Cell Surface Morphology Identifies Microglia As a Distinct Class of Mononuclear Phagocyte
The Journal of Neuroscience, November 1995, 15(11): 7712-7726
Cell Surface Morphology Identifies Microglia as a Distinct Class of Mononuclear Phagocyte
Dana Giulian,’ Jun Li,’ Sandra Bartel,’ Jennifer Broker,’ Xia Li,’ and Joel B. Kirkpatrick* ‘Department of Neurology, ‘Baylor Center for AIDS Research, ‘Department of Pathology, and 1s2Alzheimer’s Disease Research Center, Baylor College of Medicine, Houston, Texas 77030
To investigate differences among brain-derived microglia kines, brain injury, development, neurodegeneration, HIV- and other classes of immune cells, we compared the mor- 1, rat, human] phologies and growth properties of mononuclear phago- cytes isolated from tissues of the newborn rat. Scanning Microglia, a classof brain mononuclearphagocyte, are thought EM shows that microglia from postnatal rat brain are cov- to be the principal immune cells resident to the CNS (Giulian, ered with spines (typically >20 per cell body) in a distinc- 1992; Flaris et al., 1993). The functional capacitiesof microglia tive manner which contrasts the smooth surfaces of bone have received increasingattention as thesecells have beenfound marrow cells and the ruffled surfaces of tissue macro- to be the major sourceof brain cytokines (Giulian and Lachman, phages from spleen, liver, and peritoneum. The spine-bear- 1985; Hetier et al., 1990; Higgins and Olschowka, 1991) and ing surface of microglia is a specific cell marker, for it does have been implicated in the destruction of neuronsduring stroke not change with age or after exposure to cytokines or other (Giulian and Robertson, 1990; Giulian et al., 1993) trauma immunostimulants. Approximately 99% of mononuclear (Banati et al., 1993; Thanos et al., 1993), Alzheimer’s disease phagocytes cultured from normal adult rat brain are spi- (Bolsi, 1927; McGeer et al., 1987; Giulian et al., 1995) and HIV-l infection (Giulian et al., 1990). Primitive stem cells, nous microglia. Five days after injury to rat brain, cells at which give rise to microglia, are first seenin the periventricular sites of Wallerian degeneration are essentially all spinous regionsduring early embryonic developmentand eventually take ones while nearly 30% of cells found within areas of in- up residence throughout the fetal brain (Rio-Hortega, 1932; farction or penetrating trauma are invading macrophages. Ling, 1981). In the late fetal and early neonatalperiods, matur- In a similar way, nearly all cells isolated from normal, post- ing microglia differentiate from the ameboidinto ramified forms, mortem adult human brain are spine-bearing microglia characteristic of the quiescent cells found in the adult nervous (>99% homogeneity). Brains from patients with amyotro- system (Rio-Hortega, 1932). phic lateral sclerosis contain only spinous microglia Although many investigators accept the hypothesis that stem whereas cells from HIV-1 infected brains include significant cells common to all classesof mononuclearphagocytes give rise numbers of invading ruffled. macrophages. to microglia in early stagesof development(Rio-Hortega, 1932; Cultured microglia, unlike cultured bone marrow precur- Ling and Wong, 1993), there is less agreementas to the rela- sors, monocytes, or tissue macrophages, spontaneously tionships among microglia and other classesof mononuclear develop long, thin processes that extend hundreds of mi- phagocytes after birth (Imamoto and Leblond, 1977). During crons in length. Microglia retract these processes after ex- CNS pathology, microglia have been describedeither as a dis- posure to fetal bovine serum, laminin, or such immuno- tinct cell population (Hickey and Kimura, 1988) or, alternatively, stimulants as recombinant murine interferon y (rmlFNy) as a wave of invading blood monocytes (Perry et al., 1985; and lipopolysaccharide. Of all types of mononuclear phag- Jordan and Thomas, 1988). Part of this controversy arisesfrom ocytes tested, only microglia differentiate into quiescent the fact that cell markersexpressed in developing microglia are ramified cells when in contact with astrocytes. Thus, mi- often difficult to detect in mature, ramified cells (Perry et al., croglia represent a unique class of cell maintained, in part, 1993). For example, nonspecificesterase, which is expressedby by astroglia as dormant, ramified mononuclear phagocytes peripheral mononuclearphagocytes throughout development, is in mature CNS. Application of cell surface criteria de- normally only seenin microglia during fetal and early postnatal scribed here will allow study of distinct populations of development(Giulian and Baker, 1986). Becausenonspecific es- mononuclear phagocytes associated with neurologic dis- terase(+) cells exist within the newborn rat brain, Ling et al orders. (1982) concluded that blood cells invade the postnatal brain, [Key words: microglia, macrophages, monocytes, cyfo- while other investigators finding no macrophagicmakers in ma- ture brain came to an opposing conclusion (Wood et al., 1976; Oehmichen, 1983). In general, efforts to establisha panel of Received June 9, 1995; revised July 24, 1995; accepted July 31, 1995. microglia-specific markershave proved elusive, with expression The work was supported by NIH Grants NS25637, NS23 113, and MH48652, The Vivian Smith Foundation, Alzheimer’s Association, and Baylor’s Alzhei- dependentupon age, speciesand prior exposure to immunosti- mu’s Disease Research Center. mulants(Giulian, 1992; Flaris et al., 1993; Ulvestad et al., 1994). Correspondence should be addressed to Dana Giulian, M.D., Ph.D., Depart- Despitethe uncertaintiesassociated with histochemicalmarkers, ment of Neurology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. observations basedupon other techniques suggestmicroglia to Copyright 0 1995 Society for Neuroscience 0270-6474/95/157712-15$05.00/O be a distinct classof cell. Unlike differentiated blood monocytes, The Journal of Neuroscience, November 1995, 15(11) 7713 microglia may proliferate in postnatal and adult brain (Graeber A et al., 1988; Giulian et al., 1989), suggesting features of stem cells found in bone marrow. Patch clamp study of membrane EOOr conductances (Kettenmann et al., 1990) demonstrates inward di- rected rectifying potassium currents for microglia which are not found in monocytes or macrophages. Moreover, Hickey and Ki- mura (1988) note that parenchymal microglia of adult chimeric rats do not arise from marrow-derived stem cells or blood-borne monocytes. In this report, we compare brain-derived mononuclear phag- ocytes recovered from newborn rat with other classes of mono- nuclear phagocytes isolated from newborn blood, spleen, liver, marrow, and peritoneum. We find that cultured microglia display Days In Culture distinctive features including surface morphology, patterns of - Microglia differentiation, and responsiveness to cytokines. Moreover, scan- ning EM distinguishes microglia from invading blood-borne IZi Liver macrophages at sites of adult brain injury. Although astrocyte B IS8 Spleen contacts with microglia accelerate differentiation of ameboid cells into quiescent ramified ones, astroglia do not shape the 0 Marrow behaviors of other classes of mononuclear phagocytes. Further, study of human brain cells indicate scanning EM to be a useful m Blood technique to characterize inflammation associated with neuro- ETEJ Peritoneum logical disease.
