From the Departments of a Neurology (Child Neurology Division), e Pediatrics, f Microbiology and Immunology, h Pharmacology and Physiology, the University of Rochester Medical Center, Rochester, New York 14642, b ICOS Corporation, Seattle, Washington 98021, the c Department of Pediatrics, The American University of Beirut, Beirut, Lebanon, the d Department of Pharmacology, University of Connecticut, Farmington, Connecticut 06030, and the g Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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Human immunodeficiency virus type 1 (HIV-1)
infection of the central nervous system results in neuronal apoptosis.
Activated HIV-1-infected monocytes secrete high levels of the
proinflammatory cytokine tumor necrosis factor- (TNF-
) and the
phospholipid mediator platelet-activating factor (PAF). TNF-
and PAF are elevated in the central nervous system of patients with
HIV-1-associated dementia. We now demonstrate that conditioned media
from activated HIV-1-infected monocytes induces neuronal apoptosis,
which can be prevented by co-incubation with PAF acetylhydrolase, the
enzyme that catabolizes PAF in the central nervous system. Preceding apoptosis is a TNF-
-induced increase in neuronal ceramide levels. TNF-
-mediated neuronal apoptosis can also be blocked by
co-incubation with PAF acetylhydrolase, or a PAF receptor antagonist.
Blocking pathologic activation of PAF receptors may therefore be a
pivotal step in the treatment of HIV-1-associated dementia.
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INTRODUCTION |
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There is a consensus that the neuropathogenesis of
HIV-11-associated dementia
(HIV-D) is initiated by productively infected and antigenically
activated brain-resident macrophages and microglia. However, there is
not a good correlation between viral burden in the central nervous
system and neurologic disease, which is presumably secondary to
neuronal dysfunction and death. Recently, the extent of neurologic
dysfunction (i.e. dementia) has been correlated with the
total number of macrophages and activated microglia in the brain
parenchyma, rather than the number of HIV-1-infected macrophages and
microglia (1). Because direct infection of neurons with HIV-1 is
unlikely, these findings suggest that neuronal dysfunction and death
are mediated by soluble factors released by macrophages and
microglia. We and others have demonstrated that HIV-1-infected
monocytes, when activated by antigenic stimuli in vitro or
contact with neural cells in vivo, release high levels of
the proinflammatory cytokine tumor necrosis factor- (TNF-
) and
the phospholipid mediator platelet-activating factor (PAF) (2, 3).
TNF- markedly up-regulates HIV-1 production in HIV-1-infected
macrophages (4). PAF, in turn, appears to up-regulate TNF-
synthesis
in HIV-1-infected cells of monocytic lineage (5). Importantly, the
number of macrophages expressing mRNA for TNF-
is elevated in
the brains of patients with HIV-D, compared with patients with HIV-1
infection but without dementia (6). PAF is elevated in cerebrospinal
fluid of patients with HIV-1-associated dementia and immunosuppression
(7). We have previously demonstrated in both primary human neuronal
cultures and in a differentiated human neuronal cell line that TNF-
and PAF induce dose-dependent apoptosis (7-10). Taken
together, these studies suggest that reciprocal relationships between
TNF-
and PAF exist, and that TNF-
and PAF play a major role in
the pathogenesis of HIV-D. Nevertheless, the mechanisms for TNF-
-
and PAF-mediated neuronal apoptosis in HIV-D remain elusive. Because
little is known about how TNF-
and PAF interact in neuronal
cultures, we investigated cellular pathways pertinent to TNF-
- and
PAF-mediated signal transduction that results in neuronal
apoptosis.
One key element in this relationship may be the sphingomyelin ceramide,
a probable second messenger of TNF-. Activation of monocytic p55
receptors by TNF-
is thought to result in the
sphingomyelinase-mediated production of ceramide (11), which has itself
been shown to induce apoptosis (12). Consequently, we examined the
temporal interrelationship of TNF-
, ceramide, and PAF in inducing
apopotic neuronal cell death. Knowing that TNF-
induces
dose-dependent apoptosis in primary human neuronal cultures
and a differentiated human neuronal cell line (7-10), we first
investigated whether TNF-
induces time-dependent
apoptotic cell death in primary human neuronal cultures. Next, we
measured ceramide levels in a human neuronal cell line as affected by
varying lengths of TNF-
or PAF exposure. Having established both
time-dependent TNF-
-induced neurotoxicity and
time-dependent TNF-
-induced neuronal ceramide production, and because PAF receptor antagonism can down-regulate TNF-
-mediated induction of HIV-1 in a monocytic cell line (5), we
investigated whether a reciprocal relationship existed between TNF-
,
ceramide, and PAF in the brain, i.e. if PAF receptor
antagonism or catabolism of PAF would affect TNF-
- and
ceramide-mediated toxicity of primary human neuronal cultures. Finally,
to assess the relative contribution of PAF to HIV-1-induced
neurotoxicity, we used conditioned media from activated HIV-1-infected
macrophages, co-incubated with PAF-acetylhydrolase (PAF-AH), the
principle catobolic enzyme for PAF.
