v-myc carrying retrovirus

v-myc carrying retrovirus

Journal of Neuroimmunology, 27 (1990) 229-237 Elsevier 229 JNI 00935 Immortalization of murine microglial cells by a carrying retrovirus E. Blasi 1...

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Journal of Neuroimmunology, 27 (1990) 229-237 Elsevier

229

JNI 00935

Immortalization of murine microglial cells by a carrying retrovirus E. Blasi 1, R. Barluzzi

1 V.

Bocchini

2, R.

v-raf/v-myc

Mazzolla 1 and F. Bistoni 1

1 Department of Experimental Medicine and Biochemical Sciences, Microbiology and 2 Molecular and Cellular Biology Sections, University of Perugta, Perugia, Italy (Received 12 September 1989) (Revised, received 8 November 1989) (Accepted 8 November 1989)

Key words: Microglia; Phagocytosis; Cell line; Oncogenes

Summary A murine cell line (BV-2) has been generated by infecting primary microglial cell cultures with a

v-raf/v-myc oncogene carrying retrovirus 02). BV-2 cells expressed nonspecific esterase activity, phagocytic ability and lacked peroxidase activity. Such cells secreted lysozyme and, following appropriate stimulation, also interleukin 1 and tumor necrosis factor. Furthermore, BV-2 cells exhibited spontaneous anti-Candida activity and acquired tumoricidal activity upon treatment with interferon-'¢. Phenotypically, BV-2 cells resulted positive for MAC1 and MAC2 antigens, and negative for MAC3, glial fibrillary acidic protein (GFAP) and galactocerebroside (GC) antigens. Since BV-2 cells retain most of the morphological, phenotypical and functional properties described for freshly isolated microglial cells, we can conclude that J2 virus infection has resulted in the immortalization of active microglial cells.

Introduction

It is well established that cells with phagocytic properties, the so-called 'ameboid' microglia, appear in the brain during the late stage of embryogenesis. However, by the late postnatal period such cells disappear, likely giving rise to the nonphagocytic 'ramified' microglia, whose role is still unknown (Del Rio Hortega, 1932; Ling, 1981; Murabe and Sano, 1982; Oehmichen, 1982). After Address for correspondence: Dr. Elisabetta Blasi, Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Sez. Microbiologia, University of Perugia, Via del Giochetto, 06100 Perugia, Italy.

brain injury or bacteria/virus infection(s) in adult rodents, ameboid cells with phagocytic ability can be detected again. Whether these cells are bloodrecruited monocytes and/or 'reactivated' microglia is still controversial (Imamoto and Leblond, 1977; Oehmichen, 1982; Perry et al., 1987). Moreover, in humans, microglial cells are involved in pathological processes of the central nervous system (CNS), such as in demyelinating diseases and human immunodeficiency virus (HIV) infections (Esiri and McGree, 1986; Woodroofe et al., 1986; Price et al., 1988). Many attempts have been made to provide in vitro models for further studies on microglia. Using newborn as well as adult brain tissues, primary

0165-5728/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

230 cultures of microglia have been obtained in the 'active' form, as demonstrated by functional properties (Frei et al., 1986; Giulian and Baker, 1986; Suzumura et al., 1987; Hayes et al., 1988; Rieske et al., 1989). A major restriction for these approaches was the limited number of cells obtained as well as the difficult, time-consuming technical procedures involved. Since we have previously shown that a v-raf/v-myc carrying retrovirus 02) was able to immortalize phagocytic cells of the bone marrow compartment in vitro (Blasi et al., 1985), we investigated the susceptibility of microglial cells to J2 virus immortalizing capacity. Here, we show that in vitro infection of microglial cultures with J2 virus results in the generation of a cell line exhibiting morphological, phenotypical and functional properties of active microglial ceils.