Materials and Methods Isolation and identijkation of cells. Mononuclear phagocytes were iden- tified by their ability to engulf fluorescent polystyrene microspheres (0.7 pm, Covaspheres Particles, FX Green, Duke Scientific, Palo Alto, CA), by the presence of scavenger receptors (Giulian and Baker, 1985; Giu- lian et al., 1989) visualized with the fluorescent probe 1,l ‘-dioctadecyl- 1,3,3,3’,3-tetramethyl-indo-carbocyanine bound to acetylated low den- Figure I. A, Proliferation of mononuclear phagocytes isolated from sity lipoprotein (DiI-ac-LDL; obtained from Biomedical Technologies different tissues of newborn rat. When placed in chemically defined Inc., Stoughton, MA), by the presence of nonspecific esterase activity culture media free of serum, only cells isolated from brain showed spon- (Koski et al., 1981), and by immunostaining with the OX-42 antibody taneous proliferation. B, Mean frequency of process bearing cells for (Serotec, Kidlington, England) at 30 p,g/mK Astroglia were identified classes of mononuclear phagocytes was scored with a process defined bv immuno-labeling for glial fibrillarv acidic orotein (GFAP) usine rab- as >3-fold cell body diameter. As shown, about 25% microglia under- bit anti-bovine GFLP (Dako, Glostmp, Denmark) at 40 &ml & de- scribed previously (Giulian et al., 1986). went ramification by 3 d in vitro. Mononuclear phagocytes were isolated directly from various organs of rat following enzymatic/mechanical dissociation of tissues, separation by a ficol/sodium diatrizoate gradient (Histopaque 1077; Sigma, St. Louis, MO), and selective adhesion to glass or plastic (Giulian and 35% sucrose and separated by a ficol/sodium diatrizoate gradient (His- Baker, 1986). All cells were grown on glass coverslips in chemically tooaaue 1077: Sigma. St. Louis. MO). Cells were nlaced in N2 media defined N2 medium (Bottenstein and Sato, 1979) free of serum. Brain, containing gentamycin (48 kg/ml) and recovered by selective adhesion liver, and spleen were mechanically dissociated in the presence of 0.2% to plastic for 12 hr (Giulian and Baker, 1986). We typically recover l- trypsin (T-2904, Sigma) plus 0.2 mg/ml of DNase (D-5025, Sigma) for 3 X lo5 cells per gram wet weight of tissue. Amyotrophic lateral scle- 15 min at 37°C. Blood monocytes (Boyum, 1968), resident macro- rosis brains were from patients with neurogenic atrophy of skeletal mus- phages lavaged from the peritoneum (Daems, 1980), and femur marrow cles, loss of anterior horn cells, and spared intellectual function. Normal aspirates (Stewart, 1981) were mechanically dispersed without protease control brains were from adult patients with no brain pathology. HIV- by trituration for 10 min. In some experiments, highly enriched popu- l(+) brain tissues were acquired from infected adults showing micro- lations of ameboid microglia and astroglia were isolated from confluent glial nodules and giant cells but lacking evidence of other pathology dissociated brain cell cultures by rotatory shaking for 18 hr with the including parasitic infection, neoplasm, infarction, or abscess. suspension of “freed” cells collected by two rounds of selective ad- Mononuclear phagocytes were-studied after exposure to lipopolysac- hesion to plastic as described earlier (Giulian and Baker, 1986). The charide (LPS. #3120-25-O. Difco. Detroit. MI). recombinant murine tu- remaining feeder layers of astroglia were treated with carbonyl iron (1.5 mor necrosis factor 01 (rmTNFoc,‘#B3368, Genzyme, Cambridge, MA), pg per cm* of culture dish surface) once per day for 2 consecutive days recombinant murine interleukin-la (rmIL-la, #B1457, Genzyme), re- in order to eliminate phagocytic microglia by a magnet (Advanced Mag- combinant murine interferon y (rmIFNy, #B4990, Genzyme), recom- nets, Inc.; Cambridge, MA). These enriched astroglia [>99% homo- binant murine granulocyte macrophage colony stimulating factor geneous population of GFAP(+) cells] were mechanically dissociated (rmGM-CSE #B3630, Genzyme), laminin (#08-125, UBI, Lake Placid, in the presence of 0.2% trypsin and were then seeded in 100 mm culture NY), fibronectin (#F1141, Sigma), collagen IV (#CO543, Sigma), and dishes at a density of 0.5 X 10h cells per ml of chemically defined N2 fetal bovine serum (#F2442, Sigma). medium (Bottenstein and Sato, 1979). Typically, these enriched astro- Scanning electron microscopy. Cells adhering to glass coverslips glia preparations contained
Figure 2. Scanning electron photomicrographs showing the surface features of mononuclear phagocytes of the newborn rat isolated from differ rent tissues arid maintained in culture for 3 d. A, Microglia recovered from the newborn brain show both long processes and spines which radiate out from cell body. B, Peritoneal macrophages remain ameboid in shape with a ruffled surface membrane, few spines, and short stubby projections . c, Round, srmooth profiles of bone marrow mononuclear phagocytes. D, Short, stubby processes and ruffled surfaces are the main features of spleten- derived macrophages. Scale bar, 5 pm.