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EXPERIMENTAL PROCEDURES |
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Primary Neuron Cultures-- Primary human neurons were cultured from human fetal brain tissue obtained from second trimester elective therapeutic abortions in an ethical manner and in strict observation of the guidelines of the NIH and the University of Rochester. Neurons were obtained from the telencephalon with both cortical and ventricular surfaces of second trimester (13-16 weeks of gestation) human fetal brain tissue, explanted, and cultured as described previously (7). Under these conditions, cultures were comprised of 60-70% neurons, 20-30% astrocytes, ~10% macrophages and microglia, and no oligodendrocytes (7).
Monocyte Cultures and HIV-1 Infection--
Monocytes were
recovered from peripheral blood mononuclear cells of HIV- and hepatitis
B-seronegative donors after leukapheresis, purified (>98%) by
countercurrent centrifugal elutriation, and cultured as described
previously with macrophage-colony stimulating factor (2, 3). Under
these conditions, monocytes developed elongated processes and were
immunoreactive for CD68; consistent with differentiation into
macrophages cultures. 7-10 days after culturing with macrophage-colony
stimulating factor, macrophages were exposed to HIV-1ADA
(accession no. M60472) at a multiplicity of infection of 0.01 infectious virions/target cell as described previously (2, 3). The
viral inocula were free of mycoplasma contamination (Mycoplasma
Detection Kit III, Geneprobe, San Diego, CA). Under these conditions,
20-50% of the monocytes were infected 7 days after HIV-1 inoculation.
This was determined by immunofluorescent and in situ
hybridization techniques. All cultures were treated with fresh medium
every 2-3 days. Reverse transcriptase activity for HIV-1 was
determined in culture fluids as described previously (2, 3). Five to
seven days after HIV-1 infection and during the peak of reverse
transcriptase activity (1 × 107 cpm/ml), cultures of
HIV-1-infected and parallel cultures of uninfected monocytes were
stimulated with bacterial lipopolysaccharide (10 ng/ml, 30 min at
37 °C) or vehicle, then snap-frozen at 80 °C until use in
neurotoxicity assays. Lipopolysaccharide was used to "activate"
macrophages to secrete markedly increased levels of neurotoxins
including TNF-
and PAF (2, 3, 6). Conditioned medium has previously
been shown to cause a dose-dependent loss of target neurons
when applied on a volume:volume basis; a 1:10 dilution of activated
conditioned medium is sufficent to kill ~25% of the total population
of cultured neurons (2, 3).
Ceramide Assay--
Human neuroblastoma SK-N-MC cells were
obtained from the American Tissue Culture Collection, cultured, and
differentiated to a neuronal phenotype with retinoic acid as described
previously (8). SK-N-MC cells were chosen over primary neurons for
their increased homogeneity and availability in large quantities.
Primary human neuronal cultures contain at least 30% glia, which show a different pattern of ceramide production when exposed to TNF- or
PAF (data not shown). In contrast, only the neuronal phenotype of
SK-N-MC cells will undergo apoptosis after TNF-
exposure (8). For
analysis of ceramide levels, differentiated SK-N-MC cells were treated
for 2, 4, or 24 h with medium containing TNF-
(12 ng/ml)
(Genzyme, Cambridge, MA), cPAF, a non-hydrolyzable analog of PAF that
is approximately 1/10 as potent (250 ng/ml) (Biomol, Plymouth Meeting,
PA), C2 ceramide (10 µM) (Biomol, Plymouth Meeting, PA),
or vehicle, then harvested in trypsin. Total cellular lipids were
extracted via the Bligh and Dyer (14) method, then dried under nitrogen
and resuspended in 100 µl of chloroform; 20 µl were used for
phosphate measurement, and 80 µl for ceramide measurement via the
diacylglycerol kinase assay, both performed as described previously
(15). Ceramide mass was quantitated using external standards,
normalized to total phosphate, and was measured as picomoles/nmol of
phosphate.
PAF Antagonism and Catabolism--
Primary human neuronal
cultures were coincubated for 24 h with TNF- (10 ng/ml) and/or
the metabolically stable hetrazepine PAF receptor antagonist WEB 2086 (10 mM); C2 ceramide (10 mM) and/or WEB 2086 (10 mM); TNF-
(10 ng/ml) and/or PAF-AH (50 µg/ml); and
conditioned medium from activated HIV-1-infected macrophages (1:10,
vol/vol) and/or recombinant PAF-AH (50 mg/ml). Each condition was then
assayed for apoptosis as follows.
