brain macrophages in vitro

Journal of Neuroimmunology

ELSEVIER

Journal of Neuroimmunology 63 (1995) 55-61

Colony-stimulating factors regulate programmed cell death of rat microglia/brain macrophages in vitro Jochen Gehrnmnn

*

Institutes of Neuropathology and Clinical Pathology, Department of Pathology. Uniwrsity Hospital. Schmelzbergstrasse 12. CH-8091 Ziirich, Switzerland

Received 12 June 1995; revised 3 August 1995; accepted 9 August 1995

Abstract Programmed cell death of activated microglia appears to be one mechanism how steady state of microglia is achieved in vivo. Programmed cell death of microglia might result either from the downregulation of microglial mitogens/survival factors or from signals which directly induce microglial cell death. To further elucidate the mechanisms regulating programmed cell death in microglia, growth factor and cytokine dependence of microglial proliferation and cell death have been examined in vitro in microglia/brain macrophage cultures established from neonatal rat brain. Microglial proliferation was assessed by PCNA labelling and DNA fragmentation by the TUNEL technique in the presence or absence of several cytokines including IL-l, IL-6, TGFP 1, TNFcu, M-CSF and GM-CSF. Results of TUNEL labellings were supplemented by gel electrophoretic analysis of DNA extracted from cultured microglia which showed laddering of DNA fragments. Of all cytokines/growth factors tested, GM-CSF and M-CSF were not only the strongest microglial mitogens but, moreover, withdrawal of M-CSF or GM-CSF significantly enhanced rates of microglial cell death by DNA fragmentation. Expression of microglial growth factors, in particular colony-stimulating factors, may thus be instrumental in controlling steady states of microglia in the injured nervous system. Keywords: Apoptosis; Programmed cell death; Glia; Colony stimulating factor; CNS regeneration; Autoimmunity

1. Introduction Microglial proliferation and activation are common phenomena in the injured nervous system (Gehrmann et al., 1991, 1992a, b, 1995; Lawson et al., 1990; Perry and Gordon, 1988; Streit et al., 1988). Microglia become activated in many diseases of the central nervous system (CNS) such as neurodegenerative disorders, multiple sclerosis and human immunodeficiency virus infection of the CNS in AIDS (Dickson et al., 1991, Dickson et al., 1993; McGeer et al., 1993). Activated microglia may play pathophysiologically important roles in the injured CNS. They can act as antigen presenting cells, harbour infectious agents such as HIV, release cytotoxic compounds but also support repair processes by releasing growth factors and cytokines (Banati et al., 1993; Dickson et al., 1991; Matsumoto et al., 1992). Hence, information on how microglial activation is regulated in the injured nervous sys-

* Corresponding author. Phone +41 (1) 255 2595/2107; Fax f41 (1) 255 4402. 0165-5728/95/$09.50

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SSDI 0165.5728(95)00130-l

tern is important for our understanding how to reduce tissue damage in the injured CNS. In experimental models of brain injury, early onset microglial proliferation has been amply documented (Lee et al., 1994; Gehrmann and Kreutzberg, 1995). The mechanisms, however, by which the number of activated microglia is reduced following brain injury, are just beginning to be elucidated. While the classical notion of de1 Rio-Hortega (1932) that activated microglia leave the CNS through the brain vasculature has never been confirmed, it has recently been demonstrated that activated microglia are eliminated by a process which shares features with programmed cell death. Following peripheral nerve injury in the rat, synchronized proliferation of resident microglia is followed by delayed DNA fragmentation of perineuronal microglia (Gehrmann and Banati, 1995). Programmed or suicide cell death plays an important role in the maintenance of tissue homeostasis in the developing nervous system (Chu Wang and Oppenheim, 1978; Kerr et al., 1972; Wylie et al., 1980). It is under genetic control, and its pathological hallmark is intemucleosomal DNA fragmentation into fragments of approximately 180-