tive colloidal silver and viewed using a JEOL JEM-100 CX between 5 d post axotomy using standard methods as described above. Pene- 500 and 10,000 magnification. trating injury to the neocortex was produced with a microknife in adult Bruin injury models.Optic nerve transection was carried out in adult albino rats deeply anesthetized and placed in a stereotaxic device (David albino rats anesthetized by intraperitoneal injection using a combination Kopf Inc., Tujunga, CA). After the scalp was reflected, burr holes were anesthetic (0.14 mg ketamine, 0.29 mg xylazine, and 0.014 mg ace- placed 1 mm lateral to either side of the midline and rostra1 to caudal promazine per 100 gm body weight). The optic nerve was exposed by incisions were inflicted by a microknife at a depth of 3 mm. Reactive an incision placed through the lateral palebral fissure and the nerve was microglia and invading macrophages were recovered from dissociated transected while sparing the ophthalmic artery. Reactive microglia as- neocortex in the region of injury-after 5 d. Transient ischemia was sociated with degenerating axons were recovered from the optic tecta produced by occlusion of the middle cerebral artery in adult rats (Hsu et al., 1990). Using microsurgical techniques, a burr hole was made 1 mm rostra1 to the anterior junction of the zygomatic and temporal bones. Both common carotid arteries were occluded with clips, after which the Table 1. GM-CSF effect on microglial ramification right middle cerebral artery was temporarily blocked by retraction with a curved 100 pm diameter needle. After 45 min, the needle and clips Mean process lengths (pm 2 SEM) were removed and the wounds sutured. Reactive microglia and invading macrophages were recovered from cortical infarcts 5 d after injury (Giu- Control rmGM-CSF lian et al., 1993). Macrophages 38.2 + 2.7 35.5 t 2.8 Results Microglia 99.5 2 8.8 157.0 + 10.9 Distinctive microglial patterns of cell survival and Isolated peritoneal macrophages and microglia were placed in culture for 3 d diflerentiation in the presence or absence of 50 units/ml of rmGM-CSE Scanning EM was used to measure process lengths from 100 randomly selected cells defined as To assess differences among populations of mononuclear phag- process-bearing (process lengths >3X diameter of the cell body). ocytes, we compared the behaviors of cells isolated from brain The Journal of Neuroscience, November 1995, 75(11) 7715
10 15 20 25 30 % Volume Fetal Bovine Serum
130 2 25 r" m Microglia 'f 20 m Peritoneum f 15 I Marrow
2 10 m Blood 8 25 0.
be 0 control collagen laminin fibronectin
C
Figure 4. Scanning electron photomicrographs of mononuclear phag- ;z ocytes exposed to 50 units of GM-CSF for 3 d. As shown, there was 1 no changein cell shapesfor peritonealmacrophages (A) andmarrow cells(B) while significantnumbers of microgliabecome highly ramified (C). Scalebar, 5 pm. 2ROo? 30 Laminin (ug/ml)
Figure 3. A, The presenceof fetalbovine serum decreases the number with thoseof cells from other tissuesof the newborn rat. During of process-bearingmicroglia. B, Laminin(100 pg/ml) decreasesthe the first week in vitro, brain-derived microglia spontaneously numberof process-bearingmicroglia but hasno effect uponother class- proliferated while little cellular division wasnoted amongmono- es of mononuclearphagocytes. C, Lamininadded to culturemedium nuclearphagocytes from liver, spleen,peritoneum, or blood (Fig. decreasesthe numberof process-bearingmicroglia in a dosedependent 1). Bone marrow cells generally survived poorly in culture and fashion. did not proliferate in the absenceof colony-stimulating factors. Within hours after plating, microglia underwent processforma- tion while cells derived from bone marrow, liver, spleen,or peri- toneumretained ameboid shapes (Fig. 2). By 3 d in culture, 25% 7716 Giulian et al. - Properties of Microglia
A
70 - GM-CSF cu ES0 a TNF 2 2 5o z ii%?8 IFN 40 -0 81%.l 1 10 100 2 30 1 IL-1 .t GM-CSF (Units/ml) E a 20 a m LPS D 10 8 El Control 0
B * :: 10 100 1000 Interferon Gamma (Units/ml) m Micronlie E m Peritoneum
I Marrow
m Blood
m Liver
m Spleen T !inxJh I,,Control i&l-CSF * Oo? 30 Laminin (ug/ml) Figure 5. Effects of cytokines upon morphologic differentiation of mononuclear phagocytes. A, Only rmGM-CSF (50 units/ml) induces process formation, while rmIFNy (100 units/ml) and LPS (1 bg/ml) reduces it. We observed no effects of rmTNFa (100 units/ml shown here; tested from 1 to 1000 units/ml) and rmIL-lcz (10 units/ml here; tested from 1 to 100 units/ml). B, The ability of rmGM-CSF (25 units/ml) to stimulate ramification occurs only with microglia and not other classes of mononuclear phagocytes. C, Dose dependent effects of rmGM-CSF upon microglial differen- tiation D, rmIFNy decreases microglial process formation in a dose dependent manner. E, Laminin reverses the effects of rmGM-CSF (25 units/ ml) induced ramification. of all microglia were ramified (defined as cells with process reached beyond 500 pm. This pattern of process formation by lengths more than three times the diameter of the cell body) microglia in culture paralleled the pattern of differentiation seen compared with ~2% of the other tissue macrophages and none in tissues of maturing CNS as ameboid microglia transform into of the bone marrow-derived cells (Fig. 1). The mean process ramified cells (Rio-Hortega, 1932; Giulian et al., 1988; Ling and length for microglia of 100 pm (Table 1) was far greater than Wong, 1993). Cellular ramification in vitro thus serves as an that of macrophages and, in some cases, microglial branches anatomic marker for microglial differentiation. The Journal of Neuroscience, November 1995, 15(11) 7717
Signals altering microglial differentiation We next considered what signals influence differentiation of brain mononuclear phagocytes. Spontaneous process formation by microglia was reduced after cells are exposed to fetal bovine serum; the serum blockade was dose dependent and occurred when cells were plated directly onto glass or plastic (Fig. 3A). We found that commonly usedconcentrations of serum (5% to 10%) had significant impact on the ability of isolatedmicroglia to differentiate. Laminin, an extracellular matrix protein, added to culture media also markedly reduced microglial ramification (Fig. 3&C), whereas other matrix proteins such as collagen and fibronectin had no effect (Fig. 3B). Of the battery of cy- tokines tested, only recombinant murine granulocyte macro- phage colony-stimulating factor (rmGM-CSF) promoted mi- croglial ramification, with >65% of cells becoming process bearing (Figs. 4, 5) within 48 hr. This morphologic differenti- ation induced by rmGM-CSF was specific to microglia and not seenwith tissuemacrophages, marrow precursor cells, or blood monocytes (Figs. 4, 5). The mean length of microglial pro- cessesalso increased by about twofold in the presence of rmGM-CSF (Table 1). On the other hand, activators known to promote microglial secretory activity, such as LPS and rm- IFNy, elicited ameboidshapes and retraction of processes(Fig. 5A) similar to the responsesseen in tissue during reactive mi- crogliosis. Cytokine regulation of microglial’ differentiation was dosedependent for both GM-CSF stimulation and rmIFNy suppression(Fig. 5C,D). Interestingly, laminin overcame the stimulatory effect of rmGM-CSF (Fig. 5E) suggestingthat lo- Figure 6. Fluorescencephotomicrographs showing spines (arr which extend out from the surfaces of DiI-ac-LDL(+) microglia. 5 cal depositsof extracellular matrix might moderate effects of bar, 3 pm. cytokines upon microglial differentiation.
