An endogenous 55 kDa TNF receptor mediates cell death in a neural cell line

MOLECULAR BRAIN RESEARCH ELSEVIER

Molecular Brain Research 38 (1996) 222-232

Research report

An endogenous 55 kDa TNF receptor mediates cell death in a neural cell line Kimberly J. Sipe

a

Dalin Srisawasdi a Robert Dantzer c Keith W. Kelley b James A. Weyhenmeyer a,*

a Department of Cell and Structural Biology, University of Illinois, 506 Morrill Hall, 505 South Goodwin Avenue, Urbana, IL 61801, USA b Laboratory oflmmunophysiology, Department of Animal Sciences, University of Illinois, 207 Plant and Animal Biotechnology Laboratory, 1201 West Gregory Drive, Urbana, IL 61801, USA c INRA-INSERM, Unit£ de Recherches de Neurobiologie des Comportements, U767, rue Camille Saint-Saens, 33077 Bordeaux, France Accepted 31 October 1995

Abstract

Tumor necrosis factor-a (TNF) is associated with developmental and injury-related events in the central nervous system (CNS). In the present study, we have examined the role of TNF on neurons using the clonal murine neuroblastoma line, N1E-115 (N1E). N1E cells represent a well-defined model for studying neuronal development since they can be maintained as either undifferentiated, mitotically active neuroblasts or as differentiated, mature neurons. Northern and reverse transcription-polymerase chain reaction (RT-PCR) analyses revealed that both undifferentiated and differentiated N1Es express transcripts for the 55 kDa TNF receptor (TNFR), but not the 75 kDa TNFR. The biological activity of the expressed TNF receptor was demonstrated by a dose dependent cytotoxicity to either recombinant murine or human TNF when the cells were incubated with the transcriptional inhibitor actinomycin D. The lack of the 75 kDa receptor mRNA expression and the dose dependent response to rHuTNF, an agonist specific for the marine 55 kDa receptor, suggest that the TNF induced cytotoxicity is mediated through the 55 kDa receptor in both the undifferentiated and differentiated N1Es. Light microscopic observations, flow cytometric analysis of hypodiploid DNA, and electrophoretic analysis of nucleosomal DNA fragmentation of N1Es treated with actinomycin D and TNF revealed features characteristic of both necrotic and apoptotic cell death. These findings demonstrate that blast and mature N1E cells express the 55 kDa TNF receptor which is responsible for inducing both necrotic and apoptotic death in these cells. The observation that actinomycin D renders N1E cells susceptible to the cytotoxic effects of TNF indicates that a sensitization step, such as removal of an endogenous protective factor or viral-mediated inhibition of transcription, may be necessary for TNF cytotoxicity in neurons.

1. Introduction

Tumor necrosis factor-a (TNF) is a pleiotropic, pro-inflammatory cytokine capable of initiating a wide array of effects. TNF has been reported to increase proliferation of thymocytes and CT-6 cytotoxic T cells [38], change adhesion molecule expression in neuroblastoma cells [6,33], increase transforming growth factor-a and epidermal growth factor messenger RNA [22], and depending on the target cell, TNF can induce apoptotic or necrotic cell death [25]. Interestingly, evidence suggests the 55 kDa receptor also signals the production of manganese superoxide dismutase (MnSOD), which protects against the cytotoxic effects of TNF, and calbindin, which protects neurons from the effects of glucose deprivation and glutamate toxicity [38,9]. TNF initiates its effects through two transmembrane receptors. The murine receptors have been cloned and have apparent molecular weights of 55 kDa and 75 kDa [16]. Both TNF receptors belong to the family of cell surface proteins including the low affinity nerve growth factor receptor (p75LNCFR), Fas antigen/APO-1, CD40 and OX40. Members of this family characteristically contain extracellular cysteine repeats and participate in events associated with cell death [5]. While TNF induced cytotoxicity through the 75 kDa TNFR has been reported [19], this receptor does not contain

* Corresponding author. Fax: (1) (217) 244-1648; E-mail: [email protected] 0169-328X/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 01 6 9 - 3 2 8 X ( 9 5 ) 0 0 3 1 0 - X