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200 base pairs length achieved by activation of a Ca2+/Mg2+-dependent endonuclease (Arends et al., 1990). Particularly in the developing nervous system, programmed cell death is regulated by the downregulation or complete withdrawal of growth factors (Chu Wang and Oppenheim, 1978; Oppenheim, 1991; Oppenheim et al., 1995; Raff et al., 1993). In the present study, growth factor dependence of microglial/brain macrophage cell death has been examined in microglia/brain macrophage cell cultures established from neonatal rat brain. The technique of terminal transferase-mediated UTP nick end labelling (TUNEL) by Gavrielli et al. (1992) has been used to detect single-stranded DNA breaks in cultured microglia/brain macrophages. These data have been supplemented by in vitro analysis of microglial proliferation and by electrophoretic demonstration of DNA laddering. In summary, the data of this study suggest that programmed cell death of microglia/brain macrophages is growth factor dependent, and that among those growth factors studied colonystimulating factors are particularly important.

from astrocytes by short adherence on a glass surface for 15 min. As assessed by immunocytochemistry, > 98% of these cells were 0X42-, MUC 102-positive and GFAPnegative microglia/brain macrophages, while < 1% were GFAP-positive astrocytes or galactocerebroside-positive oligodendrocytes. Microglial cells were then seeded at a cell density of lo6 cells/mm2 in uncoated 35 mm2 petri dishes (Falcon) and maintained for four days in DMEM containing 5% heat inactivated fetal calf serum in a 95% sir/5% CO, humidified incubator. During this 4-day incubation period, the cells were first incubated in the presence of one of the following cytokines (concentration range tested is given in brackets): (1) murine recombinant M-CSF (100-1000 U/ml) (Genzyme, Cambridge, MA, USA); (2) murine recombinant GM-CSF (100-1000 U/ml) ipopolysaccharide (LPS) (0.1- 10 pg/ml) (Genzyme); (3) 1’ (Sigma, Munich, Germany), (4) rat interferon-y (IFN-y) (lo-200 U/ml) (Genzyme); (5) rat interleukin 1CY(IL1 CY);(6) rat interleukin-6 (IL-6); (7) rat tumor necrosis factor a (TNF-o) (lo-200 U/ml) (all three cytokines kindly provided by Dan Lindholm, Martinstied); (8) rat transforming growth factor j31 (TGF/ll) (Gentech, San Fransisco, CA, USA). After this 4-day incubation period, the cells were washed several times with DMEM to remove the individual cytokine from the culture medium. Cultures grown in parallel under identical conditions were then either continued to be grown in the presence or started to be cultivated in the absence of one of the cytokines mentioned above. Cultures grown without cytokine supplementation were grown in DMEM containing 5% heat inactivated fetal calf serum in a 95% sir/5% CO, humidified incubator. Every 24 h within this 4-day period,

2. Materials and methods 2.1. Culture protocol Rat microglial cell cultures were established from cortices of newborn rats according to Giulian and Baker (1986) and Frei et al. (1987). After lo-14 days in vitro microglia were removed from the tissue culture supernatant by gentle shaking of tissue culture flasks and centrifugation at 200 X g for 5 min. Microglia were separated

Neonatal rat brain cortices & isolation of microglia/brain macrophages after 1O-14 d.i.v. J establishment of parallel cultures at a cell density of appr. 106cells/mm2 J priming with cytokines for four days either with M-CSF, GM-CSF, LPS, IFN-)I, IL 1a, IL-6, TNPa or TGFPl -1

after extensive washings in DMEM further culture propagation L J either stimulated with or without respective cytokine L J four-day culture period with analysis of parallel cultures for proliferation (PCNA) and for apoptosis (TUNEL) every 24hrs Fig. 1. Flow chart of microglial/brain macrophage cultures.