Surface morphology of rat microglia The spinoussurface-a microglial marker for injured adult rat brain Rio-Hortega (1932) first noted, using silver carbonate staining, To determineif surfacecharacteristics could be usedto identify that the surfacesof microglia possesseddelicate spines.We also microglia in later stagesof development, we isolated mononu- observed spinesborne by fluorescently-labeledmicroglia in cul- clear phagocytes from normal brain, spleen,and liver of adult ture (Fig. 6) which were not apparentin other classesof mono- rat. Nearly all adherent cells recovered from normal adult rat nuclear phagocytes. Further study by scanning electron photo- brain (>99% of cells recovered) were microglia-like with more micrographsrevealed that microglial surfaceswere, in fact, cov- than 20 spinesper cell body and no ruffles (Fig. 9A). In contrast, ered with spinesas if protected by thorns (Fig. 7A). In contrast, liver macrophagesshowed ruffles (>90% of all cells) with few the surfacesof bone marrow-derived mononuclear phagocytes spinous surfaces(Fig. 9C). Only microglia were both ramified were generally smooth (Fig. 7B) while tissue macrophages and spine-bearing.We went on to determine whether surface showedruffling of membranes(Fig. 7C). Microglial spineswere featurescould be usedto distinguishtypes of mononuclearphag- between 2 and 4 pm in length with diametersof about 0.1 pm. ocytes cells appearingat sitesof brain damagein adult rat. We In addition, microglia also contained thread-like structures that isolated mononuclearphagocytes after inflicting different types extend several micrometersbeyond the cell surface (Figs. 7D- of brain injury and compared the numbersof spine-bearingmi- F). These threads arosefrom spinesfound on either microglial croglia to those of ruffled macrophages.As shown in Figure cell bodies or spinesfound on cell processes.Overall, microglial lOC, we recovered only microglia at sitesof axonal degeneration cellular projections (processes,spines, and thread-like networks) (i.e., from optic tectum after optic nerve transection)while 30% were strikingly different from the short, stubby processesof tis- of mononuclearcells isolated from brain damagedby stroke or sue macrophages.About 40% of these macrophagesisolated trauma were ruffled macrophages(Fig. 9B). Moreover, reactive from liver, spleen, or peritoneum of newborn rat had ruffled microglia from adult brain underwent ramification in culture membranes,in contrast to <3% of cells isolatedfrom brain (Fig. while tissuemacrophages did not (Figs. lOA,B). Thus, Rio-Hor- 8A). Nearly 95% of microglia had >20 spinesper cell (Fig. 8s) tega’s description of microglia as cells with “wavy processes while about 98% of peritoneal macrophagesshow <20 spines beset with spines” appearedto be an accurate and reliable way per cell. The differences in surface features among microglia to distinguishmicroglia from blood-bornemacrophages through- and other classesof cells persistedfor at least 30 d in culture out postnatal development. In general, CNS insults producing and were not affected by exposure to cytokines or immunosti- local hemorrhage(such as stroke and penetrating trauma) re- mulants (data not shown). Importantly, engulfment of particles cruited blood-borne macrophages,while distal injuries involving such as zymosan did not obscurethe spinoussurfaces of phago- axonal degenerationelicited only reactive microglia (Kreutzberg cytic microglia (Figs. 8C,D). and Barron, 1975; Hickey et al., 1992). 7718 Giulian et al. l Properties of Microglia
Figure 7. Scanning electron photomi- crographs at high magnification show- ing the detail of mononuclear phago- cytes. Microglia typically display 20 or more spines (A) while blood monocytes have smooth surfaces (B) and perito- neal macrophages have membrane ruf- fles (C). Scale bar, 5 km. D, Scanning electron photomicrograph of thread- like projection arising from microglial spines. Scale bar, 2.5 km. E and F, In addition to processes (0.5 km diame- ter) and spines (0.1 pm diameters), the microglia cells demonstrate a delicate network of thread-like structures (0.05 pm diameters) which spread out be- yond the cell’s surface. Scale bar, 0.5 pm.