K.J. Sipe et a l . / Molecular Brain Research 38 (1996) 222-232

223

a cell death domain homologous to that identified in the 55 kDa TNFR [36]. Lewis et al. [26] have shown that while the murine 55 kDa receptor will bind both MuTNF and HuTNF with the same affinity, the murine 75 kDa receptor preferentially binds MuTNF and has a higher affinity for TNF than the 55 kDa receptor. In addition, mice lacking the 75 kDa TNFR were less susceptible to tissue necrosis and survived low TNF concentrations better than wild type controls [12]. Consistent with the studies described above, Tartaglia and Goeddel [37] proposed a model in which the 75 kDa receptor presents TNF to the 55 kDa receptor at low TNF concentrations, making the 75 kDa receptor responsible for increased TNF sensitivity. Findings in whole animal systems and primary cultures suggest both normal and pathological roles for TNF in nervous system development and injury. Spatially and temporally regulated TNF expression have been observed in the developing chick and mouse [8,14], and in the nervous system of animals subjected to a surgical injury paradigm [39]. Schwartz et al. [35] have also shown that TNF promotes axonal extension from severed optic nerve and may therefore be involved in synaptic repair. In rat brain, substantial TNF binding has been identified in the brainstem, cortex, cerebellum, thalamus and basal ganglia [23]. TNF also acts synergistically with interleukin-1 (IL-1) to mediate 'sickness behavior', which includes the fever, increased slow-wave sleep and anorexia that accompany systemic inflammatory responses [7]. In primary cultures, TNF acts through the 55 kDa receptor to increase the frequency of miniature synaptic currents in hippocampal neurons [17]. TNF also rescues cultured neurons from glucose deprivation and glutamate induced death by increasing calbindin expression, thereby promotling calcium homeostasis [9]. Cheng et al. suggested that the effect of TNF may be attributable to action through the 55 kDa TNFR based on immunocytochemical evidence. However, their interpretation is somewhat problematic since an antibody to the human 55 kDa TNFR was used to localize the mouse protein, and there was indication of staining for the 75 kDa TNFR. Despite the evidence presented above, it remains unclear whether neurons respond to TNF directly or indirectly through neighboring cells that can subsequently influence the neuron [20]. This is particularly questionable since many of the studies done to date have used whole animal systems and primary cultures that contain non-neural cells, such as astrocytes and microglia, that might contribute to the effects of TNF on neurons. The effects of TNF on neuroblastoma cells vary. Ponzoni et al. [31] demonstrated that a combination of TNF and interferon-3/ (IFN-T) resulted in the differentiation of cultured human LAN-5 and GI-LI-N neuroblastoma cells. On the other hand Goillot et al. [15] found that TNF and insulin increased proliferation in the human SKNFI and SKNFB cells, which express both the 55 kDa and 75 kDa TNFR, as determined by immunocytochemistry. N1E-115 (N1E) murine neuroblastoma cells, originally cloned from the spontaneous mouse C-1300 neuroblastoma using the alternate animal-culture passages technique [2,3], have been used to study both neurotransmitter and channel properties. Being clonally derived, N1E cells are a pure source of neuronal cells. Importantly, N1Es differentiate into mature neurons with highly branched processes, electrical activity, and transmitter receptors, when the culture medium is switched from high serum to low serum with dimethyl sulfoxide (DMSO). As such, the N1Es are suitable for studying the differential effects of TNF on developing and mature neurons in the absence of astrocytes or microglia and provide a model system to determine the effects attributable to direct action of TNF on neurons. In the present study, we, provide evidence for the expression of functional TNF receptors on both undifferentiated and differentiated neuronal cells. While transcripts for the 55 kDa receptor were identified by Northern analysis, the 75 kDa receptor transcript could not be demonstrated by either Northern analysis or RT-PCR. In the presence of the transcriptional inhibitor actinomycin D, treatment with recombinant murine TNF (rMuTNF) resulted in a dose dependent cytotoxicity in both undifferentiated and differentiated N1Es. TNF cytotoxicity was also observed in cells treated with recombinant human TNF (rHuTNF), which preferentially binds to the 55 kDa murine receptor. DNA laddering and flow cytometry studies revealed that TNF increased the level of cytotoxicity in the presence of actinomycin D. These data demonstrate that the TNF induced cell death in undifferentiated and differentiated neurons is mediated through the 55 kDa receptor. As such, N1E cells represent a suitable model for further examining the role of the 55 kDa TNFR in neurons, and for transfecting with a 75 kDa TNFR construct to investigate potential interaction between the 55 kDa and 75 kDa TNFR's.

2. Materials and methods Cell culture reagents and medium were prepared using endotoxin free water. Fetal bovine serum (FBS) was heat-treated at 56°C for 30 min to inactivate complement. N1E-115 cells (N1Es) were generously provided by Dr. E. Richelson, Mayo Clinic, Jacksonville, FL. Undifferentiated N1Es were cultured at 37°C in a humidified chamber with 5 - 7 % CO 2 in Dulbecco's modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Passages 5 through 20 were used for experimental analyses. The medium was changed daily, beginning 48 h after passage. Cells were harvested, triturated and passaged ew;ry 3 - 4 days, as the cells reached approximately 80% confluency. For differentiation, the cells were passaged to 96-well plates or 75 cm 2 flasks coated with poly-L-lysine and allowed to grow under the conditions described above. At 50-60% confluency, the medium was switched to DMEM supplemented with 0.5% FBS, 1.5%

224

K.J. Sipe et al. / Molecular Brain Research 38 (1996) 222-232

dimethyl sulfoxide (DMSO) and antibiotics. The medium was changed after 48 h, and the cells were harvested and used for experimentation at 96 h. The WEHI 164 (clone 13) (WEHI) fibroblast cell line was obtained from Dr. Terje Espevik, Genentech, Inc., South San Francisco, CA, and the PU5-1R (PU5) macrophage cell line from ATCC (Rockville, MD). WEHI and PU5 cells were grown in RPMI supplemented with penicillin, streptomycin and 5% FBS. Cells were harvested by either mild trypsinization or scraping. Total cellular RNA was isolated using Tri-Reagent TM - RNA/DNA/protein isolation reagent (Molecular Research) based on the protocol described by Chomczynski [10]. Each sample (20 /zg) was electrophoresed on a 1% denaturing agarose/formaldehyde gel. The fractionated RNA was transferred to nylon membrane (Nytran +, Schleicher and Schuell) by capillary transfer in 20 × SSC. The membranes were hybridized with labeled DNA probes containing the complete coding sequence for either the 55 kDa or 75 kDa TNF receptor (provided by Dr. John Sims, Immunex, Seattle, WA). Probes were labeled with [32P]dCTP using the Random Primed DNA labeling kit (USB/Amersham). Membranes were hybridized in 5 ml Rapid-Hyb hybridization buffer (Amersham) at 65°C for greater than 2 h. Hybridization was performed using a minimum of 1.5 X 107 cpm. After probing for one receptor, membranes were stripped at 65°C in 55% formamide, 2 X SSPE and 0.1% SDS. Membranes were then reprobed to identify transcript for the other receptor and the housekeeping gene, glyceraldehyde phosphate dehydrogenase (GAPDH) (cDNA provided by Dr. Patrick Delafontaine, Emory University School of Medicine, Atlanta, GA). GAPDH transcript served as an internal standard for normalizing the amount of RNA loaded on the gel. For RT-PCR analysis, total RNA was again isolated from cells using Tri-Reagent TM - RNA/DNA/protein isolation reagent. Per protocol recommendations, additional centrifugation steps were included for complete removal of DNA. The reverse transcription reaction was performed at 42°C using 2 /xg total RNA, 0.5 U / m l avian myeloblastosis virus reverse transcriptase (USB/Amersham), 4 /xM random hexamer primers (Pharmacia), and 2 mM dNTP mix (Pharmacia). Polymerase chain reactions were prepared in a volume of 50 /xl with 10 /~1 of the reverse transcription reaction, 1 nM 3' primer, 1 nM 5' primer, 750/zM MgC12, and 0.025 U / m l Taq polymerase (Gibco-BRL). Reaction buffer and MgC12 were provided with the Taq polymerase. TNFR primers were based on the sequences published by Goodwin et al. [16]. GAPDH primers were designed based on the sequence published by Fort et al. [13]. Primers were: TNF-R, 75 kDa