Materials and methods

Cell cultures Microglial cells were purified by shaking and subsequent selective plastic adherence of 1-weekold mixed brain cultures obtained from C 5 7 B L / 6 mice, as detailed elsewhere (Giulian and Baker, 1986; Bocchini et al., 1988). The cultures were incubated overnight with control supernatants or J2 virus containing supernatants, fed biweekly with RPMI 1640, supplemented with 10% heat-inactivated fetal bovine serum, gentamicin (50 /~g/ml) and L-glutamine (4 mM) (complete medium). All reagents were purchased from Gibco Laboratories (Grand Island, NY, U.S.A.). The established cell line was maintained in complete medium. Biweekly passages were performed as follows: cultures were vigorously shaken/pipetted for cell detachment and fresh cultures were set at a concentration of 5 X 10 4 cells/ml. L5178Y (a chemically induced lymphoma of D B A / 2 mice) was maintained in complete medium and used as a target cell in the 51Cr release assay. Stimulating agents The stimulating agents used in the present study were: recombinant murine interferon--/ (1FN-T) (Genentech, South San Francisco, CA, U.S.A.); lipopolysaccharide (LPS) and polyinosinic-poly-

cytidylic acid (Poly I : C ) (Sigma, St. Louis, MO, U.S.A.).

Candida albicans The strain of C. albicans, used throughout this study, was the kind gift of Dr. Kerridge, Department of Biochemistry, University of Cambridge, Cambridge, U . K . C . albicans was maintained by daily passages on Sabouraud dextrose agar plates. Inactivated C. albicans (iCA) was obtained by heating the microorganisms 3 times at 121 ° C. Electron microscopy analysis BV-2 cell culture samples were processed for scanning electron microscopy (SEM) and transmission electron microscopy (TEM) as previously described (Frei et al., 1987) and photographs were made at the Center of Electron Microscopy, University of Perugia, Perugia, Italy. Colony-forming units (CFU) assay BV-2 cells were plated at different concentrations (0.1 ml/well) in 96-well plates (Corning Glass Works, Coming, NY, U.S.A.) and infected with 0.1 ml of C. albicans (5 x 104/ml). Following different times of incubation at 37 ° C, Triton X100 (0.1% final concentration) was added to the wells. Serial dilutions from each well were then made in distilled water and plated (triplicate samples) in Sabouraud dextrose agar. The number of CFU was determined after 24 h incubation at 37°C. Control cultures consisted of C. albicans incubated without effector cells. The results were expressed as percentage of CFU inhibition according to the following formula: experimentalgroup x 100. % CFU inhibition = 100- CFU CFU control cultures

Phagocytosis assay Inactivated C. albicans microorganisms (iCA) (2 X 107 cells/mi) were incubated with or without mouse serum for 15 rain and then added to the BV-2 cell cultures (10 6 cells/ml). After 2 h incubation at 37°C, the iCA excess was removed by centrifugation of the cell suspension on a Ficoll cushion at 1100 rpm for 10 rain. The cells at the interface were recovered and washed. The iCA

231 uptake was directly evaluated in Giemsa-stained cytospin preparations. A minimum of 200 cells were scored and any cell containing three or more particles was counted as phagocytic.

Supernatant production BV-2 cells were incubated for 48 h at 37 ° C at a concentration of 5 × 105 cells/ml in complete medium with or without stimuli. Cell-free supernatants from control and stimulated cells were then collected and assayed for several biological activities.

Lysozyme (LZM) assay LZM activity was determined by the modified lysoplate method of Osserman and Lawlor (1966). The LZM content of test samples was determined from the standard curve of purified egg white LZM, established under identical conditions, and expressed as # g / m l .

Interleukin 1 (IL-1) assay IL-1 activity was evaluated as incorporation of [3H]thymidine ([3H]Thy) by murine thymocytes, as described elsewhere (Mizel, 1981). Briefly, thymocytes from thymuses of 6-8-week-old C 3 H / H e J mice were cultured in complete medium (1 × 107 cells/ml) in 96-well plates. Serial dilutions of standard IL-1 or supernatants from BV-2 cells plus concanavalin A were added to the thymocyte cultures. Following 48 h incubation at 37°C, [3H]Thy (Amity, Milan, Italy) was added to each culture well for an additional 24 h. The cultures were then harvested onto glass fiber strips for scintillation counting. The results were expressed as mean cpm of triplicate samples.

