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Short communication

Time dependency of the action of nitric oxide in lipopolysaccharide–

interferon-

g

-induced neuronal cell death in murine primary neuron–

glia co-cultures

*

G.-H. Jeohn, W.-G. Kim, J.-S. Hong

Neuropharmacology Section, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences,

National Institutes of Health, P.O. Box 12233, Research Triangle Park, NC 27709, USA Accepted 18 July 2000

Abstract

We investigated the time-dependency of the action of nitric oxide (NO) on glia-mediated neuronal cell death. Cortical neuron–glia co-cultures were treated with lipopolysaccharide and interferong(LPS / IFNg). The production of NO was first detectable 9 h after the exposure to LPS / IFNgand increased for up to 48 h. A significant neuronal cell death was observed 36–48 h after treatment with LPS / IFNg. The NO generated at the initial stage of NO synthesis (about 12 h) following exposure to LPS / IFNgwas found to be critical for LPS / IFNg-induced neurotoxicity. Furthermore, the rate of NO production at the initial stage of NO synthesis was correlated linearly

2

with the extent of neuronal cell death. These findings suggest that the maximal rate of NO synthesis, instead of the accumulated NO₂ level, is a sensitive index for predicting endotoxin-induced cytotoxicity.  2000 Elsevier Science B.V. All rights reserved.

Theme: Endocrine and autonomic regulation

Topic: Neural–immune interactions

Keywords: Nitric oxide; Lipopolysaccharide; Neurons; Glia; Neurotoxicity; Primary cell culture; Cytokines

Nitric oxide (NO) has been implicated in immune-

The LPS-induced increase in the expression of inducible

mediated neurotoxicity in neuron–glia cultures [3,4,8,23]

NOS (iNOS) / NO is mediated by a series of signaling

and with various inflammation-related diseases in the CNS

events: LPS binding to a CD14-like receptor on glia [13],

[9,21,25,29]. When glial cells are exposed to endotoxins,

the stimulation of a Toll-like receptor 2 [28], and the

such as the bacterial endotoxin lipopolysaccharide (LPS)

activation of protein kinase C (PKC), protein tyrosine

and the HIV-1 coat protein gp120, they produce different

kinase C (PTK), extracellular signal-related kinase (ERK),

inflammatory modulators, including NO, tumor necrosis

and p38 kinase [2,6,10,11,18,19,27]. LPS also induces

factor

a

(TNF

a

), interleukin-1

a

(IL-1

a

), and IL-1

b

proinflammatory cytokines such as TNF

a

, IL-1

a

and

IL-[7,12,19,26]. Potentiated neurotoxicity is observed in

1

b

[5,20,22]. The LPS-induced pro-inflammatory

cyto-neuron–glia cultures when interferon-

g

(IFN

g

) is com-

kines, in combination, can re-stimulate the glia in an

bined with LPS [4,8,15]. The neurotoxicity induced after

autocrine fashion to further increase the level of NO

the treatment with LPS / IFN

g

or the combined cytokines

production [15,24], which may exacerbate the

inflamma-TNF

a

/ IL-1

a

/ IFN

g

can be suppressed by the NO synthase

tory response.

G

(NOS) inhibitor N -nitro-

L

-arginine methyl ester (

L

-

Despite the previous reports about the role of iNOS

NAME) [3,4,15,17], indicating that NO contributes to the

expression / NO production in glia- or immune-mediated

neuronal cell death induced by endotoxins.

neurotoxicity, the kinetic mechanism underlying the

neuro-toxic action of NO is unclear. In the present study, we

investigated the relationship between the rate of NO

*Corresponding author. Tel.: 11-919-541-2358; fax: 1

1-919-541-production and LPS / IFN

g

-induced neuronal cell death in

0841.

E-mail address: hong3@niehs.nih.gov (J.-S. Hong).

murine primary neuron–glia cultures. We report here that

0006-8993 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved.

the neurotoxic action of LPS was well-correlated with the

well at 9 h and 15 h after exposure to LPS / IFN

g

. Nitrite

rate of NO production during a critical period in LPS /

accumulation was measured as described above. The NO

2

IFN

g

-treated neuron–glia co-cultures.

production rate

D

[NO ] /

2

D

t was measured by calculating

2 2

The chemicals used for the cultures and treatments have

h

[NO ]

2 at 15 h

2

[NO ]

2 at 9 h

j

/(15–9 h). The neuron–glia

been described in detail [17]. Murine cortical neuron–glia

cultures were exposed to LPS / IFN

g

for 48 h. Cells were

co-cultures were made by adding E16-17 cortical neuronal

fixed and then stained with the MAP2 antibody (neuronal

cells on top of a cultured primary mixed glial layer as

cell marker). The number of MAP2-positive cells per

described previously [16,17]. The neuron–glia cultures

square millimeter was determined. The relationship

be-2

were treated in Minimum Essential Medium (MEM)

tween the calculated NO production rate

D

[NO ] /

2

D

t and

containing 2% heat-inactivated and dialyzed fetal bovine

the number of surviving neurons was analyzed for the

serum, 1 mM Na-pyruvate, 2 mM

L

-glutamine, 15 mM

treatment with

L

-NAME.