GAPDH

(5') 5'-GAGTGTGTGCTTGCGAAGCT-3' (3') 5'-CGATGTAAGGATGCTTGGAG-3' Expected product length: 383 bp (5') 5'-GGAAGCTTGTCATCAATGG-3' (3') 5'-AGATCTCGTGGTTCACACC-3' Expected product length: 225 bp

Reactions were performed using an MJ Research thermocycler. The cDNA was initially denatured for 2 min at 94°C followed by 30 cycles of the following sequence: 30 s at 94°C, 1.5 min at 55°C, and 1.5 min at 72°C. Final extension occurred for 10 min at 72°C. Product size was determined by electrophoresing the cDNA on a 1.5% agarose gel and staining with ethidium bromide. The identity of PCR generated fragments was verified by Southern blot analysis. The cDNA was denatured in the gel and immobilized on nylon membrane (Nytran ÷) by capillary transfer in 20 X SSC and ultraviolet crosslinking. A full length cDNA probe for the 75 kDa TNF receptor was labeled with [32p]dCTP using the Random Primed DNA labeling kit (USB/Amersham). Hybridization was performed in 5 ml Rapid-Hyb hybridization buffer at 65°C for greater than 2 h using a minimum of 1.5 X 10 7 cpm. The membrane was washed twice at room temperature in 2 X SSC, 0.1% SDS, 10 min/wash; at room temperature in 0.2 X SSC, 0.1% SDS for 10 min; twice at 65°C in 0.2 X SSC, 0.1% SDS, 5 min/wash; and at 65°C in 0.1 X SSC, 0.1% SDS for 15 min, and the resulting blot was exposed to film. TNF cytotoxicity was determined using a modification of the method described by Mosmann [29]. Cells were plated in 96 well plates at densities of 2 X 10 4 cells/well (WEHI 164) or 1 X 10 4 cells/well (N1E) in 100 /~1 of medium. For differentiated N1Es, cells were plated on poly-D-lysine coated wells. Cells were allowed to adhere for 12 h before the medium was removed and replaced with 50 /xl fresh medium containing 0.33 /xg/ml actinomycin D and 50/~1 of medium containing TNF (rHuTNF, 8.33 X 108 U / m l (Genzyme); rMuTNF, 1.6 X 10 9 U / m l (Genzyme)) at various concentrations. Additional experiments were carded out using 2.5 /zg/ml of the translational inhibitor, cycloheximide, instead of actinomycin D. Triton treated cells (0.01% Triton X-100) served as a 'maximum cell death' control. Triplicate wells were run for each treatment concentration. For differentiated N1Es, treatments were added in DMEM containing 0.5% FBS and antibiotics. Cells were incubated for 16-22 h, and 70 /~1 of medium was removed and replaced with 50 /zl of the organic dye, MTT (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide) at a concentration of 1 m g / m l in 1 x PBS. The cells were incubated for 3 h, MTT was carefully aspirated and dye crystals were dissolved in 150 /zl acidified isopropanol

K.J. Sipe et al. / Molecular Brain Research 38 (1996) 222-232

225

(isopropanol in 0.04 N HCI, mixed 2:1 with water). The absorbance was read on ELISA plate reader using a 540 nm filter. The cytotoxicity index was calculated as follows: (absorbance of medium control wells - absorbance of treatment wells) (absorbance of medium control wells - Triton treated wells) Electrophoretic analysis of DNA fragmentation was carded out using a modification of the technique described by Perandones et al. [30]. Undifferentiated N1E cells were plated in six well plates at 4 X 105 cells/well in 2 ml growth medium (DMEM suppleme.nted with 10% FBS and antibiotics). After 12-18 h, cells were either differentiated or treated. For experiments using differentiated cells, the cells were maintained in differentiating medium for 4 days, and treatments were added at the end of the fourth day of differentiation. Treatments included growth medium, TNF (100 U/ml), actinomycin D (0.17/zg/rnl) or a combination of TNF and actinomycin D. At 0, 6, 12 and 24 h after treatment, cells were harvested, triturated, pelleted, and resuspended in 1.0 ml medium. Aliquots (50 /xl) were counted using trypan blue exclusion to distinguish viable and non-viable cells. The remaining cells were pelleted, resuspended in 400 /xl hypotonic lysis buffer (0.2% Triton X-100, 10 mM TRIS, 1 mM EDTA, pH 8) and centrifuged at 13800 X g for 15 min. The supernatant was removed iramediately, 4 0 / z l of 5 M NaC1 and 440/zl isopropyl alcohol were added to the supernatant and the samples were stored ow~rnight at - 20°C to precipitate the low molecular weight DNA. The samples were centrifuged at 13 800 X g for 15 rain, allowed to dry at room temperature, and resuspended in 200/xl TE containing 5 0 / z g / m l RNase A. After heating for 10 min :at 75°C and incubating for an additional 45-60 min at room temperature, the samples were subjected to phenol/chloroform extraction. DNA was precipitated from the aqueous phase at - 2 0 ° C using isopropyl alcohol and NaC1, as previously described. The samples were centrifuged at 13 800 X g for 15 min and the pellets resuspended in 12 /zl TE (pH 7.4) and 3 /xl Ficoll loading dye with bromophenol blue and xylene cyanol. The samples were then incubated at 37°C for 20 min and electrophoresed on a 1% agarose gel containing 0 . 3 7 / z g / m l ethidium bromide. Staining for flow cytometric analysis was performed according to the propidium iodide (PI)/Hoechst 33342 (HO342) exclusion staining protocol described by Darzynkiewicz et al. [11]. Propidium iodide solution was prepared by dissolving 2 mg PI in 100 ml Ca 2÷, Mg 2+ free PBS. Hoechst 33342 stock solution was prepared at 0.3 mg/ml, and diluted 1:4 in PBS to obtain a working solution. Staining solutions were stored at 4°C. Undifferentiated N1Es were plated and treated as described for DNA laddering studies. At 0, 3, 6, 12 and 24 h after treatment, the cells were harvested, triturated, centrifuged at 1000 X g, and resuspended in 1 ml medium. An aliquot (50 /~1) of each cell suspension was counted using trypan blue exclusion. The remaining material was centrifuged again at 1000 X g, the medium was decanted and the pellet vortexed. After the addition of 100/xl PI, the cells were vortexed and stored at 4°C for 30 min. Fixative (1.9 ml 25% ethanol in PBS) and 50/xl HO342 working solution were added, with vortexing after each addition, and the samples were stored at 4°C for a minimum of 12 h before flow cytometric analysis. Samples were stored for up to 3 days at 4°C. The analysis was performed using a Coulter EPICS 752 flow cytometer. Ultraviolet multi-line excitation of samples was accomplished with a Coherent 90-5 argon laser set at 351 and 364 nm. A 560 nm short-pass dichroic filter was used to measure Hoechst emission while a 590 nm long-pass filter was used to measure PI emission. Parameters for G2, G 1 / M and S cell populations were determined separately for each experiment and were assigned according to the Hoechst emission pattern of untreated control cells. Necrotic cells and cells late in apoptosis were distinguished from viable and early apoptotic cells by increased membrane permeability as indicated by higher propidium iodide staining. Similarly, apoptotic cells could be separated from viable cells based on the lower Hoechst staining, due to hypodiploid DNA content, exhibited by apoptotic cells.

