Generation and characterization of mouse microglial cell lines

Journal of Neuroimmunology ELSEVIER

Journal of Neuroimmunology52 (1994) 153-164

Generation and characterization of mouse microglial cell lines Tony W. Briers *, Christel Desmaretz, Eugeen Vanmechelen Innogenetics N.V., Industriepark Zwijnaarde 7, PO Box 4, B-9052 Gent, Belgium

Received 10 February 1994; revision received and accepted 29 March 1994

Abstract

A murine cell line (MMGT1) has been established after transfection of primary microglial cell cultures with a v-myc-containing plasmid. This cell line was comparable with primary microglial cells with respect to morphology, presence of acetylated low density lipoprotein receptor, non-specific esterase, CD63, major histocompatibility complex antigens and CDll, and binding for Ricinus communis agglutinin. Primary microglia as well as MMGT1 cells were negative for glial fibrillary acidic protein. Different MMGT1 strains were obtained after subcloning, two of which resembled histiocytes (F4/80 and BM-8). These cell strains, MMGT12 and 16, were able to opsonize latex beads, and could be induced by endotoxins (LPS) to secrete TNF-a, IL-1, IL-6, TGF-/3, and EGF. The other subclones had intermediate (MCA519, ER-MP20) or mixed macrophage characteristics and did not react to endotoxin by an increase in TNF-a, IL-1, and TGF-/3. Our newly established murine microglia lines may prove to be useful models to study inflammation and repair in the brain. Key words: Brain; Glia; Inflammation; Microglia; v-myc

I. Introduction

Microglia, one of the three major glial cell types distributed throughout the central nervous system (CNS), are found in two predominant forms: the ameboid and the ramified or process-bearing type. Ameboid cells appear in the developing brain and at sites of injury, while the ramefied cells are considered as being 'quiescent' cells in mature CNS. Although the close association of microglia and CNS injury is well documented (Perry and Gordon, 1988), the role of these cells in the cellular events associated with brain pathology remains unclear. The notion of microglia as scavengers ceils in the CNS is too simple a model, and the origin of the ramified cells remains enigmatic. It has therefore been suggested that these cells are derived from the ameboid type as part of a normal differentiation process, and that upon injury these cells transform to the 'active' ameboid form (Giulian, 1987). Microglia seem to be mononuclear phagocytes that can be distinguished from blood monocytes or peritoneal macrophages (Giulian, 1987). They

* Corresponding author. Phone (09) 2410 917; Fax (09) 2410 799. 0165-5728/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165-5728(94)00055-S

are possibly the resident macrophages of the CNS that link the immune system with brain development and astroglial scar formation. Because of the importance of microglia in brain development, inflammation, and pathology, the development of continuous cell lines of such cells would permit the study of microglial function in these processes. Recently, microglia have been isolated from brain and cultured (Giulian and Baker, 1986; Suzumura et al., 1987; Gebicke-Haerter et al., 1989), but only three reports have described the establishment of microglia lines including infection with v - r a f / v - m y c and v - m i l / v - m y c retrovirus constructs (Blasi et al., 1991; Righi et al., 1991) resulting in microglial cell lines secreting oncogenic viruses (Righi et al., 1991), and by transfection with simian virus 40 large T (Hosaka et al., 1992). Because of the danger associated with the secretion of oncogenic viruses, and since large T antigen often changes the phenotype of the transfected cells (Shay, 1991), we chose to generate murine microglial lines by transfection using the v-myc gene as transfectant. For these experiments, adult brains were used because the heterogeneity of distribution and morphology of the microglial population (Lawson et al., 1990) allows the selection of specific differentiated microglial

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"ll W. Briers et al. /Journal of Neuroimmunology 52 (1904) 153 164

strains, a n d b e c a u s e t h e study of i n f l a m m a t i o n a n d b r a i n p a t h o l o g y are especially of i m p o r t a n c e in t h e adult. T h e s e e x p e r i m e n t s r e s u l t e d in e s t a b l i s h e d cell lines t h a t w e r e i n d i s t i n g u i s h a b l e from active microglia a c c o r d i n g to m o r p h o l o g i c a l , p h e n o t y p i c a l , a n d functional criteria.

2. Materials and methods 2.1. Cell cultures M i c r o g l i a l cells w e r e i s o l a t e d by a c o m b i n a t i o n of s h a k i n g a n d t r y p s i n i z a t i o n of 2 - w e e k - o l d m i x e d b r a i n cultures from newborn and adult male NMRI outbred mice, as d e t a i l e d e l s e w h e r e ( G i u l i a n , 1987). I s o l a t e d m i c r o g l i a w e r e c u l t u r e d in H a m ' s F 1 2 : D M E M ( H D ; 1 ' 1 , v / v ) s u p p l e m e n t e d with insulin (0.0005%), transferrin (0.001%), s o d i u m s e l e n i t e (30 riM), bovine s e r u m a l b u m i n (0.0003%), 2 % fetal calf s e r u m ( H D - T I S plus 2 % FCS), a n d 20% c o n d i t i o n e d m e d i u m ( C M ) o f W E H I cells ( W E H I 3, A T C C , Rockville, M D , TIB68; W E H I 3B, E C A C C , Salisbury, U K , 86013003; W E H I 3D, E C A C C , 8509403), a p r o d u c e r of IL-3 a n d G M C S F ( F r e i et al., 1986; Ihle a n d A s k e w , 1989).

