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*
Eijkman-Winkler Institute, Section of Neuroimmunology, Utrecht University, Utrecht, The Netherlands;
Laboratoire Universitaire "Virus, neurone et immunité," Université Paris-Sud, Paris, France;
Laboratory of Neuropathology, Faculté de Médecine Paris-Ouest, Garches, France;
§
Department of Neurology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One of the neurotoxins that is suggested to be involved in neuronal damage is nitric oxide (NO)4 (9). The proinflammatory cytokine IL-1ß, which is released in brain tissue of demented AIDS patients (10, 11, 12), has been shown to induce NO by up-regulating inducible nitric oxide synthase (iNOS) (13, 14, 15). Indeed, cocultures of HIV-infected macrophages and astrocytes were shown to release NO when compared with cocultures of uninfected macrophages and astrocytes (16), suggesting that interactions between HIV-infected macrophages/microglia and astrocytes are critical in the induction of factors that lead to neuronal injury (17, 18). Furthermore, it has been shown that iNOS, as well as NO, was induced in primary cultures of rat mixed brain cells in response to stimulation with gp41 (19).
Recently, evidence has been presented that direct neurotoxic effects of NO are modest but are tremendously enhanced by reacting with superoxide anion to form peroxynitrite (20, 21, 22, 23, 24). Superoxide anion is reported to be produced by myeloid-monocytic cell lines upon HIV-1 infection (25), and, to keep the concentration of this reactive free radical low, superoxide dismutase (SOD), a superoxide anion scavenger, is produced (26). Cytosolic copper-zinc SOD (CuZnSOD) is responsible for degrading reactive superoxide anion by catalyzing the dismutation of superoxide anion into molecular oxygen and hydrogen peroxide, therefore playing an important role in the defense mechanisms against oxidative stress (26). However, NO reacts with superoxide anion at a near diffusion-limited rate, and, when present in large amounts, it therefore outcompetes SOD completely (21). The reaction product peroxynitrite is a potent oxidizer that is responsible for the nitration of tyrosine residues of structural proteins (21). Neurofilament, which is a protein that provides structural stability to neurons, is one of the target proteins of peroxynitrite, and nitration will result in disrupted neurofilament assembly and thus neuronal damage (21, 26). Interestingly, this reaction is catalyzed by SOD, the enzyme that normally scavenges an excess of superoxide anion and thereby prevents the formation of peroxynitrite (21, 26, 27). This might explain the paradoxical neurotoxicity found in transgenic mice overexpressing extracellular SOD activity (28, 29).
Since the combination of NO and superoxide anion results in the generation of highly neurotoxic peroxynitrite, we here investigate their role in AIDS dementia complex. Levels of iNOS and SOD, two of the key enzymes in oxidative stress that are indicative of the presence of NO and superoxide anion, respectively, were studied in brain tissue of demented and nondemented AIDS patients. In addition, we examined the localization of these two enzymes by double immunohistochemical staining on brain slices of demented AIDS patients. To confirm our in vivo results, we investigated whether HIV-1 infection of macrophages in vitro also resulted in changes in oxidative processes, by looking at superoxide anion production and SOD mRNA expression. Finally, immunohistochemical staining for nitrotyrosine, a footprint for peroxynitrite, was performed to investigate whether peroxynitrite was present in the brains of demented and nondemented AIDS patients.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tissue specimens of the frontal cortex was obtained from
autopsied brain of thirteen HIV-1-infected and five control cases
(those who died of causes not related to HIV-1 infection). All the
HIV-1-infected individuals had developed AIDS at the time of death and
showed decreased levels of CD4+ T cells (<300). Seven of
them developed cognitive and motor impairments. None of the patients
received any antiretroviral therapy. The clinical data on all
individual patients are shown in Table I
.
AIDS or HIV-associated dementia was a premortem clinical diagnosis made
by an AIDS specialty physician or neurologist. The severity of dementia
was scored on the Memorial Sloan-Kettering scale. All demented patients
scored at least 2 or higher. In addition, from all patients, the
neurological status was determined retrospectively. Clinical premortem
diagnoses were confirmed by postmortem obduction and by staining and
neuropathological examination of frozen sections of brain tissue. The
diagnosis HIV encephalitis was made when p24 positive multinucleated
giant cells were observed. The loss of brain tissue (atrophy) was
scored using the computed tomography (CT) scan. Furthermore, all
patients showed a wide variety of opportunistic infections (Table I)
and died because of various reasons. All patients were well
characterized at the time of death, and the cause of death was never
directly associated with diseases of the central nervous system. Viral
levels were measured by RT-PCR and showed a high degree of variation
among all AIDS patients (data not shown). There was no correlation
between the presence or absence of high viral levels and the stage of
disease. When there were no differences between brain tissue of the
studied patients and normal brain tissue, it was scored as "no
significant difference."