3. Results Fig. 1 represents a Northern blot that was successively probed for the 55 kDa and 75 kDa TNF receptor transcripts. Murine PU5-1R (PU5) macrophages and WEHI 164 (WEHI) clone 13 fibroblasts were included in the analysis as positive controls. While undifferentiated and differentiated N1Es expressed only 55 kDa receptor mRNA (Fig. 1A), transcripts for both the 55 kDa and 75 kDa receptors were observed by Northern analysis of PU5 and WEHI RNA (Fig. 1A,B). The blot was also probed for the GAPDH housekeeping gene to verify equal RNA loading among the lanes (Fig. 1C). In agreement with the Northern results, RT-PCR amplification of RNA from undifferentiated and differentiated N1Es, using primers specific for tile 75 kDa murine TNF receptor, did not yield a product (Fig. 2A, lanes 2 and 3). Amplification of PU5 and WEHI RNA resulted in a single cDNA product of expected length (383 bp) that was visible on an ethidium bromide stained agarose gel (Fig. 2A, lanes 4 and 5). Products migrating faster than the 123 bp standard represent amplified primer dimers (Fig. 2A, lanes 1-5). The identity of the cDNA product was verified by Southern analysis (Fig. 2B, lanes 4 and 5). No product was detected when yeast tRNA was reverse transcribed and amplified as a negative control. Amplification using primers specific for GAPDH produced a single product in all cells tested (Fig. 2A, lanes 6-10).

226

K.J. Sipe et al. / Molecular Brain Research 38 (1996) 222-232

A

B ~z

'~

C ~

z

~z

b,.

L,,

2

1

1as

Fig. 1. Northern analyses of total RNA isolated from undifferentiated and differentiated N1E-115 neuroblastoma cells, PU5-1R macrophages and WEHI 164 (clone 13) fibroblasts reveal expression of mRNA transcripts for the 55 kD TNFR in each cell line. Lanes were loaded with 2 0 / x g total RNA. Bands were visualized using phosphorimaging. The blot was probed, in succession, with full length cDNAs for: (A) the 55 kD TNF receptor, (B) the 75kD TNF receptor, and (C) the rat GAPDH housekeeping gene.

The typical morphology of undifferentiated and differentiated N1Es is shown in Fig. 3A,B. Undifferentiated N1Es had a rounded appearance with occasional short processes, while differentiated N1Es had a fusiform appearance with extensive processes and branching. Undifferentiated N1Es treated for 15 h with 100 U / m l rMuTNF and 0.17 /zg/ml of the transcriptional inhibitor, actinomycin D, had morphological features consistent with both necrotic and apoptotic cell death (Fig. 3F). A majority of the N1Es were non-adherent, while many had cytosolic components condensed to one side and ballooned membranes. Trypan blue was excluded from a majority of these cells, consistent with the maintenance of plasma membrane integrity into late stage apoptosis. Other cells appeared flattened and enlarged with increased vacuolation,

A L

B 12345678910

123

45

Fig. 2. RT-PCR analysis for 75 kD TNFR (product length = 383 bp) (lanes 1-5) and the housekeeping gene, GAPDH (product length = 225 bp) (lanes 6-10) indicates a lack of 75 kD TNFR mRNA in N1E cells. The ladder (L) is a 123 bp standard. A: RNA was reverse transcribed from yeast tRNA (lanes 1, 6), differentiated N1E (lanes 2, 7), undifferentiated N1E ~lanes 3, 8), PU5 (lanes 4, 9) and WEHI (lanes 5,10). B: Autoradiogram of a Southern analysis for amplified 75 kD TNFR message. The portion of the gel containing 75 kD TNFR specific fragments was transferred to membrane and hybridized with full length 75 kD TNFR cDNA. Lanes 1-5 in A correspond to lanes 1-5 in B.