2.2. Transfection procedures S e l e c t e d microglial c u l t u r e s w e r e t r a n s f e c t e d with 2 /_tg pSVv-myc ( A T C C , Rockville, M D , 45014) p e r 25 cm 2 surface following the calcium phosphate (Michalovitz et al., 1987) or the m o d i f i e d lipofection p r o c e d u r e ( G i b c o B R L , G e n t , Belgium; F e l g e r et al., 1987; Briers et aI., 1993). T h e c o p r e c i p i t a t e s (both m e t h o d s ) w e r e left on t h e cells for 5 h. T h e t r a n s f e c t e d c u l t u r e s were followed daily, a n d w e r e c o m p a r e d with t h e c o n t r o l culture. S u b c u l t u r i n g a n d c h a n g i n g of m e d i u m ( H D - T I S + 2 % F C S + 20% C M of W E H I cells) w e r e p e r f o r m e d w h e n necessary.

2.3. Cell identification I m m u n o c y t o c h e m i c a l d e t e r m i n a t i o n o f the p r e s e n c e of c e l l u l a r a n t i g e n s using m o n o c l o n a l a n d p o l y c l o n a l a n t i b o d i e s ( T a b l e 1) was p e r f o r m e d using p e r o x i d a s e l i n k e d s e c o n d a n t i b o d i e s o r the A B C - e n h a n c i n g kit (Dako, Glostrup, Denmark) and 3-amino-9-ethylc a r b a z o l e as p e r o x i d a s e stain ( H e y d e r m a n , 1979; H s u et al., 1981). T h e b i o t i n y l a t e d Ricinus communis agglutinin (RCAI20, V e c t o r Lab., B u r l i n g a m e , VT), specific for the c a r b o h y d r a t e m o i e t i e s f l - g a l a c t o s e a n d fl-N-

Table 1 Primary antibodies and antisera used for immunocytochemistry Antigen !'

Antiserum

Manufacturer b

Dilution

Pan-myeloid precursor, mouse LCA, rat (OX1) LY15.2 (LFA-1), mouse MAC-l, mouse Mid-stage MQ precursor, mouse MQ mouse Macrophage Nature MQ, mouse (F4/80) Pan-tissue fixed MQ, mouse MQ aminopeptidase, mouse Class I monomorphic, rat (OX18) la monomorphic, mouse, rat (OX6) Ia polymorphic, mouse (OXI-1) Class II, mouse; MHC, rat Desmin, human Cytokeratin-Pan Vimentin, pig a-smooth muscle actine GFAP, bovine S100, bovine MAP1B MAP2 Heparan sulfate Fibroglycan (extracel. domain) Syndecan/N-syndecan Alzheimer precursor protein A4

ER-MP58 MCA43 MCA62 MCA74S ER-MP20 MCA519 HAM56 MCA497 BM-8 ER-BMDM-1 MCA51 MCA46 MCA71 MCA09 D33 C1801 5525 1A4 S 100 ! 2A1 HSM11 10E4 10H4 2E9 -

BMA Serotec Serotec Serotec BMA Serotec ENZO Serotec BMA BMA Serotec Serotec Serotec Serotec Dako Sigma Sigma Sigma Dako Dako Innogenetics Innogenetics David David David Boehringer

50 50 50 50 50 50 1000 10 50 50 50 50 50 5 50 400 400 400 250 100 50 50 500 500 500 100

a LCA, leukocyte common antigen; MQ, macrophage; MHC, major histocompatibility complex; GFAP, glial fibrillary acidic protein; MAP, microt ubule-associated protein. b Boehringer Mannheim, Germany; BMA Biomedicals AG., Augst, Switzerland; Dako A/S, Glostrup, Denmark; Dr. G. David, Center for Human Genetics, University of Leuven, Leuven, Belgium; Enzo diagnostics, New York, NY; Innogenetics NV, Gent, Belgium; Serotec, Oxford, UK; Sigma, Munich, Germany.

T.W. Briers et al. /Journal of Neuroimmunology 52 (1994) 153-164

acetylgalactosamine, was used as a microglia marker as described by Colton et al. (1992). Nonspecific esterase or a-naphthyl acetate esterase staining (aNAE-staining) was adapted for cells in culture (Rommer.ts et al.,

155

1985). Staining for the acetylated low-density lipoprotein receptor (acLDLR) was performed using an indocarbocyanate probe (DIL-Ac-LDL; Biomedical Technologies, Stoughton, CA), and the phagocytic activity

Fig. 1. Morphological properties of MMGT1 cells. (A) Phase-contrast photomicrograph of a culture of brain cells. Note the spindle-shaped and rod-like microglia (arrow). (B) Selected microglia 2 weeks after lipofection with pSVv-myc. (C) Phase-contrast photomicrograph of MMGT1 cells. (D) MMGT1 cells 24 h after LPS challenge (1 /xg/ml). (E) MMGT1 cells 24 h after seeding. Note the spindle-shaped, ameboid, ramefied and phase-contrast bright spherical cells. (F) Non-specific esterase (aNAE) staining of MMGT1 cells. (G) Opsonized latex beads (I /zm in diameter) ingested by MMGT1 cells, Giemsa staining. (H) Staining with the biotinylated Ricinus communis agglutinin. Note the strong staining of ramefied MMGT1 cells. (1) Presence of acetylated low-density lipoprotein receptor in ramefied and ameboid MMGT1 cells as shown by the fluorescent probe DIL-ac-LDL. (K) Phase-contrast control photomicrograph of (I). (A-E& It, X 100; I& K, x 400).