KCl, 50 U / ml penicillin, 50

m

g / ml streptomycin, and 50

The combined treatment of the murine primary neuron–

m

g / ml gentamicin for 48 h as follows: LPS (1

m

g / ml),

glia co-cultures with LPS (1

m

g / ml) and IFN

g

(5 U / ml)

IFN

g

(5 U / ml), and / or

L

-NAME (0.05–2.0 mM). Neurons

for 48 h resulted in more than a 90% decrease of

MAP2-were stained with an anti-microtubule-associated protein 2

positive cells (Fig. 1). The LPS / IFN

g

-induced neuronal

(MAP2) serum as described previously [17].

cell death was significantly suppressed by 1 mM

L

-NAME,

The production of NO was assessed as the accumulation

a NOS inhibitor (Fig. 1), indicating that NO was involved

of nitrite in the culture supernatants using a colorimetric

in the LPS / IFN

g

-induced neurotoxicity. A significant

reaction with the Griess reagent [14,17]. In order to study

neuronal cell death was observed 36–48 h after treatment

the kinetics of NO production following exposure to LPS /

with LPS / IFN

g

, which was significantly suppressed by

IFN

g

,

L

-NAME was added at various concentrations (0,

L

-NAME. However, a shorter period of exposure to LPS /

0.05, 0.1, 0.25, 0.5, 0.75, 1.0, or 2.0 mM). Fifty

m

l of

IFN

g

did not reduce the number of MAP2-positive cells

culture supernatant were collected twice from the same

(Fig. 2A). After the treatment with LPS / IFN

g

, the initial

or 12 h after the LPS / IFN

g

exposure, a significant

neuroprotection was still observed. However, when

L

-NAME was added after 20 h following the LPS / IFN

g

treatment, this inhibitor did not reverse the LPS / IFN

g

-induced neurotoxicity (Fig. 3A). These results suggest that

the production of NO within the first 12 h after the

LPS / IFN

g

treatment is critical for the endotoxin-induced

neurotoxicity.

In a separate study, the relationship between the rates of

Fig. 2. The time-dependency of LPS / IFNg-induced neuronal cell death and NO production in neuron–glia co-cultures. (A) Neuronal cell death. After the culture was exposed to LPS / IFNgfor the specified time with or withoutL-NAME, the cells were immunostained with the MAP2 antibody and counted. * P,0.05 and ** P,0.0001 compared to the untreated (N54). (B) NO production. The neuron–glia cultures were treated with LPS / IFNgand the culture supernatants were collected at the specified time points and the level of nitrite accumulated was measured. The rate of

2

NO production (D[NO ] /2 Dt) was calculated from the accumulated levels

of nitrite between the two time points. The experiments were repeated four times with similar results and representative data are shown (N54).

Fig. 3. Correlation between the rate of NO production and neuronal cell

2

accumulation of nitrite (NO ), a stable metabolite of NO,

₂ death. (A) Time dependency of the protective effects of L-NAME on LPS / IFNg-induced neuronal cell death. The neuron–glia cultures were

was observed at 9–12 h after the exposure, and there was a

exposed to LPS (1 mg / ml) / IFNg (5 U / ml) for 48 h. In order to

linear increase for up to 48 h (Fig. 2B). The rates of NO

determine the critical period of NO production that correlates with

production were calculated by dividing the difference of

_{LPS / IFNg-induced neurotoxicity,L-NAME (1 mM) was administered 1 h}

2 2

NO

2

levels between the two time points (

D

[NO ] /

2

D

t).

before or added at the specified time points after the treatment with

2

The analysis indicated that the rate

D

[NO ] /

₂

D

t reached

LPS / IFNg. After the 48-h exposure to LPS / IFNg, the cells were fixed and neuronal cells were immunostaining for MAP2 and counted. * P,

the maximal level 12–15 h after the exposure to LPS /

0.0001 compared to untreated group; [P,0.05 and [[P,0.0001

IFN

g

followed by a linear decline in the rate of NO

compared to the group treated with LPS / IFNgfor 48 h. (B) Correlation

production (Fig. 2B).

_{of the rate of NO production with LPS / IFNg-induced neuronal cell death.}

In order to investigate the time-dependency of the action

Neuron–glia cultures were exposed to LPS (1mg / ml) / IFNg(5 U / ml). In

2

of NO in LPS / IFN

g

-induced neurotoxicity, the NOS

order to correlate the rate of NO synthesis (D[NO ] /2 Dt) and LPS / IFNg -induced neuronal cell death, various concentrations ofL-NAME (0, 0.05,

inhibitor

L

-NAME was added at each specified time point

0.1, 0.25, 0.5, 0.75, 1.0, or 2.0 mM) were administered 1 h before

to the LPS / IFN

g

-exposed cultures. Pretreatment of the

exposure to LPS / IFNg. The culture supernatants were collected twice

culture with

L

-NAME during exposure to LPS / IFN

g

from the same wells at 9 and 12 h after the exposure to LPS / IFNgand

2

significantly suppressed the neuronal cell death. It is

the value D[NO ] /2 Dt was analyzed. After the 48-h exposure to LPS /

NO production between 9 and 15 h after treatment and the

In summary, the present findings demonstrate that the

extent of LPS / IFN

g

-induced neurotoxicity was analyzed.

rate of NO production is linearly correlated with the extent

By pretreating the cultures with different concentrations of

of neuronal cell death induced by LPS / IFN

g

and the NO

the iNOS inhibitor

L

-NAME during the exposure to LPS /

generated within a critical period plays a major role in

2

IFN

g

, different rates of

D

[NO ] /

2

D

t between the two

LPS / IFN

g

-induced neurotoxicity. These findings also

sug-2

time-points of 9 and 15 h (

D

[NO ] /

2

D

t

9|15 h

) were

gest that the maximal rate of NO synthesis, instead of the

2

achieved and the number of surviving neurons was counted

accumulated NO

2

level, is a sensitive index for predicting

at 48 h after the treatments. The concentration-dependent

endotoxin-induced cytotoxicity.

rate of NO production showed a linear correlation with the

LPS / IFN

g

-induced neuronal cell death (Fig. 3B). The

results suggest that rate of NO production at its maximal

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