K.J. Sipe et a l . / Molecular Brain Research 38 (1996) 222-232

227

Fig. 3. Photomicrographs of N1Es (200 × ; Hoffman optics) show increased cell death in the presence of actinomycin D and TNF: (A) Undifferentiated NIEs in growth medium, DMEM supplemented with 10% FBS. (B) Differentiated N1Es in differentiating medium, DMEM supplemented with low serum (0.5%) and DMSO, for 4 days. Effects of TNF and actinomycin D treatment on morphology of undifferentiated N1E's: (C) control cells; (D) N1Es treated with actinomycin D (0.17/xg/ml) for 15 h; (E) N1Es treated with rMuTNF (100 U/ml) for 15 h; (F) N1Es treated with actinomycin D (0.17/~g/ml) and rMuTNF (100 U/ml) for 15 h. Scale bar = 35 /xm.

suggesting necrotic cell death (not shown). Cells treated with actinomycin D alone also appeared both necrotic and apoptotic (Fig. 3D); however, a greater proportion of the N1Es revealed a ballooned morphology when treated with both actinomycin D and rMuTNl? as compared to actinomycin D alone. Both controls and cells treated with r M u T N F alone were typically rounded and adherent with few granules, although necrotic and apoptotic cells were occasionally observed (Fig. 3C,E). Undifferentiated and differentiated N1Es responded in a dose dependent manner to r M u T N F and rHuTNF in the presence of 0 . 1 7 / x g / m l actinomycin D (Fig. 4A,C). At 250 U / m l , r M u T N F produced similar cytotoxic responses in undifferentiated and differentiated N1Es ( 6 6 _ 12% and 54 + 12%, respectively) by one way A N O V A analysis. Recombinant H u T N F (400 U / m l ) was cytotoxic to 25 _+ 8% and 7 _+ 7% of undifferentiated and differentiated N1Es, respectively. In the presence of actinomycin D, the dose dependent response for undifferentiated N1Es to r M u T N F and rHuTNF ( P = 0.0001) and the r M u T N F and rHuTNF responses in the differentiated N1Es ( P = 0.0001 and P = 0.025, respectively) were statistically

K.J. Sipe et a l . / Molecular Brain Research 38 (1996) 222-232

228

A

B 1-

x

0.8 0.6

--

0.4

"i

0,20

"1-

J

-0.2 -0.4

0

i

0.80.6"

"o -~ 0.40.2x 0-

3"

~>, -0.2-

I

I

-0.4

I

I

o

I

I..

o

[rMuTNF]

[rMuTNF]

C 1 0.8x

0.6- ~

.~

0.4-

"~' 0.2"6 O

•

Undifferentiated

o

Differentiated

0-0.2 -0.4

,

o

i

i

i

I

~o

i

8

[d-luTNF] Fig. 4. Cytotoxicity assays measure dose dependent rMuTNF and rHuTNF cell death in undifferentiated and differentiated N1Es: (A) rMuTNF in the presence of actinomycin D (n = 8 for undifferentiated and differentiated conditions); (B) rMuTNF in the absence of actinomycin D (n = 3 for undifferentiated condition, n = 4 for differentiated condition); (C) rHuTNF in the presence of actinomycin D (n = 5 for undifferentiated condition and n = 6 for differentiated condition).

significant. In the absence of actinomycin D, undifferentiated and differentiated N1Es did not respond to rMuTNF in a dose dependent manner ( P > 0.8; Fig. 4b). WEHIs were run in parallel with the N1Es as a positive control. The translational inhibitor, cycloheximide, also increased N1E sensitivity to TNF cytotoxicity in undifferentiated cells (data not shown). Treatment with 1000 U / m l TNF and 2.5 /zg/ml cycloheximide resulted in 50% cytotoxicity in undifferentiated N1Es, versus approximately 10% cytotoxicity in the presence of cycloheximide alone. Cell viability was determined by incubating with trypan blue and counting with a hemacytometer (Fig. 5). Two way ANOVA analysis of viability indicated a statistically significant interaction between treatment and time. Post-hoe analysis indicated statistically significant differences between all actinomycin D treated conditions and control or TNF alone conditions (P---0.0001). In addition, the treatment with actinomycin D and 250 U / m l TNF was found to be significantly different from the conditions with actinomycin D alone or actinomycin D and 100 U / m l TNF ( P = 0.003 and 0.01, respectively). 100

75-

[]

Control

•

1COU/mlTNF

•

250U/mlTNF

•

ActD

[]

ActD+100U/mlTNF

•

Act D + 250U/mlTNF

50-

25-

0-

Time

Fig. 5. Viability of undifferentiated N1Es after treatment, as determined by trypan blue analysis. Cells were plated at 4X 105 cells per well in a six well plate and allowed to adhere a minimum of 12 h prior to treatment additions. Treatments were added by replacing the medium with fresh medium containing treatments. Cells were harvested and counted at 6, 12 and 24 h after treatment. Graph depicts mean + SEM (n = 4).

K.J. Sipe et al. / Molecular Brain Research 38 (1996) 222-232

229

A

B

t~

40

[]

Control

•

100U/mlTNF

•

250U/mlTNF

•

ActD

[]

ActD+100U/mlTNF

•

ActD+250U/mlTNF

i m

O

T

30

i m

O O

20 ~TT 10

6

12

24

Time Fig. 6. A: Electrophoretic analysis of DNA showing nucleosome fragmentation, indicating apoptotic cell death in undifferentiated NIE cells. Treatment conditions included actinomycin D, rMuTNF (100 U/ml) or a combination of actinomycin D and rMuTNF. Laddering is most evident in lanes representing actinomycin D or actinomycin D and rMuTNF treated cells. B: Dual-label flow cytometric analysis of DNA and plasma membrane integrity in undifferentiated N1Es detects an increase in apoptotic cell death across time and treatment. Cells were stained with propidium iodide prior to ethanol fixation and the addition of Hoechst 334. Graph depicts the percentage (mean _+ SEM, n = 4) of apoptotic cells across time and treatment.