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the medium was removed from cultures grown in parallel, cultures rinsed three times in 25 mM HEPES, pH 7.4, cells fixed in 4% ice-cold paraformaldehyde for 20 min, and processed for PCNA or for TUNEL labelling as described below. The culture procedure is summarized in Fig. 1. 2.2. PCNA labelling for cell proliferation Proliferation was assessed in vitro by immunocytochemistry for proliferating cell nuclear antigen (PCNA) with a mouse monoclonal IgG,, antibody against PCNA (DAKO, Hamburg, Germany). PCNA staining was performed as follows: After blocking the fixed cells with 2% normal horse serum for 30 min at room temperature, they were incubated for 1 h at room temperature with the primary anti-PCNA monoclonal antibody diluted 1: 100 in PBS. After washing the cells 2 X 5 min in 0.1 M PBS (pH 7.4) containing 1% BSA, they were incubated for lhr at room temperature with the secondary antibody, i.e. a biotinylated anti-mouse IgG antibody (Serotec, UK, diluted 1: 100) followed by incubation with the ABC vectastain kit (Burlingame, CA, USA) for 1 h at room temperature with 3,3’-diaminobenzidine (Sigma) as substrate. Controls included omission of the primary or the secondary antibody or of the color substrate and replacement of the primary antibody by a control IgG,, antibody. Programmed cell death was evaluated as described below by the TUNEL method. To confirm cellular identity of proliferating and apoptotic microglia/brain macrophages, double-labellings for PCNA or TUNEL staining respectively and OX-42 immunostaining were additionally performed. In this case, OX-42 immunocytochemistry was performed using an alkaline phosphatase-conjugated secondary antibody with fast blue salt as the color substrate. All experiments were repeated in triplicate in three independent assays. 2.3. In situ end labelling of fragmented

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the ABC Elite kit (Vector Labs., Burlingame, CA, USA) for 45 min at room temperature and developed with 3,3’diaminobenzidine as substrate. Negative controls included omission of either terminal transferase, biotinylated UTP, the avidin biotin complex or the color substrate. As a positive control, labelling almost all nuclei, slides were pre-treated with DNAase I (20 pg/ml; Sigma, Munich, Germany). As a further positive control, paraffin sections of mouse embryos, gestational age E17, were included in each TUNEL run. In these embryonic mouse sections numerous labelled apoptotic nuclei were routinely detected for example in liver, intestine and dorsal root ganglia.

100

0

Control

LPS

IL-I

TNF

M-CSFGM-CSF

A

looI “,

=

z

80

DNA

For in situ detection of nuclear DNA fragmentation the TUNEL method (terminal transferase-mediated UTP nick end labelling) by Gavrielli et al. (1992) was used. TUNEL labelling was performed as following: cell cultures grown on glass cover slips were fixed for 20 min in 4% ice-cold paraformaldehyde. Endogenous peroxidase was inactivated by incubating the sections in 3% H,O, for 10 min at room temperature. After pre-incubation in TdT buffer (30 mM Tris-HCl, pH 7.2, 140 mM sodium cacodylate, 1 mM cobalt chloride) for 5 min at room temperature, the sections were incubated in TdT buffer containing 0.5 U/p1 terminal transferase (Boehringer Mannheim, Germany) and 0.5 pmol biotinylated 16-dUTP (Boehringer Mannheim) in a humid atmosphere for 1 h at 37°C. The reaction was stopped by transferring the coverslips into 300 mM sodium chloride, 30 mM sodium citrate for 15 min at room temperature. After rinsing the sections for 10 min in 2% bovine serum albumin (BSA), they were incubated with

Control

LPS

IL-I

TNF

M-CSFGM-CSF

B

Fig. 2. Quantitative analysis of microglial/brain macrophage proliferation in vitro with (A) or without (B) cytokine stimulation. The proliferation index is expressed as the percentage of PCNA + cells per OX42 + microglia/brain macrophages. Measurements were performed from 1 up to 4 days in vitro (div). Values represent means * S.E.M. (n = 3). Abbreviations (cytokine concentrations given in brackets): LPS, lipopolysaccharide (1 pg/ml); IL-l, interleukin-ln (100 U/ml); TNF, tumor necrosis factor-a (100 U/ml); M-CSF, macrophage colony-stimulating factor (200 U/ml); GM-CSF, granulocyte-macrophage colony-stimulating factor (200 U/ml). * : values are statististically different from control values (P < 0.01).

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For quantification of PCNA- and TUNEL-labellings, PCNA and OX-42 double-labelled cells and TUNEL and OX-42 double-labelled cells were counted twice in at least 10 randomly chosen areas of 0.71 mm’. Values are given as means _+ standard deviation per 10 mm2. Statistical analysis was performed using Student’s r-test. 1198 -

2.4. Gel electrophoresis

676 -

DNA was extracted from cultured microglial cells/brain macrophages as described by Miller et al. (1988). As the starting material for DNA extraction, freshly isolated microglial cells/brain macrophages, microglial cells/brain macrophages after stimulation with GM-CSF or cells after

Fig. 4. Gel electrophoresis of DNA extracted from cultured rat microglia/brain macrophages (lane 1, molecular weight markers; lane 2, freshly isolated microglial cells; lane 3, rat microglial cells/brain macrophages analysed after 4 days in vitro in the presence of GM-CSF (200 U/ml); lane 4, rat microglial cells/brain macrophages analysed after 4 days in vitro following withdrawal of GM-CSF).