Surface morphology of human microglia comparedcell types isolatedfrom humanbrains known to contain As noted above, the classesof reactive mononuclearphagocytes reactive mononuclearphagocytes. Neocortical mononuclearcells which appearwithin damagedbrain dependupon the nature of found in amyotrophic lateral sclerosis(ALS) were essentiallyall the injury. This problem becomesmore pressingin the study of spine-bearingmicroglia (Fig. 1lD; Table 2) while significant human pathology since cell specificmarkers are not available to numbersof ruffled macrophageswere found in HIV-l infected distinguishreactive microglia from macrophages.Accordingly, brains (Fig. llF, Table 2). The patternsof appearancefor these we applied cell surface criteria to cells isolated from human cell populationssuggest that slow, degenerativedisorders such as tissueswithin a 6 hr post mortem interval. In a pattern similar ALS recruit reactive residentmicroglia while HIV-l infection in- to rat, we found recovered ruffled macrophagesfrom human volves compromisedblood-brain barrier and increasedtrafficking liver and from blood (Fig. 11E) and spine-bearingmicroglia of monocytesinto the CNS (Flaris et al., 1993). from normal adult human brain (Fig. 11A). Human microglia developed processesin vitro as found for the rodent cells and Astroglial interactions with mononuclearphagocytes theseprocesses were covered with spines(Figs. 1lC,D). Quan- As describedhere, different classesof isolatedmononuclear phag- titation of suchcells indicated that >99% of mononuclearphag- ocytes behaved in distinct ways when grown in culture. It re- ocytes recovered from normal human brain possessedspinous mained possible,however, that blood monocytes,bone marrow morphology (99.0 + 0.3%; n = 300). precursors,or tissuemacrophages might take on featuresof mi- We extended these observationsto neurologic diseasesand croglia after exposure to a “brain environment.” To determine, The Journal of Neuroscience, November 1995, 75(11) 7719
B
m Microglia EEI Peritoneum m Microglia
Di Liver 70 v) 60 = iSI Spleen 0* 50 40 El Marrow ‘iij z 30 I- m Blood 20 28 @II Peritoneum 10 0 O-9 10-19 20-29 *30 Spines per Cell Body
Figure 8. Surfacemorphological features for differentclasses of mononuclearphagocytes recovered from newbornrat. A, Microglia seldomshow ruffled membranesin contrastto about40% of tissuemacrophages. B, Histogramshows the frequencyof numbersof spinesfor microgliaand liver macrophages.As shown,most macrophageshave few,spines(
C
100 - 90 - 80 - m Stroke 70 - Normal 60 - 50 - 0 Trauma 40 - 30 - m Axotomy 20 - 10 0 t - Figu, re 9. Microglia isolated from adult rat brain can be distinguish1 :d from tissue macrophages by scanning EM. As shown in A, nearly i Ill Figure 10. Reactive microglia recovered from axotomized adult optic mien 3glia (99%) isolated from normal adult brain contain numeral US tectum (B) show process bearing cells which are clearly different from spine:s. In contrast, invading macrophages which appear in the brain 5 the liver macrophages shown in A. Scale bar, 20 km. The histogram d after penetrating trauma (B) or liver macrophages (C) have ruffle:d (C) confirms that these populations of mononuclear phagocytes can be surfa ces with no spines. Scale bar, 5 pm. distinguished in adult tissues by scanning EM with >99% microglia found in normal or axotomized tissue whereas about 30% of mononu- clear cells isolated 5 d after ischemic or traumatic injuries to brain are invading macrophages with ruffled surfaces.
Figure II. Scanning electron photomicrographs of mononuclear cells isolated from human brain after 3 d in culture. A, Nearly all cells recovered from normal adult brain are spine-bearing microglia. B, Human microglia from brains of patients with amyotrophic lateral sclerosis appear identical to those of normal brain. C, Adult human microglia underwent ramification in vitro with processes that are covered with spines (D). E) Human blood monocytes in culture for 5 d take on the appearance of ruffled macrophages as noted for liver macrophages. F, At least 5% of cells recovered from HIV-l infected brains of adults appear as ruffled macrophages. Scale bars: A, B, and D, 1.2 pm; C, 7.0 pm; E and F, 1.25 pm.
7722 Giulian et al. l Properties of Microglia
Figure 12. Scanningelectron photographs of mononuclearphagocytes placed in coculturewith astrogliafor 7 fl. No processformation is noted for periltoneal macrophages (A), marrowcells (B), liver macrophages(C), or spleenmacrophages (D). Scale bar, 5 brn. Micro@ :lia (E) retainspine bearing, surfaces while peritonealmacrophages show ruffled surfaces(F; scalebar, 0.5 pm). or homogeneouscell populationsphysically separatedfrom one lial ramification by nearly fivefold (Fig. 14DJ’). Thus, contact another by filter-bottomed chambers.When isolated microglia with astrogliawas necessaryfor microglia to undergo the accel- were grown in 10% fetal bovine serum,we found Table 2. Mononuclear phagocytes isolated from human brain Table 3. Morphology of mononuclear phagocytes in culture with astrocytes % Ruffledmacrophages PeritoneumSnleen Liver Marrow Microglia Normal 0.3 2 0.3 ALS 0.7 2 0.5 % Ruffled 62 + 3 58 +- 2 46 2 2 21 + 4 6kl HIV- 1 5.3 t 1.3** % Spinous Ok0 Ok0 o-co Ok0 9421 % Smooth 38 + 2 41 t 2 54 t 3 78 t 4 Olrl Randomly selected mononuclear cells isolated from human brains within a 6 hr post-mortem interval were monitored for the presence of ruffled macro- % Branched 121 1t1 2t1 oto 5723 phages. Nearly all cells cultured from normal adult brain as well as cells from Features for different classes of mononuclear phagocytes placed in coculture brains with amyotrophic lateral sclerosis (ALS) were spine-bearing microglia. with astroglia for 7 d. Microglia properties such as branching (defined as cells However, HIV-l infected adult brain contained a significant increase in the with process lengths >3X diameter of cell body) and spinous surfaces (defined number of ruffled macrophages when compared to normal brain (**, Student’s as >20 spines per cell) were not detected in other classes of mononuclear test = 3.74, df = 598; p < 0.0002). Data are expressed as percentages + SEM phagocytes, all of which maintained a ruffled surface morphology despite con- based upon 300 cells randomly selected from each diagnostic category. tact with astroglia. brain cell with two or more branchedprocesses and