156

T.W. Briers et al. /Journal ~)f Neuroirnmunology 52 (1994) 153-164

was measured using latex beads (1 p~m) both following the procedure of Giulian and Baker (1986). 2.4. Cell activation and biological assays

Tumor necrosis factor-a (TNF-a) activity was measured using L929 cells (ECACC, 85011425) in a standard cytotoxicity assay (Fransen et al., 1986) using Crystal violet staining for the estimation of the surviving viable cells (Gillies et al., 1986). Interleukin-1 (IL-1) activity was scored with MG63 human osteosarcoma ceils (ATCC, CRL1427; Van Damme et al., 1988). IL-6 activity was scored using an IL-6 growth-dependent mouse hybridoma line, 7TD1 (ATCC, CRL1851; Mosmann, 1983). The transforming growth factor-/3 (TGF/31 & 2) bioassay is based on its ability to inhibit the growth of mink lung cells (MvlLu; ATCC, CCL64; Cone et al., 1988), with inhibition scored by measuring tritiated thymidine incorporation (Briers and Desmaretz, 1993). TGF-/3 samples were activated by treatment at 95°C for 10 min. Heparin binding growth factor (HBGF) assay was performed with fetal bovine heart endothelial cells (FBHE; ATCC, CRL1395) which are fibroblast growth factor (FGF)-dependent (Van Zoelen, 1990). The growth stimulatory effect of HBGF on the endothelial cells (64-h induction) was measured by the incorporation of tritiated thymidine. Basic FGF and acidic FGF in the absence and presence of heparin were used as controls for this assay. The epidermal growth factor (EGF) assay is based on its growth inhibitory effect on human epidermoid carcinoma cells (A431; ATCC, CRL1555; Van Zoelen, 1990). The dose-response effect of EGF samples was monitored by the inhibition of tritiated thymidine uptake in exponentially growing A431 cells. Sonicated (1 rain) lipopolysaccharide (LPS; Escherichia coli O55:B5, Difco, Detroit, MI), used at a concentration of 1 /zg/ml, served as an activator of microglia-like cells, and induced the release of inflammatory cytokines. Activation was performed over a 24-h period under serum-free conditions (HD-TS, with the omission of the insulin component). 2.5. Northern and Western blot analysis

RNA preparations of the monolayers were made using the guanidinium hydrochloride method (Chomczynski and Sacchi, 1987). Purification of poly-A RNA was performed as described by Aviv and Leder (1972). The separation on formaldehyde agarose gels and the transfer of the RNA to nylon filters was performed according to Maniatis (1982). The cDNA probes used in this study were for amyloid precursor protein (APP) an E c o R I fragment from pCD9 (Kang et al., 1987), kindly provided by Dr. K. Beyreuther (Zentrum fiir Molekulaire Biologie, Heidelberg, Germany).

For Western blot analysis, whole-cell lysates were separated on 10% polyacrylamide gels (Laemmli, 1970) and transferred to nitrocellulose (Towbin et al., 1979). The monoclonal antibody to APP (clone 22Cll, Boehringer Mannheim) was used to detect APP in the Western blots.

3. Results 3.1. Characterization of a microglia line, MMGT1

Selected microglia were transfected (calcium phosphate precipitation and lipofection) with a r-myc-containing plasmid (pSVv-myc). Several days after transfection, a large portion of the cells died, and only small colonies of quiescent cells remained (Fig. 1B). The medium was changed weekly with HD-TIS plus 2% FCS and 20% CM of WEHI cells. Only one out of four transfections (lipofection) resulted in an immortalized line, MMGT1. All control cultures and other transfected cultures were discarded 8 months after transfection. The MMGT1 cell line was derived from a few remaining plaques which remained dormant for several months. The morphology of MMGT1 cells included spindleshaped, ameboid, ramified, and polynucleated cells (Fig. 1E). Small spherical, phase contrast bright cells were also seen to be floating or adhering to the plastic. These spherical cells could be observed in several transitional stages evolving from the round type to the spindle-shaped, ameboid or ramified type (during the transitional phase the phase contrast brightness of the cell disappeared, Fig. 1E). Thus, the morphology of the transfected cell line, MMGT1, resembled that of the selected microglial cells in primary culture (Fig. 1A, B). The MMGT] cell line has been maintained in culture for more than 1 year, and was subcultured more than 100 times. The doubling time of the cell line decreased over the culture time (passage 8, 103 + 2 h vs. passage 54, 22 + 1 h), and remained constant at _+22 h from the 50th passage onwards. The cell line can be stored under frozen conditions. MMGT1 cells were positive for macrophage and microglia-specific markers: acLDLR (Fig. lI, K), aNAE (Fig. 1F), RCA120 (Fig. 1H), class II (Ia), C D l l a and CDllb, and were negative for the astrocyte marker GFAP, S100, and peroxidase as for selected microglia in primary culture (Table 2). RCA reactivity was found especially in ramified and spindle-shaped cells, whereas ameboid forms showed only weak staining. By contrast, aNAE-staining was strongest for the ameboid cell type. MMGT1 cells were able to opsonize latex beads (Fig. 1G). Further characterization showed positivity for the macrophage markers (Table 3), MHC class I (OX18, Table 4), as well as vimentin and actin (Table 5).