0 Control

LPS

IL-l

TNF

M-CSFGM-CSF

A 100 m

?I 0

80

+ 7 ::

60

; d

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withdrawal of GM-CSF were used. DNA was extracted by phenol/chloroform 1: 1, precipitated overnight in - 20°C ethanol containing 0.3 M sodium acetate and then centrifuged for 30 min at 4°C at 12000 X g. The pellet was resuspended in 0.1 M Tris-HCl, pH 8.0, 10 mM EDTA (TE buffer) and the DNA treated with RNAase (100 pg/ml) for 1 h at room temperature and subsequently with proteinase K (100 pg/ml) at 37°C overnight. DNA was finally re-extracted with phenol, phenol/chloroform, chloroform, precipitated with ethanol, and resuspended in TE buffer. DNA samples (0.2 pg each) were electrophoretically separated on 1.8% agarose gels containing ethidium bromide (0.4 pg/ml). DNA was visualized by a UV transilluminator and the gels photographed with a polaroid camera.

s 0

3. Results Control

LPS

IL-l

TNF

M-CSFGM-CSF

B

Fig. 3. Quantitative analysis of microglial/brain macrophage apoptosis in vitro with (A) or without(B) cytokine stimulation. The apoptosis index is expressed as the percentage of TUNEL + cells per OX42 + microglia/brain macrophages. Measurements were performed from 1 up to 4 days in vitro (div). Values represent means f S.E.M. (n = 3). Abbreviations (cytokine concentrations given in brackets): LPS, lipopolysaccharide (1 pg/ml); IL-l, interleukin-1 (Y (100 U/ml); TNF, tumor necrosis factor-a (100 U/ml); M-CSF, macrophage colony-stimulating factor (200 U/ml); GM-CSF, granulocyte-macrophage colony-stimulating factor (200 U/ml).

3.1. Microglial/ brain macrophage proliferation In vitro, microglia/brain macrophages showed a base line level of proliferation which was strongly enhanced in the presence of GM-CSF (200 U/ml) and to a lower extent in the presence of M-CSF (200 U/ml) (Fig. 2A). These effects were dose-dependent (data not shown) and the percentage of proliferating microglia/ brain macrophages increased over the incubation period (Fig. 2A). In addition, IL-l and TNFo had some weak mitogenie effect on microglia/brain macrophages although this

J. Gehrmann / Journal of Neuroimmunology 63 (199.5) 55-61

effect was not statistically significantly different from control values (Fig. 2A). In cultures treated without M-CSF or GM-CSF, the number of proliferating, PCNA-positive microglia/brain macrophages decreased over time (Fig. 2B) paralleling an increase in the number of TUNEL-positive microglia/brain macrophages (Fig. 3B). 3.2. Programmed macrophages

cell

death

of

microglia / brain

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ever, the number of TUNEL-labelled microglia/brain macrophages was strongly increased over the incubation periods (Fig. 3B). Of ail other cytokines tested, only IL-l (Y and TNFo marginally increased the number of TUNELlabelled cells if the cells were grown without these cytokines (Fig. 3B). After 4 days in vitro the percentage of TUNEL-labelled cells per OX-42-positive microglia/brain macrophages was statistically significantly higher in IL-

In vitro, microglia/brain macrophages underwent programmed cell death and incorporated labelled DNA fragments. If cells were grown in the presence of M-CSF or GM-CSF, the number of TUNEL-labelled microglia was low and did not differ significantly from control cultures without cytokine supplementation (Fig. 3A). In parallel cultures treated without M-CSF or without GM-CSF, how-