spines.Rio- vated mononuclearbrain cells might be quite different-the re- Hortega further described microglia during embryonic devel- sult of either trafficking of blood monocytes across the blood opment as ameboidcells showing such macrophagicproperties brain barrier or the recruitment of quiescentresident microglia. as migratory capacity and phagocytosis. During CNS injury, Our finding that cell surface features viewed by scanningEM ramified microglia retract processes,become more ameboid-like, can distinguish human microglia from invading macrophages and transform into a third form, the reactive microglia (Rio- provides a new method for classification of brain inflammatory Hortega and Penfield, 1927). After Rio-Hortega, the art of silver responses.As describedhere, ALS is one type of neurodege- carbonatestaining, and thus the ability to identify microglia, was nerative disorder which doesnot involve blood cell invasion. In lost. Many investigators came to believe that microglia were contrast, recovery of significant number of macrophagesfrom simply blood monocytesinvading acutely injured tissue and not a distinct classof brain cells (Konigsmark and Sidman, 1963; Perry et al., 1985). This problem hasbeen compoundedby con- tradictory reportson the presenceor absenceof macrophagecell markers as viewed by histological study of brain tissuestaken from different stagesof development or CNS injury (Wood et al., 1976; Ling et al., 1982). Currently, there are no unique his- tochemical markers that distinguish activated or ameboid mi- croglia from invading brain macrophages(Flaris et al., 1993; Ulvestad et al., 1994). This inability to distinguishcell types has led to much confusion regarding the presenceand function of microglia in brain pathology. As described here, microglia cultured from postnatal brain possessedspecific characteristicsindicating this cell population to be a distinct classof mononuclearphagocyte. We found, for example, that microglia of rat showedproliferative capacity, pat- terns of differentiation, surfac? morphologies,responses to cy- tokines, and interactions with astrocytes that were clearly dif- ferent from macrophagesof spleen,liver, and peritoneum,from monocytes of blood, and from bone-marrowderived stemcells. Importantly, thesedifferences persisted into adulthood suchthat spine-bearingmicroglia were readily distinguishedfrom ruffled macrophagesby scanningEM. Scanning EM provided, more- over, a method to distinguishinvading macrophagesfrom resi- dent microglia at CNS sites of trauma, stroke, and axonal de- generation.The types of inflammatory cells identified by scan- ning EM in such models of brain injury generally agree with the descriptionsfrom other laboratoriesusing bone marrow chi- meras(Hickey and Kimura, 1988; Hickey et al., 1992; Flaris et al., 1993). Our data suggestthat CNS injury which produced local hemorrhagegave rise to macrophageinvasion of the brain whereasdistal axotomy elicited only reactive microgliosis. The lack of unique microglia identifiers has been a limiting factor when assessingtypes of inflammationin human brain pa- Figure 13. Fluorescencephotomicrographs of mononuclearphago- thology. A growing body of evidence now showsthat cytokines cytesafter 30 d in contactwith astrogliaDiI-ac-LDL(+) liver macro- phagespersist atop feeder layers of astrogliaand retain ameboid shapes and cytotoxins releasedby reactive mononuclearphagocytes af- (A). In contrast, all microgliaundergo differentiation with strikingpro- ter brain injury will influence survival of neurons (Giulian, cessformation (B). Thesecells intertwine with astroglialprocesses and 1992; Giulian et al., 1995). However, the origin of these acti- integrate within the brain environment. Scale bar, 20 pm. 7724 Giulian et al. * Properties of Microglia E F tl 5.0 p 60 ..- 2 IL ZJ 4.0 50 ti = a 40 6 3.0 m Microglia Only v) i = Q) 30 0 Astro Media 0 2 20 *- tz*a Astro Chamber z E 10 )II Astro Contact is 0 ap Figure 14. Fluorescence photomicrographs of DiI-ac-LDL(+) microglia after exposure to astroglia. All microglia were grown at low density in the presence of fetal bovine serum. Microglia control cultures were grown in the absence of astrocytes (Microglial Only; A) or in the presence of astroglia but separated by filtered bottom chambers (Astro Chamber; B). There is an increase in cell number (mitogenic effect) but no effect upon process formation. Microglia exposed to 30% culture media conditioned by astrocytes (Astroglial CM; C) also shows an increase in cell number but not process formation. When in direct contact with astroglia, microglia both increase in number and increase in process formation (Astro Contact; D, scale bar, 20 km). Quantitative measurements of different coculture conditions indicate that astroglia-contacts promote microglia differentiation (F) whereas secreted astroglial factors were mitogenic (E; see Giulian et al., 1991). Neither mitogenic nor differentiation effects were noted with other classes of mononuclear phagocytes placed in culture with astroglia. The Journal of Neuroscience, November 1995, 75(11) 7725 HIV-l infected brain points to another type of pathology with peritoneum, gut, liver, spleen, and lung. We suggest that astro- an altered flow of blood-borne cells into the CNS. Further stud- glial contacts help to maintain microglia in a differentiated, qui- ies employing morphological markers may help to delineate the escent state in order to restrict such microglial capacities as syn- subpopulations, locations, and neurotoxic capacities of viral in- aptic stripping (Blizinger and Kreutzberg, 1965) and release of fected cells within the brain. neurotoxins (Giulian et al., 1993). Thus, astroglia provide a sta- Microglia could be distinguished from bone marrow-derived bilizing influence upon microglia to reduce the likelihood of mononuclear cells, which have been considered the peripheral immune attack upon neurons. This dampened microglial re- precursor cells for adult brain microglia. For example, under the sponse might protect brain from disruptive effects of immuno- culture conditions used here, only microglia showed spontane- regulators produced during systemic immune activation and ous mitosis, while the precursor cells of bone marrow survive might be one reason the CNS rejects tissue transplants slowly poorly. To determine if a “brain cell environment” altered