T.W. Briers et al. /Journal of Neuroimmunology 52 (1994) 153-164

157

Table 2 F u n c t i o n a l a n d a n t i g e n i c m a r k e r s in s e l e c t e d p r i m a r y m i c r o g l i a a n d a v-myc t r a n s f e c t e d cell line, M M G T 1

Table 4 A n t i g e n i c i n t e g r i n a n d M H C m a r k e r s in v-myc t r a n s f e c t e d M M G T cell lines

Cell m a r k e r

Primary microglialike cells

Cell line

aNAE

MMGT-1

a

LFA-1 a

MAC-1

Class I

+

++

++

+++

+ + + + + + -

+ + + ND + + + + + +

+ + + + + + + +

+ + + + + + +

+ q._ _}_ _[_ b

-I- -'}- -}- -}-

MMGT1

+++

acLDLR Peroxidase P h a g o c y t o s i s (latex)

+ + + Yes

+ + + Yes

RCA120 MAC-l, CDllb GFAP

+ + + + -

+ + + + + -

MMGT11 MMGT12 MMGT13 MMGT14 MMGT15 MMGT16 MMGT17 MMGT18

+ + + + + + + + + +

a a N A E , a - n a p h t y l a c e t a t e e s t e r a s e ; a c L D L R , a c e t y l a t e d low d e n s i t y l i p o p r o t e i n ; R C A , Ricinus communis a g g l u t i n i n ; G F A P , glial fibrillary acidic protein. b--, no staining; +, weak staining and sparse; + +, moderate s t a i n i n g a n d focal; + + + , s t r o n g s t a i n i n g a n d n o t u n i f o r m ; + + + + , strong staining and uniform; ND, not determined.

Activation of MMGT1 cells with 1 /xg/ml LPS for 24 h resulted in a proliferation arrest (seeded ceils 4 x 106 cells; 24 h post-stimulation 3 + 0.3 x 106 ceils for the LPS-treated vs. 6.4 + 0.2 x 106 cells for the controls, n = 3) and the cells changed morphologically from a more rounded form to ameboid, spindle-shaped, and ramified types (Fig. 1C vs. 1D). The stimulated cultures clearly produced T N F - a (9107 + 3036 U / m l vs. < 16 U / m l for controls) and IL-1 (1280 U / m l vs. < 20 U / m l for controls). Activation was only possible when MMGT1 cultures were firmly adherent before LPS challenge.

3.2. Characterization of MMGT1 strains The MMGT1 celt line was subcloned by limited dilution, when the culture was not yet stabilized (subculture 11). At a dilution of 3 cells/well, colonies were formed in 28% of the wells. At lower dilutions 1 and 0.3 cells/well, no colonies were formed, and at higher dilutions, colonies were present in all wells. Only those

b

Ia mono

+ +

+ + + +

Ia poly

a L F A - 1 , C D l l a ; M A C - l , C D l l b ; Class I m o n o m o r p h i c , O X 1 8 ; Ia m o n o m o r p h i c , O X 6 ; Ia p o l y m o r p h i c , O X I - 1 . b - - , n o s t a i n i n g ; + , w e a k s t a i n i n g a n d s p a r s e ; + + , m o d e r a t e staining a n d focal; + + + , s t r o n g s t a i n i n g a n d n o t u n i f o r m ; + + + + , strong staining and uniform; ND, not determined.

Table 5 A n t i g e n i c c y t o s k e l e t a l a n d p r o t e o g l y c a n m a r k e r s in u-myc t r a n s f e c t e d M M G T cell lines a Cell line

Vimentin

Actin

Heparan sulfate

Fibroglycan

APP b

MMGT1 MMGTll MMGT12 MMGT13 MMGT14 MMGT15 MMGT16 MMGT17 MMGT18

+ c + + + + + + + + + + +

+ + + + + + + + + + + + +

ND + + + + + + + + + + + + + +

ND + + + + + + + + + + + + + + + +

ND + + + ND ND

+ + + + + + + +

+ + + + + + + +

All cell lines w e r e n e g a t i v e f o r d e s m i n , c y t o k e r a t i n ( p a n ) , glial fibrillary a c i d i c p r o t e i n ( G F A P ) , m i c r o t u b u l e - a s s o c i a t e d p r o t e i n ( M A P I B & 2), S100 p r o t e i n , a n d s y n d e c a n . b APP, amyloid precursor protein. c_, no staining; +, weak staining and sparse; + +, moderate s t a i n i n g a n d focal; + + + , s t r o n g s t a i n i n g a n d n o t u n i f o r m ; + + + + , strong staining and uniform; ND, not determined. a

