CNS act as a chemotactic signal for the monocytes, which stimulates their recruitment. The fact that macrophages phagocytose dying cells in the developing brain suggests that they play a role in tissue modelling. However, the distribution of macrophages or immature microglia in the brain of newborn animals does not appear to be solely related to the distribution or numbers of degenerating cells and their processes. For example, in the white matter of the developing cortex when axons from cortical cells are degenerating, macrophages are concentrated in discrete regions and are not found uniformly throughout the white matter 11. Since the monocytes are delivered by the vascular supply, these local concentrations suggest that there may be some specialization of the blood vessels at these sites, but the problem of just how and where monocytes cross the intact blood-brain barrier has not been explored. It is known that macrophages can secrete a variety of growth and synergistic factors including intedeukin1 and tumour necrosis factor that can themselves stimulate angiogenesis 15'16 and glial proliferation 17'18. Both angiogenesis and gliogenesis occur pre- and post-natally in the rodent brain and it is possible that macrophages could contribute to these processes. Macrophages isolated from newborn rat brains have been shown to secrete factors that promote glial proliferation 18. The immature brain has a number of widely distributed extraceUular matrix components which are not found at later ages 19. Macrophages secrete several neutral proteinases that could degrade these substances once they had served their developmental purpose. It has been suggested that the plasminogen activator-plasmin system is involved in neuronal migration 2° and it is well established that recently recruited macrophages are a potent source of plasminogen activato& 1. The extent to which the diverse potential of macrophages is expressed during the development of the CNS is unknown.

Microglia in the adult CNS Following their migration from the blood vessels into the retina and brain and the phagocytosis of dying cells, the macrophages become distributed and persist in the tissue in regular arrays. The regular distribution of these microglia is most easily seen in the retina (Fig. 3A) but can also be observed in other brain structures (Fig. 3B). The nature of the cell-cell Macrophages in the developing CNS interactions that results in this specialized macIt is important here to distinguish between the rophage morphology, which is similar to that of macrophages in the parenchyma of the developing and Langerhans cells of the skin 1, is unknown. The fine-

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scale distribution of microglial processes within the neuropil has not been systematically mapped and there are no quantitative studies on the distribution of microglia throughout the brain. We cannot readily relate their distribution to some other cell type or transmitter which might allow us some insight into their function. The microglia are ubiquitous, more common in grey than in white matter, and there is some evidence that there are regions, for example the substantia nigra, where the microglia appear to be more numerous than in surrounding structures. The present evidence suggests that microglia in the adult may turn over rather slowly22'~3. Ceil surface antigens that have been demonstrated on microglia in normal brain include the Fc and complement type-three (CR3) receptors n and so in principle these ceils might play a role in the defence of the CNS. The brain has been thought of as an immunologically privileged site - it lacks a lymphoid system and there is no evidence for a population of dendritic cells. Dendritic cells are important for 'antigen presentation' in lymphoid tissues, as they constitutively express high levels of class II molecules, are able to stimulate a mixed lymphocyte reaction and have a distinct morphology24. Class II molecules of the major histocompatability complex are only expressed on some specialized ceils (including dendritic cells and macrophages) and these molecules are important in the activation of helper T (Th) lymphocytes. The expression of class II antigens on ceils in the CNS, which might then act as antigenpresenting cells, does not occur in normal rodents 2s but is variable in studies of human CNS 26. The expression of class II antigens has been demonstrated on microglia in a variety of pathological conditions. In addition to the expression of class II antigens on microglia, it has been shown that astrocytes in vitro can also express class II antigens and present antigen to T lymphocytes27. The important question as to whether the microglia or the astrocytes can trigger an immunological reaction in the CNS is unanswered. It is interesting to note that astrocytes share a number of secretory products with macrophages, for example apolipoprotein-E, prostagiandins and interleukin-128. The significance of the resemblance in phenotype between astrocytes and macrophages is unclear. On a topical note, the microglia in the rat express low levels of the CD4 antigen 14. In man this antigen serves as an entry receptor for HIV-1, the virus involved in AIDS. (Macrophages in rat and human express the CD4 antigen as do the Th ceils.) The expression of this antigen on the microglia is regulated during differentiation and is also found at increased levels on microglia around a site of injury, so-called activated microglia. There is evidence that the bloodbrain barrier plays a role in the regulation of CD4 antigen expression. The microglia in structures outside the barrier that are exposed to plasma, for example in the subfornical organ and median eminence, express higher levels than those lying within the barrier and isolated from plasma proteins. The expression of CD4 is in marked contrast to that of the CR3 receptor, which is present at high levels on all microglia in and outside the blood-brain barrier, although it can be down-regulated on tissue macrophages outside the CNS. It has been demonstrated TINS, Vol. 11, No. 6, 1988

Fig. 2,

in the brains of AIDS patients that the virus is localized within macrophages/microglia 29. Whether the extensive and prevalent neuropathology3° associated with AIDS can be linked to abnormal macrophage/microglia function within the CNS is not yet known. A possible role for microglia in neurotransmitter metabolism has been suggested 31. Although direct evidence for this proposal is lacking, it is interesting to note that macrophages have receptors for a number of known and putative neurotransmitters (e.g. Ref. 32), contain enzymes that degrade various neurotransmitters u3 and have high levels of~ enzymes involved in the metabolism of amino acid neurotransmitters 34.