mor- (Medawar, 1947). Through such inhibitory mechanisms, the po- phologies of mononuclear phagocytes, we placed cells in co- tentially disruptive activities of reactive microglia are narrowly culture among highly enriched populations of astrocytes. Mi- limited by astrocytes to specific sites and locations of injured croglia established complex branches that entwined astroglial brain. processes while bone marrow cells retained ameboid shapes and Microgha have distinctive morphologies and behaviors in cul- did not interact with astroglia. Although it has long been pro- ture when compared to cells from spleen, liver, peritoneum, posed that bone marrow-derived stem cells were a source of blood monocytes, and marrow. The surface features seen by microglia, we found no in vitro evidence that postnatal marrow- scanning EM distinguish microglia from populations of all types derived cells transform into microglia. Such observations are of tissue macrophages in the postnatal animal. Importantly, mi- consistent with the in vivo findings of Hickey and Kimura (1988) croglia respond differently to contact with astroglia by under- in which no exchange between blood cells and parenchymal going differentiation. Despite testing a variety of culture con- microglia occurred in normal adult brain. In view of the fact that ditions, we did not find any evidence that brain provides an the survival or proliferation of bone marrow cells, unlike mi- environment to transform macrophages, monocytes, or primitive croglia, were not in any way influenced by astroglia, we con- marrow cells of postnatal rat into microglia. Cultured microglia clude that microglia are uniquely adapted to the CNS. Perhaps eventually transform into quiescent cells, as observed in vim, primitive stem cells common to microglia of the CNS and to with reduction of surface receptors, loss of phagocytic activity, macrophages of other organs exist-but only during early pre- and reduced secretory activity (data not shown). Reactivation of natal development. Our observations contrast a recent report by ramified microglia involves the upregulation of surface receptors Sievers et al. (1994) which describes the appearance of process- in these cells, the reappearance of secretory capacity, and new bearing cells after blood monocytes are added to heterogeneous migratory activity-all necessary as microglia engage in neu- cultures of brain glia. Through mixing experiments (coculture rotoxic or wound healing behaviors described in a variety of with different strains of murine cells or exposure of human disorders including stroke, trauma, HIV-l infection, and Al- monocytes to rat astrocytes) these authors conclude that blood zheimer’s disease. Chronic microgliosis may depend, therefore, monocytes take on microglia-like shapes when exposed to as- upon the persistence of activating signals and the nature of mi- trocytes. Further work using more carefully controlled in vitro croglia-astroglia interactions. conditions and selective cell markers will be needed to compare the monocyte-astrocyte interactions reported here with those References seen in culture systems described by Sievers et al. (1994). In order to organize and maintain a distinct population of Banati RB, Gehrmann J, Schubert P, Kreutzberg GW (1993) Cytotox- icity of microglia. Glia 7: 11 l-l 18. mononuclear cells within the CNS, specific signals must act Blinzinger K, Kreutzberg G (1968) Displacement of synaptic terminals upon developing microglia to induce cells to mature and grasp from regenerating motoneurons by microglial cells. Z Zellforschung the tissues of the brain. In general, we found that the number 85:145-157. and length of processes formed by microglia in vitro were de- Boje KM, Arora PK (1992) Microglial-produced nitric oxide and re- pendent upon the culture environment. Fetal bovine serum, for active nitrogen oxides mediate neuronal cell death. Brain Res 587: 250-256. example, substantially inhibited microglia from forming pro- Bolsi D (1927) Placche senile e microglia. Riv Patol Nerv Mental 32: cesses, as did laminin. Immuno-activators such as LPS and 65-74. rmIFNy drove microglia to retract processes whereas such cy- Bottenstein JE, Sato GH (1979) Growth of a rat neuroblastoma cell tokines as rmIL-lo and rmTNFa did not alter microglial shapes. line in serum-free supplemented medium. Proc Nat1 Acad Sci USA Although both microglia and bone marrow-derived cells prolif- 76514-517. Boya J, Carbonell AL, Calvo J, Borregon A (1987) Ultrastuctural study erated in response to rmGM-CSF, only microglia underwent cy- on the origin of rat microglia cells. Acta Anat 130:329-335. tokine-induced ramification. The GM-CSF released by brain tis- Boyum A (1968) Isolation of leucocytes from human blood. A two- sues during early postnatal development (Giulian et al., 1991) phase system for removal of red cells with methylcellulose as eryth- might promote microglial populations to mature and, thus, serve rocyte-aggregating agent. Stand J Clin Lab Invest Suppl 97:9-29. as an endogenous regulator of microglial differentiation. Daems WT (1980) Peritoneal macrophages. In: The reticuloendothelial system (Carr I, Daems WT, eds), pp 57-127. New York: Plenum. Why are differences in the behaviors of microglia and mac- Flaris NA, Densmore TL, Molleston MC, Hickey WF (1993) Char- rophages important? Unlike other tissues, the mature CNS dem- acterization of microglia and macrophages in the central nervous sys- onstrates an immune “privilege” which reflects, in part, the fact tem of rats: definition of the differential expression of molecules us- that adult brain is populated by a quiescent class of mononuclear ing standard and novel monoclonal antibodies in normal CNS and in four models of parenchymal reaction. Glia 7:34+0. phagocytes. Quiescent microglia, with marked reduction in Giulian D (1987) Ameboid microglia as effecters of inflammation in MHC class II expression, reduced capacity to present antigen, the central nervous system. J Neurosci Res 18: 155-171. and limited secretory function, are in marked contrast to the Giulian D, Baker TJ (1986) Characterization of ameboid microglia active phagocytic macrophages (Giulian, 1992) which guard the isolated from developing mammalian brain. J Neurosci 6:2163-2178. 