Table 3 A n t i g e n i c m a c r o p h a g e m a r k e r s in a c-myc t r a n s f e c t e d M M G T cell lines Cell line

LCA a

Pan-myeloid

Mid-stage

MQ

MQ MMGT1 MMGT11 MMGT12 MMGT13 MMGT14 MMGT15 MMGT16 MMGT17 MMGT18

ND b + ++ + + + ND ND

+ ++ + +++ + +

_ + + + + + + + + +

+ + + ++ . + + + + +++ + + +

Mature

Pan-tissue

MQ

MQ

+ +++

+ ++++

+ + + ++++

++++ + + -

_ _ ++++ + + + + +

.

. +++ + + -

AP

.

a L C A , O X - 1 ; P a n - m y e l o i d p r e c u r s o r ; M Q , m a c r o p h a g e ; M i d - s t a g e m a c r o p h a g e p r e c u r s o r ; M a t u r e m a c r o p h a g e , F 4 / 8 0 ; P a n - t i s s u e fixed m a c r o p h a g e ; A P , m a e r o p h a g e a m i n o p e p t i d a s e ; H A M 5 6 , m a c r o p h a g e w a s n e g a t i v e f o r all cell lines. b - - , n o s t a i n i n g ; + , w e a k s t a i n i n g a n d s p a r s e ; + + , m o d e r a t e s t a i n i n g a n d focal; + + + , s t r o n g s t a i n i n g a n d n o t u n i f o r m ; + + + + , s t r o n g staining and uniform; ND, not determined.

158

T W. Briers et al. /Journal of Neuroimmunology 52 (1994) 153-164

wells were collected in which only a single colony was visible (n = 8). The morphology of the cells differed considerably between cell strains. M M G T l l , 13, 14 and 18 showed mostly a spindle and ramified appearance, often with long processes. The rounded form was also observed as well as multi-nucleated cells (Fig. 2A, C, D, I); all these MMGT strains had doubling times in excess of 20 h (range 22-44 h). In strains MMGT12 and 16, the cells resembled the rounded and rod-like types (Fig. 2B, F);

~-~,

these cells had doubling times of less than 20 h (MMGT12, passage 12 and 61, 18 ± 1 h; MMGT16, passage 9, 19 + 2 h, passage 60, 16 + 1 h). MMGT17 cell cultures exhibited all types and forms including those present under activated (LPS) conditions (Fig. 2H; doubling time 29 h). Lastly, MMGT15 had a spindle-shaped appearance (Fig. 2E; doubling time 38 h). These MMGT1 strains were positive for the typical microglial markers: o~NAE, acLDLR, and RCA120.

,o

Fig. 2. Morphological properties of MMGTI cell strains presented as phase-contrast photomicrographs. (A) MMGTll. (B) MMGT12; a normal cell culture is depicted in the upper panel, while a cell culture 24 h after LPS challenge (1 /zg/ml) is depicted in the lower panel. (C) MMGT13. (D) MMGTI4. (E) MMGT15. (F) MMGT16. (G) MMGT16 cells 24 h after LPS challenge (1 /~g/ml), (H) MMGT17. (I) MMGTI8. (A-l, x 100).

T.W. Briers et al. / Journal of Neuroimmunology 52 (1994) 153-164

159

Q

Fig. 3. Immunocytochemical staining of MMGT1 strains. (A) Marker LY15.2, MMGTll. (B) Marker mouse macrophage (MCA519), MMGT16. (C) Marker fibroglycan (10H4), MMGT14. (D) Marker class I monomorphic (OX18), MMGT15. (E) Marker mid-stage macrophage precursor (ER-MP20), MMGT18. (F) Marker heparan sulfate (10E4), MMGT14. (G) Marker Ricinus communis agglutinin, MMGT13. (H) Marker mature macrophage (F4/80), MMGT12. (I) Marker macrophage aminopeptidase (ER-BMDM-1), MMGT12. (K) Marker class II MHC (Ia polymorphic, OXI-1), MMGT16. (L) Marker vimentin, MMGT16. (M) Marker pan-tissue fixed macrophage (BM-8), MMGT16. (N) Marker MAC-1 (CDllb), MMGT16. (O) Marker pan-cytokeratin, MMGT16. (A-G, and I-O, X 200; H, × 400).