Tumor necrosis factor (TNF) assay The quantitation of T N F activity was performed by a bioassay using L929 cells, as described (Rubin et al., 1985). Briefly, L929 cells were seeded into 96-well flat-bottom plates (4 × 104 cells/well) and incubated for 24 h at 37°C. The spent medium was then removed and replaced with test sample or standard T N F preparations containing actinomycin D (3 ttg/ml). After 20 h of incubation, plates were stained with 0.5% crystal violet in 20% methanol for 15 min and washed in tap water. After drying, absorbance was

determined at 540 nm using a Titertek Multiskan platereader (Flow, Rockville, MD, U.S.A.). All determinations of T N F activity in test samples were compared to commercially available preparations of T N F with known titers and the results were expressed as U / m l .

51Cr release assay The assay was performed as previously described (Blasi et al., 1987). Briefly, BV-2 cells (2 × 106/ml) were plated in 96-well plates with various stimuli. After overnight incubation, the cells were tested against 51Cr-labeled L-5178Y target cells in a 24 h release assay, at an effector to target ratio of 20 : 1. The spontaneous release (SR) from target cells alone was between 10 and 20% of the total radioactivity incorporated. The results were expressed as percent of cytotoxicity, calculated from the average of triplicate samples according to the following formula: cprn with effector cells- SR %cytotoxicity- total cpm incorporated in target cells- SR

Flow cytometry analysis BV-2 cells were washed twice before being resuspended (106/ml) in phosphate-buffered saline containing 0.5% bovine serum albumin and 0.1% sodium azide. The cell suspensions were mixed with 10/xl of antibodies (anti-MAC1, anti-MAC2 and anti-MAC3 antibodies were purchased from Hybritech, San Diego, CA, U.S.A.) and incubated for 45 min. The cells were then washed 3 times, and 10 ~tl of the fluoresceinated isothiocyanate conjugate anti-immunoglobulin (FITC-anti-Ig) (Cappel, Malvern, PA, U.S.A.) was added for an additional 45 rain. All staining procedures were carried out at 4 o C. The cells were washed 3 times and analyzed for cell surface phenotype on a fluorescein-activated cell sorter (FACS) (FACSTAR, Becton Dickinson, Erembodegem, Belgium).

Statistical analysis The significance of the data was evaluated by the Student t-test. All experiments were repeated at least 5 times and representative experiments are depicted in the Figures and Tables.

232

Results Primary microglial cell cultures, essentially composed of esterase-positive (> 95%) adherent cells, were exposed to the 52 virus. Uninfected controls received complete medium. Infected and uninfected cultures were fed at regular intervals and examined for morphology. Within 3-4 weeks, foci of proliferating cells (BV-2 cells) were observed in the infected cultures, whereas the uninfected cells gradually lost adherence properties and eventually died. As shown in Fig. lA, BV-2 cell cultures appeared as a population composed of adherent, variably shaped cells. When assessed for histochemical markers, BV-2 cells exhibited a high degree of homogeneity, being > 90% positive for nonspecific esterase activity. All cells lacked peroxidase activity (data not shown). Since morphology and phagocytic ability are crucial parameters for discriminating between microglia and other glial cells (Saito et al., 1986; Bocchini et al., 1988), we performed electron microscopy analysis on BV-2 cells cultivated on glass coverslips in the presence of iCA microorganisms. BV-2 cells, which displayed a large number of elongated filopods (Fig. lB), appeared overengulfed with C. albicans particles (Fig. 1C). In an attempt to better define the phagocytic properties of BV-2 cells, phagocytosis experiments were carried out by incubating BV-2 cells and iCA microorganisms, which had or had not been preopsonized with fresh mouse serum. As shown in Table 1, 81% of the cells were phagocytic, whereas the percentage increased to 93% when opsonized C. albicans microorganisms were used. Moreover, by opsonization the number of iCA ingested per cell also augmented, as indicated by the increased phagocytosis index (Table 1). In order to determine whether BV-2 cells exhibited anti-Can&a activity against viable microorganisms, BV-2 cells were incubated with C. albicans for 4 h and then the CFU assay was performed. Time- and E: T ratio-dependent antiCandida activity was observed, the maximal levels being reached after 24 h of co-culture at an E: T ratio of 20 : 1 (Table 2). The secretory properties of BV-2 cells were also evaluated. As depicted in Fig. 2, unstimulated BV-2 cells did not secrete significant levels of IL-1