T h e results o f D N A laddering analysis are s h o w n in Fig. 6. Undifferentiated N 1 E s first displayed laddering 12 h after treatment with either a c t i n o m y c i n D alone or in c o m b i n a t i o n with T N F . L a d d e r i n g was still present after 24 h o f treatment. S o m e laddering was also apparent in 12 and 24 h control and T N F treated cells. S u b s e q u e n t f l o w c y t o m e t r i c analysis was p e r f o r m e d to quantitate necrotic and apoptotic cell death f o l l o w i n g the various treatments. D N A laddering was apparent for all the differentiated N 1 E treatment conditions, including samples collected before treatments w e r e added (data not shown). This observation agrees with an earlier report o f apoptosis in N 1 E s differentiated by serum starvation [24].

230

K.J. Sipe et al. / Molecular Brain Research 38 (1996) 222-232

Results of flow cytometric analysis are presented in Fig. 6B. At 12 h, N1Es treated with actinomycin D exhibited more apoptosis than either controls or cells treated with TNF alone. Increased apoptosis was more apparent at 24 h. Two way ANOVA analysis indicated a statistically significant interaction between treatment and time. Post hoc analysis revealed that the levels of apoptosis in control cells and those treated with 100 U / m l TNF and 250 U / m l TNF did not differ significantly from each other, but were significantly different from cells that were treated with actinomycin D. In addition to the difference between actinomycin D treated and untreated cells, cells treated with both 250 U / m l TNF and actinomycin D exhibited a statistically significant increase in apoptosis relative to cells treated with actinomycin D alone ( P = 0.04). The degree of apoptosis measured in N1Es treated with both 100 U / m l TNF and actinomycin D is between that observed in cells treated with actinomycin D alone and in cells treated with a combination of actinomycin D and 250 U / m l TNF. While the difference is not statistically significant, it suggests that TNF is acting in a dose dependent manner to increase apoptosis in N1Es.

4. Discussion In the present study, we have shown that undifferentiated and differentiated N1E cells exclusively express the 55 kDa TNF receptor transcript. We have also demonstrated that this receptor is coupled to the dose dependent cytotoxic effects of acute rMuTNF treatment, in the presence of the transcriptional inhibitor actinomycin D, in both undifferentiated and differentiated N1Es. In addition, the translational inhibitor, cycloheximide, increased TNF sensitivity in undifferentiated N1Es. The fact that actinomycin D is required for TNF induced cell death in N1Es indicates the possibility that a protective factor or cell death inhibitor must be removed for N1E sensitization to TNF cytotoxicity. Our light microscopic findings suggest that N1Es treated with actinomycin D alone or in combination with TNF become both necrotic and apoptotic, the latter exhibiting a condensation of intracellular components and membrane blebbing. Although cells with a similar appearance were seen in control cultures, the numbers were significantly less. Our DNA laddering and flow cytometric data provide additional support for the cytotoxic effect of actinomycin D and TNF. Laddering was seen in all differentiated N1E samples, regardless of type or length of treatment. This agrees with an earlier study by Kruman et al. [24] indicating that differentiation of N1Es by serum starvation results in DNA laddering. In addition, we utilized a flow cytometric technique which is based on differences in cellular DNA content and membrane permeability to quantitate the apoptotic cell population. Analysis of control and treated N1Es indicated that actinomycin D alone is capable of increasing the apoptotic population above control levels, and that the number of apoptotic cells is further increased in a dose dependent manner by TNF. While flow cytometry provides an approximation of the size of the apoptotic population, there are significant limitations to the interpretation of these findings. Cells dying in G 2 / M phase of the cell cycle will not necessarily lose enough DNA to be identified as hypodiploid, i.e. apoptotic. In addition, plasma membrane permeability increases late in apoptosis, making it difficult to distinguish late apoptotic cells from necrotic cells. As both of these caveats would lead to a more conservative estimate of the number of apoptotic cells in a sample, the ability of TNF to affect apoptosis in N1E cells may be more pronounced than indicated by our results. Based on the observations that the transcriptional inhibitor, actinomycin D, is required for TNF mediated apoptosis in N1E cells, it could be argued that actinomycin D increases N1E susceptibility to TNF cytotoxicity by inhibiting the production of one or more protective factors. While we cannot exclude the possibility that cell replacement may account for the undifferentiated N1Es' lack of sensitivity to TNF in the absence of actinomycin D, the difference in TNF sensitivity between post-proliferative, differentiated cells in the presence or absence of actinomycin D suggests that differentiated N1Es may be producing one or more protective factors. In addition, undifferentiated cells do not exhibit DNA laddering until more than 6 h after treatment with TNF and actinomycin D, which may represent the period of time required to degrade existing protective factors. The relative susceptibility of cells to TNF cytotoxicity, as determined by the level of protective factor production or differentiative state, may play a significant role in TNF's involvement in nervous system pathology and development. For example, some viruses are capable of inhibiting cellular RNA synthesis [21]. This represents a situation analogous to actinomycin D treatment in the N1E system and could explain how virally infected neurons might become more sensitive to the cytotoxic effects of TNF. Lower transcription levels would lead to the production of fewer protective factors making the neurons more susceptible to cytotoxicity. In a non-neuronal model, melanoma cells infected with Newcastle Disease virus (NDV) are sensitized to TNF cytotoxicity [28]. It should be noted that NDV is also cytotoxic to some neuroblastoma cells in vivo [27]. The increased TNF cytotoxicity in the presence of NDV is believed to be mediated by increased IFN-c~ levels and may be attributable to the ability of IFN-a to disrupt cell cycle mechanisms and activate enzymes which destroy single-stranded RNA. Vesanen et al. [40] have shown that TNF activates the expression of incorporated human immunodeficiency virus (HIV) in neuroblastomas, suggesting a role for inflammatory cytokines in the progression of AIDS associated CNS pathology, i.e. AIDS dementia. It has also been reported that circulating levels of IFN-a are increased in AIDS patients [18] possibly creating a situation in which neurons could be sensitized to the cytotoxic effects of TNF.