7: W. Briery et al. /Journal of Neuroirnmunology 52 (1994) 153-164

160

They were further tested for markers of the macrophage lineage, cytoskeleton, and proteoglycan. One group of antigens revealed macrophage characteristics (Table 3), thereby permitting a classification according to the differentiation stage as indicated by these markers. Two strains, MMGT12 and 16, were strongly positive for mature (F4/80) and pan-tissue macrophage (BM-8) markers as well as for aminopeptidase, pointing to their histiocytic nature. Examples are shown for M M G T I 6 cells (pan-tissue macrophage, Fig. 3K) and for MMGT12 ( F 4 / 8 0 and aminopeptidase, Fig. 3H, I). Four strains, M M G T l l , 14, 15 and 18, showed characteristics of mid-stage macrophage precursor (positive for MCA519 and / o r ER-MP20; Fig. 3E). Of these, two were still positive for aminopeptidase ( M M G T l l and 18). MMGT17 cells had mixed characteristics. One cell strain, MMGT13, seemed to have no macrophage characteristics, although up to 50% of the cultures were RCA- (Fig. 3G) and a N A E positive. All the strains displayed antigens for the MHC class 1 and class II (Fig. 3D, K), but to different degrees (Table 4). The histiocyte-like strains, MMGT12 and 16, were also positive for the integrins LFA-1 and MAC-1 ( C D l l a and b; Table 4, Fig. 3L). The strains were also positive for the cytoskeletal markers vimentin and actin, but negative for cytokeratin, desmin, MAP 1B and 2, and G F A P (Table 5, Fig.

Percentlge ol l h y m l d l n e Incotporillon 160

.A

80

60

40 • 20

MMGT12

~ ' MMGT16 MMGT13

0 0.01

0.10

1.00

5.0

'~0

100

10oo

LPS u o n c o n t r l t i o n (055:B5). ng/rnl

Perc:enllge c01thymi~line incorporation 120

B

IOO( 80

6O

¢. 41)

0

1

2

kDa

0

"0PU5-1.8 l 0

o.01

0 1

~ O0

500

10

100

I000

LPS concen~fehov, I055 flS) n O'mt

kDa 285

200

-

APP 97.4

-

185 68-

A

B 43-

Fig. 4. Presence of amyloid precursor protein (APP) in M M G T 1 cells. (A) Northern blot showing a m R N A signal at 3.2 kb; lane 1, m R N A isolated from mouse brain, lane 2, total R N A isolated from M M G T 1 cells, (B) Western blot showing an immunoreactive band at a molecular mass of 130 kDa.

Fig. 5. Effect of endotoxin challenge on tritiated thymidine incorporation into DNA of MMGT cells. LPS (O55:B5) was used at

concentrations from 0.01 up to 1000 ng/ml. Tritiated thymidine incorporation was measured (6 h) after an overnight induction (18 h). Thymidine incorporation into DNA is expressed as the percentage of the counted tritiated thymidine after LPS challenge vs. the counted tritiated thymidine of the HD-TS control. (A) MMGT12 (I), MMGT16 (zx), MMGT13 (v). (B) MMGT1 ( i ) , mouse monocyte/macrophage cell line, PU5-1.8 (0). Mean + SD, n = 3; SD smaller than data points not shown. 3L, O). All but the histiocytic strains showed strong staining for heparan sulfate (Table 4, for MMGT14 Fig. 3C, F), and all were negative for syndecan. Only the strains M M G T l l , 13, and 15 stained positively for APP using immunocytochemistry, although all were positive when analyzed by Northern or Western blot (Fig. 4). Northern blot analysis demonstrated a signal at 3.2 kb, and a Western blot showed an immunoreactive band at a molecular mass of 130 kDa. When LPS was added to the culture medium, MMGT12 and MMGT16 reacted by proliferation arrest (Fig. 5, Table 6); a change in morphology of

T.W. Briers et al. /Journal of Neuroimmunology 52 (1994) 153-164

20-40% of the cells in culture from ramified, rod-like, and rounded to a more flattened ameboid appearance (Fig. 2B, F, G); and by the secretion of TNF-a, IL-1, IL-6, EGF, and TGF-/3 (Table 6). Thymidine incorporation experiments showed that the inducible cell lines (MMGT1, 12 and 16) were very sensitive to LPS; the LPS concentrations for the dose-response curve were found between 0.1 and 5 ng/ml. At LPS concentrations greater than 5 ng/ml LPS, the activation process was completed. In cell strains MMGT12 and 16, thymidine incorporation into DNA dropped to very low levels (MMGT12, 641 + 85 cpm for 1 /zg LPS/ml vs. 112035 + 11956 cpm for the HD-TS control; MMGT16, 477 + 153 cpm for 1 /zg LPS/ml vs. 13 649 + 2254 cpm for the HD-TS control). This was not the case for the mother line, where about 10% of the initial thymidine incorporation remained after an LPS challenge of 1/xg/ml (8471 + 747 cpm vs. 91337 + 8883 cpm for the HD-TS control). By contrast, a mouse monocyte-macrophage cell line (PU5-1.8) showed a more gradual sensitivity to LPS, starting from 0.01 ng/ml but going up to 1000 ng/ml. MMGT12 and MMGT16 reacted to LPS by the secretion of inflammatory cytokines (Table 6). The other strains did not react by a proliferation arrest (Table 6, Fig. 5) or by the secretion of inflammation-related cytokines (Table 6), except for the M M G T l l strain that secreted IL-6

161

upon induction. However, these strains (MMGTll and 15) secreted compounds with HBGF activity, and this activity decreased after LPS challenge. The strains MMGT12 and 16, that reacted to endotoxins, were able to opsonize latex particles.