Theappearanceof immunohistochemicWlylabelled macrophages (arrows) in the choroid plexus. The cells were labelled with the monoclonal antibody 0X42 directed against the iC3b receptor. Note that the cellshave a relatively simple stdlate morphology. Bar = 25 #m.

Macrophage response to CNS injury The response to injury of different ceils in the CNS is an area of intensive research because of the interest in the limited capacity of the CNS for regeneration. A specific question is which of the different glia, astrocytes, oligodendrocytes or microglia, retain the capacity to proliferate, and give rise to the characteristic increase in non-neuronal ceils, known as gliosis. It has been established that some astrocytes, in particular the type 1 astrocytes, retain the abilty to proliferate 35. The question as to whether the microglia can divide is at the moment open. It is clear that following an injury such as a stab wound, which damages the blood-brain barrier, circulating monocytes are recruited in large numbers. Whether any resident microglia divide in these circumstances is TABLE I. Phenotype of macrophages in the developing rodent CNS, and microglia in the normal adult rodent CNS Antigen

Developing CNS AdultCNS Function

F4/80

+

Fc CR3

+ +

+ + +

CD4

+

+/-

+ ND

+/+/-

ND

-

Leucocyte common antigen Transferrin receptor CLass II MHC (la)

? Ig binding Complement binding and adhesion to endothelium and other ligands ? Other than HIV entry ?

Transferrin uptake Cellular recognition in immune response

+ indicates that the antigen is readily detectable. + / - indicates that the antigen is weak or limited in its expression- the antigen is undetectable. (ND = not done.)

It is of interest to know whether the increase in astrocyte number and their hypertrophy is stimulated by an astroglial growth factor released by the recruited macrophages 18. Recent evidence suggests that the oligodendrocytes play a critical inhibitory role in CNS regeneration a7, since neurites growing in vitro appear actively to avoid oligodendrocytes in their path. A feature of Wallerian degeneration in the CNS is that the recruitment of macrophages to the distal segment away from the injury is pooraS. The poor recruitment correlates with the persistence of myelin and axonal debris in Wallerian degeneration within the CNS. This is in marked contrast to the brisk macrophage recruitment and rapid removal of debris in the PNS that accompanies PNS regeneration. The removal of debris and re-establishment of a normal blood supply is an important component of inflammation and repair in other sites of the body and we would expect macrophages to play a similar role in the nervous system. It has recently been demonstrated that the inflammatory response in the CNS can produce unwanted side effects39. Lesions produced by the injection of ibotenic acid, which are intended to destroy neuronal cell bodies but leave axons en passage intact 4°, are accompanied by a dramatic inflammatory reaction at the site of the lesion within a few days. If this lesion is made in a region of the brain where the fibre fascicles are small and well Fig. 3. (A) Microglia in the outer plexiform layer of a retina dispersed, such as in the septum, the intense stained immunohistochemically with the antibody F4/80 inflammation can be sufficient to demyelinate or even and viewed in a wholemount. (B) Microglia in the cortex immunohistochemically labelled with 0X42. Note that in destroy the normal axons en passage 39. The fact that both the retina and cortex the cells are arrayed in a regular stimulated machrophages secrete myelinolytic profashion and have long, branched, fine processes. Compare teinases is well documented 41. There is evidence that post-traumatic inflammation in the spinal cord may these cells with those shown in Fig. 2. Bar = 25 i~m. lead to demyelination of axons that had survived the initial injury 42. technically a difficult question to resolve since we have to distinguish between recruited and dividing Macrophage response in PNS injury resident cells. The contribution that recruited monoA small number of macrophages is found in the cytes or resident non-neuronal cells make to the normal peripheral nerve. Although these macrophaincreased cell numbers after stab wounds and other ges are stellate in form they do not have the elaborate types of CNS injury is controvers~ (reviewed in Ref. processes of CNS microglia. The possible importance 36). of macrophages in peripheral nerve regeneration was The functions of astrocytes, oligodendrocytes and highlighted in recent experiments by Beuche and macrophages in the repair processes at a site of injury Friede 4a. They placed fragments of sciatic nerve in or neuronal degeneration in the CNS are not well miilipore chambers within the peritoneal cavity of defined. On the one hand it has been suggested that rats. The millipore chambers were designed so that astrocytes may provide an inhibitory barrier to CNS they would either exclude or allow the entry of nonregeneration, on the other they may release trophic resident cells. In the chambers from which nonfactors which modify neuron extension and survival36. resident ceils were excluded, the myelin sheaths of TABLE II. Possible functions of macrophages in the nervous system