Fig. 1. Photomicrographs of BV-2 cells. A: Photomicrograph of viable BV-2 cell cultures, bar = 30 pm. E: SEM appearance of fixed BV-2 cells cultivated on glass coverslides, bar = 5 pm. C: Portion of BV-2 cell cytoplasm engulfed with iCA as assessed by TEM, bar = 3 pm.

activity. Following a dose-dependent whereas treatment

treatment with LPS, we found induction of such activity, with Poly 1: C or IFN-y had

233 TABLE 1

80

P H A G O C Y T I C ABILITY O F BV-2 CELLS iCA pretreatment a

% Phagocytic cells b

Phagocytosis index c

None Mouse serum

81 93

8.3 16.7

g

a iCA (2X107/ml) were incubated for 15 rain without or with fresh mouse serum before being used as targets for phagocytosis. b The phagocytosis assay was performed as detailed in Materials and Methods. total number of phagocytized iCA Phagocytosis index = total n u m b e r of BV-2 phagocytic cells

60

g 40

20

TABLE 2 KINETICS CELLS

OF

ANTI-CANDIDA

ACTIVITY

BY

BV-2

BV-2 cells, plated at various concentrations (106/ml, 2 × 105/ml and 4 x 104/ml), were incubated with C. albicans (5 x 104/ml). At the indicated times, C F U assays were performed and the results expressed according to the formula depicted in Materials and Methods. Standard deviations less than 5% have been omitted. Time

% CFU in~bition

(h)

20:1

4:1

0.8:1

2 4 8 24

26 35 70 97

24 32 69 94

16 23 54 NT

LPS wg/mb

POLY I:C (t,9,'rnl)

Fig. 2. Effect of LPS, Poly I : C or IFN-y treatment on IL-1 activity by BV-2 cells. BV-2 cells were plated (5 × 105 cells/ml) and incubated in medium or in the presence of Poly I : C, LPS, or IFN- T. Following 48 h culture, the cell-free supernatants were harvested and assayed for IL-1 activity, as detailed in Materials and Methods. The values represent the mean cpm of triplicate samples and the bars the standard deviations. * Significantly different from controls at p < 0.01.

TREATMENT

NT, not tested.

I FNy tu/mb

30

TN]: ACTIVITY (tgMt) 20 10

LZM ACTIVITY G,G:ML) 20 40

TABLE 3 INDUCTION CELLS

O F T U M O R I C I D A L ACTIVITY IN BV-2

___+

NONE

I

~CA

BV-2 cells (2X 106/ml) were incubated overnight with the agents described below before being assessed for tumoricidal activity. The cytotoxicity assay was performed against 51Crlabeled L5178Y target cells at an effector to target ratio of 20 : 1. The results are expressed as percent cytotoxicity according to the formula depicted in Materials and Methods.

LPS 0,1 (~C/ML) 1 10

Treatment

Concentration

% Cytotoxicity

ICFU~tdL, 420

None iCA (cells/ml) Poly I : C (/~g/ml)

4 x 107 100 10

IFN-y (U/ml)

100 20 4 10

4.7 5.1 6.6 4.8 5.7 44.5 18.1 1.9 4.6 5.7 4.1

1

LPS (/~g/ml)

1

0.1

* Significantly different from controls at p < 0.01.

POLYIC 1 (uG/Mt) 10 100





÷*

,p

100

30

2~)

1'0

~

~o

Fig. 3. T N F and L Z M activity of BV-2 cells following iCA, Poly I : C, LPS or IFN-y treatment. BV-2 cells (5 x l0 s cells/ml) were plated and incubated in medium or in the presence of iCA, Poly 1 :C, LPS or IFN-y. Following 48 h culture, the cell-free supernatants were harvested and assayed for T N F and L Z M activity, as detailed in Materials and Methods. The values represent the mean of quadruplicate samples. The bars represent the standard deviations. * Significantly different from controls at p < 0.01.