Cell Death Assays and Quantitation-- For Fig. 1 only, a previously described (13) double stain methodology was used, in which neuronal cultures were first stained for membrane permeability (i.e. necrosis) with trypan blue, then fixed in 4% paraformaldehyde and stained for fragmented nuclear DNA (i.e. apoptosis; TUNEL method) using an adaptation of the commercially available ApopTagTM kit (ONCOR, Gaithersburg, MD), modified to incorporate an alkaline phosphatase secondary antibody visualized by New Fuchsin (Dako, Carpinteria, CA). For all other figures, neuronal cultures on coverslips were fixed in 4% paraformaldehyde, and analyzed exclusively for DNA fragmentation via the TUNEL method, using the ApopTagTM kit and methodology. For morphometrical analyses of neuronal apoptosis, fixed neuronal coverslip cultures were examined in the following manner: digitized images of 15 microscopic fields, pooled from two identically treated coverslip cultures, were analyzed for number of apoptotic neuronal nuclei/total number of neurons (TUNEL positive/TUNEL positive and negative) per 66× field using computerized morphometry (MCID, Imaging Research, St. Catherines, Ontario, Canada). Data from each of the 15 microscopic fields are compiled and expressed as the mean percentage of apoptotic (TUNEL positive) neuronal nuclei per field ± standard error of the mean. Because gestational ages of neuronal cultures varied between 11 and 16.5 weeks at the time of explantation, TUNEL staining in control cultures varied between 2 and 18% of the total neuronal population. Tests of statistical significance between control and experimental treatments were determined by analysis of variance or paired t tests. Results were judged significant at p < 0.05.
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RESULTS |
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TNF--induced Time-dependent Neuronal
Apoptosis--
A 1 ng/ml fixed dose of TNF-
applied to primary
human neuronal cultures caused a time-dependent increase in
neuronal apoptosis over time periods between 30 min and 18 h
(Fig. 1).
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Ceramide Levels--
Ceramide levels increased after 2 h of
TNF- exposure and peaked by 4 h (Fig.
2). In contrast, cPAF treatment caused a
decrease in ceramide production at 4 h, and an even greater
decrease at 24 h. Treatment of primary human neuronal cells with
C2 ceramide, a cell-permeable analog of ceramide, mimicked
TNF-
-mediated neurotoxicity (Fig.
3).
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PAF Antagonism and Catabolism--
PAF receptor antagonism by WEB
2086 (16) was able to completely reverse TNF--mediated neurotoxicity
and by itself had no neurotoxicity (Fig. 3A). C2
ceramide-mediated neurotoxicity could also be completely reversed by
co-application of WEB 2086 (Fig. 3B). Catabolism of PAF by
recombinant human PAF-AH, the principle catabolic enzyme for PAF in the
central nervous system (17), similarly reduced TNF-
-mediated
neurotoxicity (Fig. 4A).
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PAF in HIV-1-induced Neurotoxicity--
Conditioned medium from
activated HIV-1-infected macrophages was used at 1:10 vol/vol dilution
(Figs. 4, B-F), which induced neuronal cell death in nearly
25% of the total population of neurons. Co-incubation with PAF-AH was
able to reduce neuronal cell death to nearly control levels. PAF-AH by
itself was not neurotoxic. These results demonstrate that, although
conditioned medium from activated HIV-1-infected macrophages contains a
number of candidate neurotoxins, including TNF-, PAF, leukotrienes,
and lipoxins (2, 3), the majority of the 25% neurotoxicity
demonstrated here is likely due to PAF receptor activation.
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DISCUSSION |
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The results described here imply a novel relationship between the
proinflammatory cytokine TNF- and PAF receptor activation that
results in neuronal cell death. Neuronal dysfunction and death in HIV-D
is thought to be due to the presence of HIV-1 gene products, increased
amounts of proinflammatory cytokines, TNF-
, PAF, and other products
of arachidonic acid metabolism (18). Here we establish a
TNF-
-induced neurotoxicity that increases proportionally with length
of exposure. Our data further suggest that this TNF-
-induced
neurotoxicity occurs via production of the second messenger ceramide
and requires activation of PAF receptors, since ceramide levels were
elevated in response to TNF-
exposure (Fig. 2), and PAF receptor
antagonism abrogated both TNF-
- and ceramide-mediated neurotoxicity
to a similar degree (Fig. 3). PAF catabolism also reduced
TNF-
-mediated neurotoxicity (Fig. 4A). A comparison of
the data in Figs. 1 and 2, showing maximal ceramide levels present
before the majority of apoptosis has occurred, lends further
support to the theory that TNF-
-induced ceramide production may
serve as an initiator of apoptotic neuronal cell death. Furthermore,
the findings that overexpression of the anti-apoptosis gene crmA
inhibits ceramide formation in response to TNF-
(19), and
protects cells from the cytotoxic action of TNF-
(8, 19), but not
from ceramide-induced cytotoxicity (19), provide strong additional
support for the role of ceramide as a mediator of TNF-
-induced neuronal apoptosis. Further, if elevated ceramide levels do indeed lead
to neurotoxic activation of PAF receptors, our finding that direct PAF
receptor activation decreased neuronal ceramide levels prior to cell
death (Fig. 2) suggests a potential compensatory neuroprotective
mechanism against PAF receptor-mediated cell death.