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. HIV-1 infection of macrophages
PBMC were isolated from heparinized blood from HIV-1-, HIV-2-,
and hepatitis B-seronegative donors and obtained on Ficoll-Hypaque
density gradients. Cells were washed twice, and monocytes were purified
by countercurrent centrifugal elutriation. Cells were >98% monocytes
by criteria of cell morphology on May-Grünwald-Giemsa-stained
cytosmears and by nonspecific esterase staining using
-naphtylacetate (Sigma, St. Louis, MO) as substrate. Monocytes were
cultured in suspension at a concentration of 2 x 106
cells/ml in Teflon flasks (Nalgene, Rochester, NY) in DMEM with 10%
heat-inactivated human AB serum negative for anti-HIV Abs, 10 mg/ml
gentamicin, and 10 mg/ml ciprofloxacin (Sigma). As previously
described, HIV-1 infection of nonadherent macrophages, especially when
using a low multiplicity of infection, appears much more reproducible
than infection of macrophages that were first allowed to adhere (30).
After 7 days, monocyte-derived macrophages (MDM) were recovered from
the Teflon flasks and infected with HIV-1Ba-L at a
multiplicity of infection of 0.01. Two hours later, macrophages were
removed from the Teflon flasks, washed twice to remove unbound virus,
and used for studies to determine levels of superoxide anion production
by chemiluminescence and SOD expression by RNA PCR in HIV-infected
macrophages.
Chemiluminescence
Cells were recovered from Teflon flasks and washed twice with HBSS containing 1% FCS (Life Technologies, Grand Island, NY). A total of 2 x 105 cells was exposed to an equal volume of HBSS in the presence of 250 nM bis-N-methylacridinum (Lucigenin, Sigma). Superoxide dismutase (SOD, Sigma) was added to HIV-infected macrophages to demonstrate that chemiluminescence was superoxide anion specific. Lucigenin reacts with superoxide anion (31), and this reaction is accompanied by photon emission. The number of photons emitted during stimulation of the macrophages was measured as light emission in a luminometer (Packard Instruments, Brussels, Belgium) and expressed as cpm, as described previously (32).
RNA PCR detection of cytokines and enzymes
Brain tissue and 2 x 106 macrophages were
homogenized and lysed, respectively, in 1 ml and 0.4 ml TRIzol (Life
Technologies) according to the manufacturer’s guidelines. In
experiments where the levels of SOD expression of macrophages were
determined, the lysed cells were stored in Trizol at -70°C. When
lysates of all time points were obtained, total RNA was isolated. Total
RNA was dissolved in diethylpyrocarbonate (DEPC)-treated water, and 1
µg of RNA was used for the synthesis of complementary DNA. The RNA
was previously heated for 5 min at 70°C, chilled on ice, and added to
a mixture containing 1x reverse transcriptase (RT) buffer (Promega,
Madison, WI), 200 U of reverse transcriptase, 0.1 M DTT (Life
Technologies), 2.5 mM dNTP (Boehringer Mannheim, Indianapolis, IN), 80
U random hexamer oligonucleotides (Boehringer Mannheim), and 10 U
RNAsin (Promega). The complete mixture was now incubated for 60 min at
37°C and then heated for 5 min at 90°C. The final reaction volume
was diluted 1:8 by adding distilled water. Amplification of the cDNA
was accomplished using one primer biotinylated on the 5' terminal
nucleotide to facilitate later capture using streptavidin. The PCR
primer pair was chosen to span at least one intron. To the PCR reaction
mixture the following components were added: 0.25 mM dNTP mix
(Boehringer Mannheim), 1 x PCR buffer (50 mM KCl, 10 mM Tris-HCl,
1.5 mM MgCl2; Promega), 0.2 µM sense and antisense
primers (Table II), 5 µl cDNA, and 1 U
Taq polymerase (Promega). Denaturation, annealing, and
elongation temperatures for PCR were 94°C, 60°C, and 72°C for 1,
1, and 2 min each, using a DNA thermal cycler (Perkin-Elmer, Norwalk,
CT). Negative controls were included in each assay to confirm that none
of the reagents were contaminated with cDNA or previous PCR products.