234

little or no effect. Different patterns of results were observed when TNF or LZM activities were assayed (Fig. 3). In particular, BV-2 cells secreted no detectable levels of TNF and none of the agents, with the exception of Poly I : C , was able 3~B

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rLl

2~0

8

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301~

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to induce such activity. In contrast, high levels of LZM were found in supernatants from unstimulated cells ( > 50/~g/ml), whereas treatment with each of the agents resulted in a dose-dependent inhibition of LZM activity. When tested against L5178Y target cells, we found that BV-2 cells were not constitutively cytotoxic. Following overnight exposure to iCA, Poly I : C , or LPS their basal cytotoxicity was also unaffected. In contrast, exposure of BV-2 cells to IFN-T resulted in a dose-dependent induction of tumoricidal activity (Table 3). Furthermore, upon co-stimulation with IFN-T plus LPS (100 ng/ml), a synergistic effect was also observed (data not shown). Phenotypical analysis was performed on BV-2 cells by flow cytometry. We found that staining with anti-MAC1 antibody resulted in a weak but consistent shift of the fluorescence intensity profile above the negative controls (Fig. 4A). BV-2 cells expressed high levels of MAC2 (Fig. 4B), whereas no detectable MAC3 antigens were found (Fig. 4C). Furthermore, BV-2 cells were negative for glial fibrillary acidic protein (GFAP) and galactocerebroside (GC) antigens (data not shown), which are specific markers for astrocytes and oligodendrocytes, respectively (Bignami et al., 1972; Raff et al., 1978). Upon continuous in vitro culture (> 18 months), morphological, phenotypical and functional properties of BV-2 cells remained unchanged and, as assessed by Northern blot analysis, such cells constitutively expressed high levels of J2 virus m R N A (data not shown). Discussion

O

''l''''l loft

....

I .... 20~

FLI Fig. 4. Surface phenotype of BV-2 cells. Flow cytometric histograms of cells stained with anti-MAC1 (A), anti-MAC2 (B) or anti-MAC3 (C) antibodies plus FITC-anti-Ig are indicated with dotted lines. Continuous lines represent cells stained with FITC-anti-Ig.

Suitable tissue culture techniques for in vitro isolation and propagation of microglial cells have proved to be difficult. Only recently, it was possible to obtain primary cultures of microglia by plastic adherence (Giulian and Baker, 1986) or explantation of axotomized rat facial nuclei (Rieske et al., 1989). Nevertheless, time-consuming techniques a n d / o r difficulties in isolating a sufficient number of ceils were a major restriction(s). In an attempt to obtain a new in vitro model for further studies on microglia, we investigated whether the recombinant J2 retro-

235

virus, which carries v-tar and v-myc oncogenes, could immortalize microglial cells in vitro, without affecting their morphological, phenotypical and functional properties. Our data show that this goal has been achieved. We initially reported that infection of fresh murine bone marrow cells from adult mice with the J2 virus results in the selective immortalization of macrophage-like cells, the phenomenon being restricted to the bone. marrow compartment (Blasi et al., 1985). With the exception of growth factorindependent proliferation in vitro, such macrophage cell lines maintain the morphological, phenotypical and functional characteristics of normal syngeneic tissue macrophages (Blasi et al., 1987). The data presented here, together with those recently published on the in vitro generation of fetal liver cell lines by J2 virus infection (Cox et al., 1989), indicate that compartments other than bone marrow are accessible to the J2 virus immortalizing capacity, providing that non-adult tissues are used. It remains to be clarified whether this is an age-related phenomenon. Alternatively, differences on the frequency of J2 'sensitive' progenitor cells, within the tissues assessed, may account for the results obtained. Morphological studies on the established BV-2 cell line show the presence of adherent ceils which possess many spinous processes, similar to in vitro cultured microglia (Giulian and Baker, 1986; Frei et al., 1987). Such morphological properties are maintained by BV-2 cells during the time in culture and are also observed in ten out of ten clones (cloning efficiency > 40%) (unpublished results). Phenotypical analysis of BV-2 cells indicates the presence of a homogeneous population (single-peak fluorescence profiles), which exhibits weak but consistent positivity for MAC1 but lacks MAC3 antigen. BV-2 cells express high levels of MAC2 antigen, which is also detectable in primary microglial cells (data not shown). Moreover, BV-2 cells are negative for GFAP and GC, specific markers for astrocytes and oligodendrocytes (Bignami et al., 1972; Raft et al., 1978). Furthermore, most of the cells, similarly to primary newborn microglia (Giulian and Baker, 1986), possess nonspecific esterase activity (> 90%) and lack peroxidase activity. These findings allow us to conclude that BV-2 is a microglial cell line.