The data presented here also presents the question of whether a glial
cell intermediary is involved in TNF--mediated neurotoxicity. The
answer to this question remains uncertain, but may well be species-dependent, because several important
species-specific differences exist in the biological actions of TNF-
in the central nervous system. Several reports have emphasized the
neuroprotective role of TNF-
in vitro and in
vivo in the murine central nervous system in acute brain injury
such as stroke or head trauma that may involve excitotoxicity resulting
in necrotic neuronal death (20). However, in human primary neurons or
neuronal cells, exposure to TNF-
results in apoptosis in most, but
not all experimental systems (8-10, 21). Our confirmation here of
TNF-
-induced neuronal apoptosis in vitro is consonant
with findings of TUNEL-stained neuronal nuclei with chromatin
condensation adjacent to focal inflammatory infiltrates of activated
microglia and reactive astrocytes present in postmortem brain tissue
from patients with HIV-D (22-24). TNF-
may initiate a sequence of
events resulting in neuronal damage and death in a chronic setting of
low level inflammation, a concept that is strengthened by the
observation that levels of TNF-
mRNA in brain tissue of patients
with HIV-D correlate with dementia (6).
Additional studies may provide insight into how PAF receptor activation
may lead to downstream neuronal death. Previous experiments from this
laboratory have established that NMDA receptor channel antagonists
including MK 801 and memantine substantially ameliorate PAF-induced
neurotoxicity in both human and rat culture systems (7). It is unknown
whether PAF can directly activate NMDA receptors with subsequent
excitotoxic damage. One study has shown that PAF can increase
intracellular Ca2+ in a population of rodent hippocampal
neurons with NMDA receptors (25). PAF can also lead to excitatory
neurotransmission through increased glutamate release (26-28).
TNF-, at levels present in conditioned medium from activated
HIV-1-infected macrophages, decreases the Vmax
by 30% for high affinity uptake in human, but not rat astrocytes (2,
3, 29). It is also possible that during conditions that promote chronic
inflammation in the brain, including HIV-1 encephalitis, PAF can
activate microglia to release arachidonic acid in a
Ca2+-dependent manner, and arachidonic acid, in
turn, can down-regulate high affinity glial glutamate uptake (30-32).
Although the mechanisms are different, both TNF-
and PAF disrupt
homeostasis for glutamatergic transmission, and thus may ultimately
increase ambient glutamate to levels sufficient to cause NMDA
receptor-mediated apoptosis in vulnerable neurons (33). Finally, we
have previously demonstrated that levels of PAF in cerebrospinal fluid
correlate well with neurologic dysfunction (i.e. dementia in
adults and progressive encephalopathy in children) and
immunosuppression (7). In the experiments reported here, we extend
these findings to show that PAF acetylhydrolase, the catabolic enzyme
for PAF in the central nervous system, can almost completely prevent
the neurotoxicity induced by exposure to neurotoxins from activated
HIV-1-infected macrophages. This suggests that PAF may be the principle
initiator of neuronal dysfunction and death in the clinical setting of
HIV-D, and further, that PAF-AH, or PAF receptor antagonists, may
ameliorate or prevent the neuronal damage associated with HIV-D.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants RO1 MH56838 (to H. A. G.) and PO1 MH57556 (to S. D., L. G. E., and H. A. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
i To whom correspondence should be addressed: Harris A. Gelbard, M.D., Ph.D., University of Rochester Medical Center, Box 631601 Elmwood Ave., Rochester, NY 14642. Tel.: 716-275-4784; Fax: 716-275-3683; E-mail: hgelbard{at}mail.neurology.rochester.edu.
1
The abbreviations used are: HIV-1, human
immunodeficiency virus type 1; HIV-D, HIV-1-associated dementia;
TNF-, tumor necrosis factor-
; PAF, platelet-activating factor;
AH, acetylhydrolase; NMDA,
N-methyl-D-aspartate.
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REFERENCES |
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