Development and remodelling

Homeostasis

Lipid turnover

Inflammation and repair

Immune responses

276

Phagocytosis (direct and opsonin mediated), extracellular matrix catabolism (neutral proteinase secretion), production of angiogenesis and other growth factors (e.g. GM-CSF) and inhibitors (e.g. TGF-13) Neurotransmitter and hormone processing and catabolism (e.g. purines, amino acids [glutamine], peptides [neurotensin, enkephalin], glucocorticoids, opioids, adrenergic, cholinergic) Ganglioside and phospholipid catabolism, apolipoprotein binding and secretion Release of mediators (e.g. interleukin-1, tumour necrosis factor, prostaglandins, leukotrienes), neutral proteinases (e.g. plasminogen activator and other myelin degrading enzymes), cytotoxic agents (oxidative radicals) and growth factors (e.g. gliogenesis) Antigen processing (? MHC class II-dependent) and macrophage activation by lymphokines (,/-interferon) with resultant priming of respiratory burst

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TINS, VOI. 11, NO. 6, 1988

the sciatic nerve segments survived for several weeks and the Schwann cells did not proliferate. If leucocytes were allowed access to the nerve segments then the myelin sheaths were rapidly removed and the Schwann cells proliferated. The conclusion was that macrophages may be necessary for the removal of the debris from a degenerating nerve and that a signal from the macrophage may be necessary for Schwann cell proliferation. Since Schwann cell proliferation is an important component of peripheral nerve repair the results suggest that macrophages may be an important element in the repair process. Immunocytochemical evidence shows that macrophages are recruited to the damaged sciatic nerve in situ and the time course of this recruitment is compatible with the role suggested aboveaa. A rather different line of research has also suggested a role for macrophages in peripheral nerve regeneration. Following a crush injury to the sciatic nerve there is a dramatic increase in the synthesis of apolipoprotein-E in the distal segment of the nerve ~. It is proposed that the apolipoprotein-E is secreted by recruited macrophages and involved in the redistribution of lipid from the degenerating to the regenerating axons. It has been shown that growth cones of PC12 cells have receptors for apolipoprotein-E 4~. The macrophage possibly plays an important role in lipid turnover acting as an intermediary between the degenerating and regenerating axons.

Summary Macrophages play a part in normal CNS development during the period of naturally occurring cell death, but their role in the adult CNS is at present a list of possibilities rather than proven (Table II). There is evidence that macrophages may be involved in peripheral nerve repair but in the damaged CNS they may either respond poorly or too actively. The microglia of the adult CNS are an extreme macrophage specialization and it seems likely that their distinctive morphology is accompanied by specialization of function. Macrophages and microglia have the potential to interact with all the other cell types of the CNS and PNS.

Selected references 1 Gordon, S. (1986) J. Cell 5ci. (Suppl. 4), 267-286 2 Gordon, S., Crocker, P. R., Lee, S-H., Morris, L. and Rabinowitz, S. (1986) in Host-Resistance Mechanisms to Infectious Agents, Tumours and AIIografts (Steinman, R. M. and North, R. J., eds), pp. 121-137, Rockefeller University Press 3 Crocker, P. R. and Gordon, S. (1986) J. Exp. Med. 164, 18621875 4 Wright, S. D. and Silverstein, S. C. (1986) in Handbook of Experimental Immunology (Vol. 2) (Weir, D. M., ed.), pp. 41. 1-41.14, Blackwell 5 Nathan, C. F. (1987) J. Clin. Invest. 79, 319-326 6 del Rio Hortega, P. (1932) in Cytology and Cellular Pathology of the Nervous System (Penfield, W., ed.), pp. 482-534, Paul B. Hoeber 7 Ling, E. A. (1981)in Advances in Cellular Neurobiology (Vol. 2) (Federoff, S. and Hertz, L., eds), pp. 33-82, Academic Press 80emichen, M. (1982) in Recent Advances in Neuropathology (Vol. 2) (Smith, W. T. and Cavanagh, J. B., eds), pp. 83-107, Churchill Livingstone 9 Austyn, J. M. and Gordon, S. (1981) Eur. J. Immunol. 10, 805-811 10 Hume, D. A., Perry, V. H. and Gordon, S. (1983) J. Cell. Biol. 97, 253-257 TINS, Vol, 11, No. 6, 1988

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Acknowledgemen~ We thank Dr M. C. Brown for his commentson the manuscript. Thiswork wassupportedby the MRC (UK). VHPis a Senior Wellcome ResearchFellow.

Errata In the article 'Atrial natriuretic factors and the brain: an update' by R~mi Quirion (February 1988, Vol. 11, pp. 58-62), Fig. 1 showing ANFimmunoreactive neurons in the frog preoptic nucleus was unfortunately printed upside-down. Also, in the article 'Is there a correlation between continuous neurogenesis and directed axon regeneration in the vertebrate nervous system?' by Nigel Holder and J. D. W. Clarke (March 1988, Vol. 11, pp. 94-99), the word 'not' was omitted from the last sentence of the article. The sentence should read: 'This view suggests that the ability to regenerate is an inevitable consequence of continuous growth and not a process selected for in its own right'. We apologize for these errors.