4. Discussion

Several microglial cell strains of mouse origin were generated. The full characterization study encompassed macrophage and cytoskeletal markers as well as endotoxin challenge. The morphology and marker characteristics accord with the observations for primary microglial cell cultures. Microglia have been extensively investigated immunohistochemically with a wide variety of markers. Major histocompatibility complex (MHC) antigens have been well studied; class I (CD64, OX18) and class II antigens (OX6; OXI-1) are solely found in microglia mostly in activated ceils - in sections of the brain as well as in primary culture of brain cells (Akiyama et al., 1988; McGeer et al., 1988; Poltorak and Freed, 1989; Streit et al., 1989; Peudenier et al., 1991). Microglia were also shown to express integrins (CDll, Giulian and Baker, 1986; Alliot et al., 1991; Peudenier et al., 1991), vimentin (Graeber et al., 1988; Streit et al.,

Table 6 C h a l l e n g e of M M G T 1 clonal cell lines with 1 / . t g / m l l i p o p o l y s a c c h a r i d e (LPS) a Treatment MMGT11 Control

N u m b e r of cells ( × 107)

TNF (U/ml)

1

_

LPS MMGT12

1

-

Control LPS MMGT13 Control LPS MMGT14 Control LPS MMGT15 Control LPS MMGT16

4 2

IL-6 (U/ml) c

5546 + 411 *

IL-1 (U/ml)

_

760 +

183 *

107 + 0.1 6984 + 1261 *

5 5 4 6

-

1 1

-

-

106_+

32 *

TGF-/3 (pg/ml)

-

-

-

-

1467 + 231 *

EGF (ng/ml)

HBGF (ng/ml)

< 0.13 < 0.13

254 + 29 *

1.8 + 0.3 8.7 + 1.9 *

6.5 + 4 2.1 + 0.1 < 0.25 < 0.25

-

-

ND

ND

-

-

ND

ND

-

-

ND

ND

-

-

ND

ND

-

-

1.4

-

1.0

-

4 1

3200 + 277 *

10 + 2.6 7065 +_ 1481 *

1

24

ND

-

-

ND

ND

LPS MMGT18 Control LPS

1

28

ND

-

-

ND

ND

-

-

ND

ND

-

-

ND

ND

-

-

317 _+ 80 *

1.8 _+ 0.2 3.0 + 0.7 *

3.3 + 0.6 1.4 + 0.1

Control LPS MMGT17 Control

3 3

4800 + 226 *

_+ 0.4 + 0.1

*

< 0.25 < 0.25

a T h e cells w e r e s t i m u l a t e d for 24 h in H D T S w i t h o u t W E H I - 3 c o n d i t i o n e d m e d i u m . b Significant differences b e t w e e n c o n t r o l and i n d u c e d s a m p l e s (* P < 0.05); o n e - w a y analysis of variance. c T h e results are e x p r e s s e d as m e a n s + SD; e x p e r i m e n t s w e r e run in triplicate. N D , not d e t e r m i n e d ; - , cytokine s e c r e t i o n not p r e s e n t or below the d e t e c t i o n limit: for T N F , IL-1 a n d IL-6 1 U / m l , for TGF-/3 10 p g / m l , for E G F 0.5 n g / m l , and for H B G F ( F G F ) 2 n g / m l .

162

T. [J4 Briers" et al. /Journal o f Neuroimmunology 52 (1994) 153-104

1989) and mature macrophage markers (F4/80, Lawson et al., 1990; Alliot et al., 1991). Microglia were negative for T-cell markers (Streit et al., 1989), astrocyte markers (GFAP, Giulian and Baker, 1986; S1OOb, Akiyama et al., 1988), and oligodendrocytic markers (galactocerebroside; Giulian, 1987). They were also shown to have receptors for RCA and ac-LDL (Giulian and Baker, 1986; Streit et al., 1988; Colton et al., 1992). The immunocytochemical profile of the microglial strains developed by us fully covers these microglial characteristics. Indeed, the set of macrophage markers selected for this study allowed the classification of the strains into three groups: (i) cells not reactive for the markers; (ii) cells with intermediate characteristics; and (iii) cells with full histiocytic characteristics. The two cell strains with histiocyte characteristics, MMGT12 and 16, closely resemble the reactive microglial cell type. However, these cell strains as well as the mother cell line still had to be challenged with endotoxins to obtain the production of inflammationlinked cytokines. The fact that not all the thymidine incorporation into D N A was blocked by endotoxin for MMGT1 cells, but virtually blocked for MMGT12 and 16 cells, points to the existence of non-inducable population(s) in the original microglial cell line. It was also remarkable that only those cells tightly associated with the plastic surface (before endotoxin challenge) could be induced with endotoxins, and that the induction resulted in an even more stringent adhesion accompanied by a change in morphology to a more flattened and ameboid form. This observation contrasts with those in mouse monocytic-macrophage cell lines like PU5-1.8 or J774, which are found in suspension or loosely adherent before LPS challenge, and become tightly adherent thereafter. Upon induction with endotoxins, MMGT1, 12, and 16 secrete large amounts of TNF, IL-1, and IL-6, as has been shown for primary cultures of microglia (Giulian, 1987; Gebicke-Haerter et al., 1989). When challenged, these cell lines secrete increased amounts of E G F and TGF-/3, also shown for primary microglia (Plata-Salamfin, 1991; Lindholm et al., 1992). These findings are in agreement with the proposed functions of microglia in brain homeostasis, defence and repair (Perry and Gordon, 1988; Rogers and Rovigatti, 1988; Streit et al., 1988). H B G F activity was only found for non-inducible strains ( M M G T l l and 15); the only effect observed after LPS challenge was a reduction of the H B G F secretion. Basic FGF, a compound of the H B G F group, is produced in cultured rat microglia (Shimojo et al., 1991; Araujo and Cotman, 1992), and has a t r o p h i c effect on neurons and glia (Baird and Wallicke, 1989). Together with IL-1, it is also known to stimulate the production of /3APP m R N A (Bauer et al., 1991; Rothwell, 1991). It has been suggested that ameboid microglia con-