K.J. Sipe et al. / Molecular Brain Research 38 (1996) 222-232

231

It is also possible thai: differentiative state could determine susceptibility to TNF cytotoxicity. Studies from the hematopoietic system indicate TNF has a differential effect on lymphocytes depending on the stage of differentiation [34,38]. Similarly, the temporal expression of TNF in the developing CNS [8,14] could differentially affect subsets of neurons based on their stage of maturation. Developmentally, a neuron with a lowered capacity to produce protective factors (e.g., superoxide dismutases, calbindin, peroxidases, Bcl-2, baculovirus p35 protein), such as one deprived of trophic factor support, could be killed by TNF, while a neuron with an increased level of a protective factor could survive [1,4,32]. This scenario would be analogous to the situation in B-cell development, where Bcl-2 expression apparently spares B-cells from death during receptor re~rangement, and is consistent with the suggestion that viral infection may sensitize neurons to cytotoxic agents by limiting the production of protective factors at the level of transcription a n d / o r translation [28,27,21]. In summary, we have shown that undifferentiated and differentiated N1E neuronal cells exclusively express the 55 kDa TNFR transcript. In addition, agonist binding to this receptor, alone or in combination with the transcriptional inhibitor actinomycin D, results in neuronal cell death. Our results indicate that N1Es not previously exposed to TNF express only the 55 kDa receptor, and as such provide a model system for studying the relative contributions 75 kDa TNF receptors on the 55 kDa receptor expression a n d / o r function following transfection with the 75 kDa TNFR construct. The present findings support and extend earlier studies suggesting a role for TNF on neuronal differentiation [31] and proliferation [15].

Acknowledgements We would like to thank: S. Arkins, C. Minshall, D. Reese and A. Wonderlick for their assistance with the cytotoxicity and molecular studies, G. Durack and K. Magin in the UI Biotechnology Flow Cytometry Facility for their assistance in the flow cytometric analyses, and D. Essex-Sorlie for her assistance with the statistical analysis. This work was supported by NIH Grant DK49311 and NSF Grant IBN-9320158. K.J.S. was supported by NIH-CMB Training Grant GM07238.

References [1] Allsopp, T.E., Wyatt, S., Paterson, H.F. and Davies, A.M., The proto-oncogene bcl-2 can selectively rescue neurotrophic factor-dependent neurons from apoptosis, Cell, 73 (1993) 295-307. [2] Amano, T., Richelson, E. and Nirenberg, M., Neurotransmitter synthesis by neuroblastoma clones, Proc. Natl. Acad. Sci. USA, 69 (1972) 258-263. [3] Augusti-Tocco, G. and Sato, G., Establishment of functional clonal lines of neurons from mouse neuroblastoma, Proc. Natl. Acad. Sci. USA, 64 (1969) 311-315. [4] Batistou, A., Merry, D.E., Korsmeyer, S.J. and Greene, L.A., Bcl-2 affects survival but not neuronal differentiation of PC12 cells, J. Neurosci., 13 (1993) 4424-4428. [5] Beutler, B. and van Huffel, C., Unraveling function in the TNF ligand and receptor families, Science, 264 (1994) 667-668. [6] Birdsall, H.H., Land, C., Ramser, M. and Anderson, D.C., Induction of VCAM-1 and ICAM-1 on human neural cells and mechanisms of mononuclear leukocyte adherence, J. Immunol., 148 (1992) 2717-2731. [7] Bluth~, R.M., Pawlowski, Ivl., Suarez, S., Pamet, P., Pittman, Q., Kelley, K.W. and Dantzer, R., Synergy between tumor necrosis factor a and interleukin-1 in the induction of sickness behavior in mice, Psychoneuroendocrinology, 19 (1994) 197-207. [8] Bums, T.M., Clough, J.A., Klein, R.M., Wood, G.W. and Berman, N.E.J., Developmental regulation of cytokine expression in the mouse brain, Growth Factors, 9 (1993) 253-258. [9] Cheng, B., Christakos, S. and Mattson, M.P., Tumor necrosis factors protect neurons against metabolic-excitotoxic insults and promote maintenance of calcium homeostasis, Neuron, 12 (1994) 139-153. [10] Chomczynski, P., A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples, BioTechniques, 15 (1993) 532-537. [11] Darzynkiewicz, Z., Li, X., Gong, J., Hara, S. and Traganos, F., Analysis of cell death by flow cytometry. In: G. Studzinski (Ed.), Cell Growth and Division, A Practical Approach, Oxford University Press, Oxford, 1995. [12] Erickson, S.L., de Sauvage, F.J., Kikly, K., Carver-Moore, K., Pitts-Meek, S., Gillett, N., Sheehan, K.C.F., Schreiber, R.D., Goeddel, D.V. and Moore, M.W., Decreased sensitivity to tumour-necrosis factor but normal T-cell development in TNF receptor-2-deficient mice, Nature, 372 (1994) 560-563. [13] Fort, P., Marry, L., Piechaczyk, M., Sabrouty, S.E., Dani, C., Jeanteur, P., Blanchard, J.M., Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate-dehydrogenase multigenic family, Nucleic Acids Res., 13 (1985) 1431-1442. [14] Gendron, R.L., Nestel, F.P., Lapp, W.S. and Baines, M.G., Expression of tumor necrosis factor alpha in the developing nervous system, lnt. J. Neurosci., 60 (1991) 129-136. [15] Goillot, E., Combaret, V., Ladenstein, R., Baubet, D., Blay, J., Philip, T. and Favrot, M.C., Tumor necrosis factor as an autocrine growth factor for neuroblastoma, Cancer Res., 52 (1992) 3194-3200. [16] Goodwin, R.G., Anderson, D., Jerzy, R., Davis, T., Brannan, C.L., Copeland, N.G., Jenkins, N.A. and Smith, C.A., Molecular cloning and expression of the type 1 and type 2 murine receptors for tumor necrosis factor, Mol. Cell. Biol., 11 (1991) 3020-3026. [17] Grassi, F., Mileo, A.M., Monaco, L., Punturieri, A., Santoni, A. and Eusebi, F., TNF-a increases the frequency of spontaneous miniature synaptic currents in cultured rat hippocampal neurons, Brain Res., 659 (1994) 226-230.