Discussion

Fig. 5. Examples DNA fragmentation macrophages four days (A) prior to withdrawal bodies

rat microglia/brain (C) Apoptotic 400

X

nuclear

Programmed cell death of activated microglia has recently been identified as one mechanism to eliminate activated microglia and to control steady state of microglial cell numbers in the injured CNS. Perineuronal microglia undergo DNA fragmentation following peripheral nerve injury (Gehrmann and Banati, 1995). In experimental autoimmune encephalomyelitis, perivascular macrophages have been shown to undergo apoptosis (Nguyen et al., 1994) in addition to infiltrating, encephalitogenic T lymphocytes (Schmied et al., 1993). In the normal mouse brain, microglial apoptosis can be induced by intravenous administration of a monoclonal antibody against complement receptor type three (Reid et al., 1993). In Alzheimer‘s disease, DNA fragmentation has been detected in microglia in addition to oligodendrocytes and neurons (Lassmann et al., 1995). In HIV encephalitis, single-stranded DNA breaks have recently been shown to occur in microglia/perivascular cells (Petit0 and Roberts, 1995). Programmed cell death of microglia as indicated by the presence of single-stranded DNA breaks appears to be under the control of several factors. In vivo and in vitro, programmed cell death of microglia may either result from a signal which directly induces microglial cell death and/or from the downregulation of expression of maintenance or survival factors (Schwartz and Osborne, 1993; Martin et al., 1994; McConkey et al., 1990). The results of the present study, provide evidence in support of the latter hypothesis. Withdrawal of microglial mitogens, i.e.

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colony-stimulating factors, enhances programmed cell death in microglia in vitro. Colony-stimulating factors indeed prevent programmed cell death of human monocytes (Brach et al., 1992; Mangan and Wahl, 1991). In addition, interleukins, particularly IL-3 and IL-4, have been shown to reduce apoptosis of bone marrow-derived mononuclear phagocytes (Baffy et al., 1993; Lotem and Sachs, 1992; Rodriguez-Tarduchy et al., 1992). The data of this study therefore point to autocrine and paracrine control of microglial turnover in vivo. Colony-stimulating factors are potent mitogens for microglia in vitro and in vivo (Giulian and Ingeman, 1988; Suzumura et al., 1990). Following peripheral nerve injury, activated, proliferating microglia newly express receptors for M-CSF and GM-CSF but this expression is again downregulated by day seven post-lesion (Raivich et al., 19911, i.e. at a time when nuclear DNA fragmentation begins to occur in microglia in vivo (Gehrmann and Banati, 1995). Downregulation of colony-stimulating factor expression during CNS injury may thus provide a stimulus for inducing programmed cell death in activated microglia. This assumption is supported by the observation that rates of microglial programmed cell death are enhanced after withdrawal of M-CSF or of GM-CSF. These effects are fairly specific since other cytokines, e.g. TGFP 1, do not influence to a great extent rates of microglial/brain macrophage cell death. Conclusions drawn from these in vitro studies, however, can only be extrapolated with caution to the in vivo situation. Firstly, changes in CSF receptors and CSF ligands must not be necessarily linked with each other, and thus may have different effects on the net outcome of for example microglial apoptosis. Secondly, microglial/brain macrophage cultures established from neonatal rat brain reflect the in vivo situation only to a very limited extent (Frei et al., 1987; Giulian and Baker, 1986). Importantly, cultured microglia/brain macrophages show an activated phenotype which does not match the downregulated properties of resting microglia in vivo (Graeber et al., 1988, 1989). In this respect, cell death of microglia/brain macrophages appears to be regulated by growth factors similar to what has previously been shown for resident macrophages in peripheral organs (Munn et al., 1995). In summary, the data of the present study suggest that withdrawal of microglial survival factors, i.e. colonystimulating factors, is involved in switching activated microglial from a proliferative state into a state of de-activation whereby they undergo cell death by DNA fragmentation. Which functional implications may a cytokine control of microglial proliferation/cell death have in CNS diseases? In gliomas, cytokine-controlled induction of microglial cell death may be one mechanism how brain neoplasms escape immune surveillance by activated microglia (Morioka et al., 1992). In infectious diseases of the CNS, cytokines released by infected cells may control steady state of host microglia similar to what has been shown for other resident mononuclear phagocytes (Zych-

linsky et al., 1992). Since microglial activation is part of such an intrinsic CNS immune defence system (Graeber and Streit, 1990), information on how these processes are regulated in vivo may contribute to our understanding how immune and repair processes can be influenced in the injured CNS.

Acknowledgements

I thank

Wey and Hannes Nef for photographi-

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