vert into ramified microglia during the postnatal period (Ivy and Killacky, 1978), and that the ramified cells are able to transform into reactive microglia upon certain stimuli (Giulian, 1987). Therefore, the ameboid microglia are considered to be a developmental precursor of the ramified cell. The ramified microglia, being derived from the ameboid form and having a lower level of phagocytotic activity, are thought to be quiescent precursors of the reactive cells, but the morphology of microglia remains variable (Jordan and Thomas, 1988; Perry and Gordon, 1988; Lawson et al., 1990). Reactive microglia appear distinct from ramified and ameboid cell forms. While they possess some established features in common with the ameboid form such as dense bodies, lipid vacuoles, and lysosomes, the reactive cells are small, and round to rod-shaped (Murabe and Sano, 1981; Murabe et al., 1981). While devoid of processes, they exhibit no pseudopodia or philopodia but sometimes have membrane ruffels. This cell type is associated with injury or damage to brain tissue and also corresponds to a transient population. They appear in the tissue near the site of injury within a day after trauma, and persist for several days depending on the extent of tissue damage (Akiyama et al., 1988). These cells seem to be capable of developing at any time after the early postnatal period and in any area of brain tissue. Under culture conditions, we were able to observe: bright-rounded cells to rod-like, spindle-shaped, ameboid, and ramified. In the early stages of implantation, spindle-shaped cells have been shown to predominate over ramified forms, and these cells gradually develop ramified processes (Rieske et al., 1989). Isolated microglia undergo in vitro transformations to ameboid, rod-shaped, and ramified forms in response to several cytokines, or factors which affect macrophage function, and these transformations are reversible (Suzumura et al., 1991). Our observations on morphological transformations of microglia in vitro are fully corroborated by the studies of other groups. Cloned macrophages, derived after immortalization by infection with u-myccontaining retroviruses, have differentially regulated expression of cytokine genes, and this heterogeneity seems to be derived from differentiation-related mechanisms (Pirami et al., 1991). Our observation of microglia-like strains with intermediate characteristics having a more spindle-shaped morphology and a different cytokine profile may point to the existence of specific microglial subpopulations, and possibly to the existence of a microglial lineage. Recruitment of these cells after brain injury or infection may therefore be more complex because LPS or other compounds on their own are not able to induce this transformation. We have been able to develop cell lines that retain the typical morphology of microglia in primary culture, as well as their physiological capacities. V-myc was

T.W. Briers et aL /Journal of Neuroimmunology 52 (1994) 153-164

selected (the suitability of v-myc for immortalization has been reviewed thoroughly by different authors: Weinberg, 1989; Zelenka, 1990; Bishop, 1991; Shay et al., 1991; Prendergast and Ziff, 1992), since it was already used successfully for immortalization of several cell types without loss of the primary phenotype (Hurlin et al., 1989; Birren and Anderson, 1990; Briers et al., 1993), and we preferred transfection because of the possible secretion of oncogenic viruses after infection (Righi et al., 1991). In conclusion, the transfected cell line MMGT1, and the selected strains (11-18) resemble microglial cells in primary culture. MMGT12 and 16 show clear 'tissuespecific macrophage' characteristics and resemble reactive microglia. The rounded form has hitherto not been described using in vitro culture systems, but it has been shown to be present in brain tissue (Jordan and Thomas, 1988). Our results point to the existence of a heterogeneous microglial population containing cells with different properties dependent of their differentiation stage. MMGT1 cell line and cell strains are safe and suitable for in vitro studies of the process of microglial activation. The cell lines show rather uniform properties and therefore can be used for biochemical and cell biological analysis. Such studies may shed some light on the microglia enigma: its embryological origin, its normal function, and its role under pathological conditions.

Acknowledgements We wish to thank Dr. G. David for providing us with monoclonal antibodies to glycosaminoglycan (Center for Human Genetics, University of Leuven, Leuven, Belgium), and Dr. K. Beyreuther for the cDNA probe for APP, pCD9 (Zentrum fiir Molekulaire Biologie, Heidelberg, Germany). We thank S. Pattijn, M. Defruyt, and P. De Corte for their technical assistance, and F. Shapiro for manuscript editing.

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