7726 Giulian et al. - Properties of Microglia Giulian D, Lachman LB (1985) Interleukin-1 stimulation of astroglial Koski IR, Poplack DG, Blaese RM (1976) A nonspecific esterase stain proliferation after brain injury. Science 228:497-499. for the identification of monocytes and macrophages. In: In vitro Giulian D, Woodward J, Young D, Krebs JE Lachman LB (1988) In- methods in cell-mediated and tumor immunity (Bloom BR. David terleukin-1 iniected into mammalian brain stimulates astrogliosis and IR, eds), pp 3.59-362. New York: Academic. neovascularization. J Neurosci 5:2485-2490. Kreutzberg GW, Barron KD (1978) 5’-Nucleotidase of microglial cells in the facial nucleus during axonal reaction. J Neurocytol7:601-610. Giulian D. Chen J., Inzeman” JE. GeorgeVI J. NononenI M (1989)\ I The role of mononuclear phagocytes in wound healing after traumatic Ling EA (1981) The origin and nature of microglia. In: Advances in injury to adult mammalian brain. J Neurosci 9:4416-4429. cellular neurobiology (Fedoroff S, Hertz L, eds), pp 33-82. New Giulian D, Robertson C (1990) Inhibition of mononuclear phagocytes York: Academic. reduces ischemic injury in the spinal cord. Ann Neurol 27:3342. Ling EA, Wong WC (1993) The origin and nature of ramified and Giulian D, Vaca K, Noonan CA (1990) Secretion of neurotoxins by amoeboid microglia: a historical review and current concepts. Glia mononuclear phagocytes infected with HIV- 1. Science 250: 1593- 7:9-18. 1596. Ling EA, Kaur C, Wong WC (1982) Light and electron microscopic Giulian D, Johnson B, Krebs JE George JK, Tapscott M (1991) Mi- demonstration of non-specific esterase in amoeboid microglial cells croglial mitogens are produced in the developing and injured mam- in the corpus callosum in postnatal rats: a cytochemical link to mono- malian brain. J Cell Biol 112:323-333. cytes. J Anat 135:385-394. Giulian D (1992) Microglia and diseases of the nervous system. Cur-r McGeer PL Itagaki S, Tago H, McGeer EG (1987) Reactive microglia Neurol 12:23-54. in patients with senile dementia of the Alzheimer type are positive Giulian D, Corpuz M, Chapman S, Mansouri M, Robertson C (1993) for the histocompatibility glycoprotein HLA-DR. Neurosci Lett 79: Reactive mononuclear phagocytes release neuron killing factors after 195-200. stroke and trauma, J Neurosci Res 25:227-233. Medawar PB (1948) Immunity to homologous grafted skin. III. The Giulian D, Vaca K, Corpuz M (1993) Brain glia release factors with fate of skin homografts transplanted to the brain, to subcutaneous opposing actions upon neuronal survival. J Neurosci 13:29-37. tissue, and to the anterior chamber of the eye. Br J Exp Path01 29: Giulian D. Haverkamn LJ. Li J. Karshin WL. Yu J. Tom D. Li X. 58-69. Kirkpatrick JB (1995) Senile plaques stimulate midroglia to’release Oehmichen M (1983) Inflammatory cells in the central nervous system. a neurontoxin found in Alzheimer’s brain. Neurochem Int 27: 119- A integrating concept based on recent research in pathology, immu- 137. nologv, and forensic medicine. Prog Neurooathol 5:277-335. Graeber MB, Tetzlaff W, Streit WJ, Kreutzberg GW (1988) Microglia Perry VH, Hume DA, Gordon S (1985) Immunohistochemical local- cells but not astrocytes undergo mitosis following rat facial nerve ization of macrophages and microglia in the adult and developing axotomy. Neurosci Lett 85:317-321. mouse brain. Neuroscience 15:313-326. Hetier E, Ayala J, Bousseau A, Denefle P Prochiantz A (1990) Amoe- Perry VH, Matyszak MK, Fearn S (1993) Altered antigen expression boid microglial cells and not astrocytes synthesize TNFol in Swiss of microglia in the aged rodent CNS. Glia 7:60-67. mouse brain cell cultures. Eur J Neurosci 2:762-768. Pulliam L, Herndier BG, Tang NM, McGrath MS (1991) Human im- Hickey WE Kimura H (1988) Perivascular microglial cells of the CNS munodeficiency virus-infected macrophages produce soluble factors that cause histological and neurochemical alterations in cultured hu- are bone marrow-derived and present antigen in vivo. Science 239: man brains. J Clin Invest 87:503-512. 290-292. Rio-Hortega P de1 (1932) Microglia. In: Cytology and cellular pathol- Hickey WE Vass K, Lassmann H (1992) Bone marrow-derived ele- ogy of the nervous system (Penfield W, ed), pp 481-584. New York: ments in the central nervous system: an immunohistochemical and Hoeber. ultrastructural survey of rat chimeras. J Neuropathol Exp Neurol 51: Rio-Hortega P del, Penfield W (1927) Cerebral cicatrix: the reaction 246-256. of neuroglia and microglia to brain wounds. Johns Hopkins Hosp Higgins GA, Olschowka JA (1991) Induction of interleukin-1E mRNA Bull 41:278-303. ?n adult rat brain. Mol Brain Res 9:143-148. Sievers J, Parwaresch R, Wottge U (1994) Blood monocytes and spleen Hsu CY. Yona YH. Ping-Keung Y. Luk YO. Chen ST. Hogan ET (1990) macrophages differentiate into microglia-like cells on monolayers of A stroke model’ de&red f& preclinical trial. In: ‘Cerebral i&hernia astrocytes: morphology. Glia 12:245-258. and resuscitation (Surr A, Rigor BM, eds), pp 47-59. Boca Raton: Stewart CC (1981) Murine mononuclear phagocytes from bone mar- CRC. row. In: Methods for studying mononuclear phagocytes (Adams DO, Imamoto K, Leblond CP (1977) Presence of labeled monoctyes, mac- Edelson PJ, Koren HS, eds),-pp 5-20. Newkotk: Academic. rophages, and microglia in a stab wound of the brain following an Thanos S. Mev J, Wild M (1993) Treatment of the adult retina with injection of bone marrow cells labeled with ‘H-uridine into rats. J microglia-suppressing factors retards axotomy-induced neuronal deg- Cbmp Neurol 174:255-280. radation and enhances axonal regeneration in vivo and in vitro. J Jordan FL. Thomas WE (1988) Brain macroohanes: auestions of origin Neurosci 13:455466. and interrelationship. Brain ‘Res 472:165-i787 ’ Ulvestad E, Williams K, Mork S, Ante1 J, Nyland H (1994) Phenotypic Kettenmann H, Hoppe D, Gottmann K, Banati R, Kreutzberg G (1990) differences between human monocytes/macrophages and microglial Cultured microglial cells have a distinct pattern of membrane chan- cells studied in situ and in vitro. J Neuropathol Exp Neurol 53:492- nels different from peritoneal macrophages. J Neurosei Res 26:278- 501. 287. Wood GW, Gollahon KA, Tilzer SA, Vats T, Morantz RA (1979) The Konigsmark SW, Sidman RL (1963) Origin of brain macrophage in failure of microglia in normal brain to exhibit mononuclear phagocyte mouse. J Neuropathol 22:643-676. markers. J Neuropathol Exp Neurol 38:369-376.