Phagocytosis studies using C. albicans microorganisms (5 gm) strengthen this conclusion. In fact, it is well-known that astrocytes and oligodendrocytes have phagocytic ability to some extent (Noske et al., 1982; Saito et al., 1986; Bjerkness et al., 1987); however, only microglial cells are able to ingest large-size particles such as Candida (Bocchini et al., 1988). Thus, also in this respect, BV-2 cells and primary microglial cells are indistinguishable. Upon opsonization the percentage of phagocytic cells as well as the phagocytosis index are enhanced, indicating that a functionally active Fc receptor system is expressed on BV-2 cells. Similarly to primary microgha (Giulian et al., 1986; Frei et al., 1987), BV-2 cells retain constitutive as well as inducible secretory functions. In particular, BV-2 cells respond to LPS or Poly I : C as IL-1- or TNF-inducing factors, respectively. Furthermore, our data provide the first evidence that LZM activity is constitutively expressed at high levels in a microglial cell line. Interestingly, the susceptibility to LPS, as an LZM down-regulating signal, is shared by BV-2 cells and tissue macrophages (Warfel and Zucker-Franklin, 1986). However, unlike the previously described J2 virus-derived macrophage cell line (Pitzurra et al., 1989), BV-2 cells show reduced LZM activity upon exposure to IFN-y. At the present time, it remains to be established whether the few phenotypical a n d / o r functional differences observed between BV-2 cells and microglia/tissue macrophages may be due to the different experimental conditions used. Alternatively, it is possible that the stage of activation/differentiation of BV-2 cells is responsible for the pattern of biological properties observed. The proposed 'scavenger' role of microgha in vivo (Ling, 1981; Oehrnichen, 1982) is once again supported by our in vitro data showing high phagocytic ability as well as constitutive antiCandida activity of BV-2 cells. The kinetics and E : T ratio dependency closely resemble those previously shown for body macrophages (Fromtling and Shadomy, 1986) and the J2 virus-derived bone marrow cell line (Blasi et al., 1990). Moreover, BV-2 cells acquire tumoricidal activity upon treatment with IFN-7 at doses as high as 20 U/ml. In this respect, BV-2 cells exhibit susceptibility to

236

IFN-7 slightly lower than peritoneal macrophages, which become tumoricidal following exposure to 3 U / m l (Varesio et al., 1984). Nevertheless, BV-2 cells respond to the synergistic action of IFN-3, plus LPS (data not shown) as peritoneal macrophages and microglia (Varesio et al., 1984; Frei et al., 1987). Overall, these findings demonstrate that primary microglial cultures are susceptible to the J2 virus immortalizing capacity. The J2 virus-derived cell line retains most of the immunological properties ascribed to the active microglia, both as secretory and effector cells. Therefore, the BV-2 cell line provides a useful model for in vitro studies on the molecular mechanisms controlling induction and/or expression of biological activities in microglia. A better understanding of the microglia biology may, in turn, represent a precious step in the struggle against microglia-involved pathologies.

Acknowledgements We wish to thank E. Rossodivita and A. Nuti for performing electron microscopy and flow cytometry analysis. Our acknowledgements are also extended to E. Mahoney for editorial and secretarial assistance.

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