232

K.J. Sipe et al./ Molecular Brain Research 38 (1996) 222-232

[18] Grunfeld, C., Kotler, D., Shigenaga, J.K., Doerller, W., Tierney, A., Wang, J., Pierson, R.N. and Feingold, K.R., Circulating interferon-a levels and hypertriglyceridemia in the acquired immunodeficiency syndrome, Am. J. Med., 90 (1991) 154-162. [19] Heller, R.A., Song, K., Fan, N. and Chang, D.J., The p70 tumor necrosis factor receptor mediates cytotoxicity, Cell, 70 (1992) 47-56. [20] Hopkins, S.J. and Rothwell, N.J., Cytokines and the nervous system I: expression and recognition, Trends Neurosci., 18 (1995) 83-88. [21] Huang, A.S., Wagner, R.R., Inhibition of cellular RNA synthesis by non-replicating vesicular stomatitis virus, Proc. Natl. Acad. Sci. USA, 54 (1965) 1579-1584. [22] Kalthoff, H., Roeder, C., Brockhaus, M., Thiele, H. and Schmiegel, W., Tumor necrosis factor (TNF) up-regulates the expression of p75 but not p55 TNF receptors, and both receptors mediate, independently of each other, up-regulation of transforming growth factor a and epidermal growth factor receptor mRNA, J. Biol. Chem., 268 (1993) 2762-2766. [23] Kinouchi, K., Brown, G., Pasternak, G. and Donner, R., Identification and characterization of the receptors for tumor necrosis factor-a in the brain, Biochem. Biophys. Res. Commun., 181 (1992) 1532-1538. [24] Kruman, I., M.A., Gordon, R.Y., Popov, V.I. and Umansky, S.R., Differentiation and apoptosis of murine neuroblastoma cells N1E-115, Biochem. Biophys. Res. Commun., 191 (1993) 1309-1318. [25] Laster, S.M., Wood, J.G. and Gooding, L.R., Tumor necrosis factor can induce both apoptotic and necrotic forms of cell lysis, J. Irnmunol., 141 (1988) 2629-2634. [26] Lewis, M., Tartaglia, L.A., Lee, A., Bennett, G.L., Rice, G.C., Wong, G.C.H., Chen, E.Y. and Goeddel, D.V., Cloning and expression of cDNAs for two distinct murine tumor necrosis factor receptors demonstrate one receptor is species specific, Proc. Natl. Acad. Sci. USA, 88 (1991) 2830-2834. [27] Lorence, R.M., Reichard, K.W., Katubig, B.B., Reyes, H.M., Phuangsab, A., Mitchell, B.R., Cascino, CJ., Walter, R.J. and Peeple, M.E., Complete regression of human neuroblastoma xenografts in athymic mice after local newcastle disease virus therapy, J. Natl. Cancer Inst., 86 (1994) 1228-1233. [28] Lorence, R.M., Rood, P.A. and Kelley, K.W., Newcastle disease virus as an antineoplastic agent: induction of tumor necrosis factor-a and augmentation of its cytotoxicity, J. Natl. Cancer Inst., 80 (1988) 1305-1312. [29] Mosmann, T., Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. lmmunol. Methods., 65 (1983) 55-63. [30] Perandones, C.E., Illera, V.A., Peckham, D., Stuntz, L.L. and Ashman, R.F., Regulation of apoptosis in vitro in mature murine spleen T cells, Z Immunol., 151 (1993)3521-3429. [31] Ponzoni, M., Casalaro, A., Lanciotti, M., Montaldo, P.G. and Cornaglia-Ferraris, P., The combination of y-interferon and tumor necrosis factor causes a rapid and extensive differentiation of human neuroblastoma cells, Cancer Res., 52 (1992) 931-939. [32] Rabizadeh, S., LaCount, D.J., Friesen, P.D. and Bredesen, D.E., Expression of the baculovirus p35 gene inhibits mammalian neural cell death, J. Neurochem., 61 (1993) 2318-2321. [33] Rozzo, C., Ratti, P., Ponzoni, M. and Comaglia-Ferraris, P., Modulation of og1 ill, 0/2 /~1, anc 0/3 ~1 integrin heterodimers during human neuroblastoma cell differentiation, FEBS Lett., 332 (1993) 263-267. [34] Rusten, L.S., Jacobsen, F.W., Lesslauer, W., Loetscher, H., Smeland, E.B. and Jacobsen, S.E.W., Bifunctional effects of tumor necrosis factor 0/ (TNF0/) on growth of mature and primitive human hematopoietic progenitor cells: involvement of p55 and p75 TNF receptors, Blood, 83 (1994) 3152-3159. [35] Schwartz, M., Solomon, A., Lavie, V., Ben-Bassat, S., Belkin, M. and Cohen, A., Tumor necrosis factor facilitates regeneration of injured central nervous system axons, Brain Res., 545 (1991) 334-338. [36] Tartaglia, L.A., Ayres, T.M., Wong, G.H.W. and Goeddel, D.V., A novel domain within the 55 kDa TNF receptor signals cell death, Cell, 74 (1993) 845-853. [37] Tartaglia, L.A. and Goeddel, D.V., Two TNF receptors, lmmunol. Today, 13 (1992) 151-153. [38] Tartaglia, L.A., Weber, R., Figari, I.S., Reynolds, C., Palladino, M.A. Jr. and Goeddel, D.V., The two different receptors for tumor necrosis factor mediate distinct cellular responses, Proc. Natl. Acad. Sci. USA, 88 (1991) 9292-9296. [39] Tchelingerian, J., Quinonero, J., Booss, J. and Jacque, C., Localization of TNF0/ and IL-1 0/ immunoreactivities in striatal neurons after surgical injury to the hippocampus, Neuron, 10 (1993) 213-224. [40] Vesanen, M., Wessman, M., Salminen, M. and Vaheri, A., Activation of integrated human immunodeficiency virus type 1 in human neuroblastoma cells by the cytokines tumour necrosis factor alpha and interleukin-6, J. Gen. Virol., 73 (1992) 1753-1760.