PCR was also performed on RNA samples to exclude genomic DNA
contamination. To confirm single band product, positive reactions were
subjected to 40 cycles amplification and electrophoresis, followed by
ethidium bromide staining. Then, for semiquantification, every primer
pair was tested at different cycle numbers to determine the linear
range. GAPDH, SOD, and IL-1ß mRNA levels were high, and 20 cycles was
enough to measure the PCR product in its linear range, whereas iNOS
cDNA had to be subjected to 30 cycles and IL-10 even to 40 cycles to be
in the linear range.
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Statistical analysis
Data were compared, and a two-tailed Student’s t test was used to determine p values.
Immunohistochemical analysis of brain tissue
Frozen sections of brain tissue were analyzed for SOD, iNOS, nitrotyrosine (NT), and HIV p24 Ag expression. Brain slices were first incubated for 18 h at 4°C with the first Ab (anti-SOD human liver, Calbiochem, San Diego, CA; anti-iNOS mac NOS, Transduction Laboratories, Lexington, KY; anti-HIV-1 p24, Dupont-NEN, Boston, MA; anti-NT polyclonal, Upstate, Biotechnology, Lake Placid, NY). Astrocytes and microglial cells were stained with anti-glial fibrillary acidic protein (GFAP; Amersham Life Science, Rainham, England) and anti-human phagocyte macrophage/microglia CD68/Ki-M7 (BMA, Valbiotech, France), respectively. The binding was subsequently revealed after another incubation of 45 min at 18°C with a corresponding alkaline phosphatase conjugated anti-IgG Ab and fast red substrate (Boehringer Mannheim).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A semiquantitative fluorescence assay was used to study the levels
of expression of IL-1ß, IL-10, iNOS, and SOD in brain tissue of the
frontal cortex of the individual patients described in Table I. The
mRNA levels of all gene products, expressed in RFU, are depicted in
Fig. 1, A-D. To detect all PCR
products in their linear range, cDNA was subjected to 30, 40, 20, and
20 cycles for iNOS, IL-10, SOD, and IL-1ß, respectively, indicating
that high levels of IL-1ß and SOD were present in all patients.
IL-1ß, a proinflammatory cytokine, was detected in the HIV-demented
group at significantly higher levels than in the group of nondemented
HIV patients (p < 0.025) and in the control
group (p < 0.025). This finding confirms that,
also in these demented patients, cerebral immune activation seems to
occur (10, 11, 12), which may eventually prove to be a crucial event in the
neuropathogenesis of HIV-1 infection (33, 34, 35, 36, 37).
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). With the exception of case 12, it was
found that the moderate to high levels of SOD in patients 7, 8, 9, and
11 (Fig. 2A) correspond to moderate and high levels of iNOS
(Fig. 2B). This trend is not observed in the nondemented
patients or in the control group. Brain specimens of most nondemented
patients even do not have detectable SOD levels. In the two cases
(cases 3 and 6) where SOD expression was detected, only in one (case 3)
iNOS was found. Thus, these data suggest that the presence of both SOD
and iNOS seems to be highly associated with the occurrence of clinical
dementia.
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The expression of SOD, iNOS, and HIV-1 p24 Ags was analyzed in
frozen sections of frontal lobes of five of the studied patients
(patients 1, 3, 7, 9, and 14). Expression of SOD and iNOS Ags was
detected in patients 3, 7, and 9, whereas they were barely detectable
in patient 1 and control patient 14, a result that paralleled that of
the RNA detection method. Since staining patterns between the different
patients did not differ substantially, only single stains of patient 9
for SOD and iNOS are shown in Fig. 3,
A and C, respectively. In general, SOD labeling
was more intense than that of iNOS, which was also observed by the RNA
detection method. SOD was found in cells of both perivascular and
parenchymal areas. To better define the SOD-positive cells, double
immunohistochemical staining was performed on cases 3 and 9. SOD
immunoreactivity patterns did not differ for both patients, except for
minor differences in staining intensity. Therefore, only the result of
the double staining of brain tissue of patient 9 is shown in Fig. 3B. SOD expression was mostly localized in CD68-positive
microglial cells in the parenchyma (Fig. 3B) and in the
perivascular areas where the frequency of SOD-positive cells was less
(data not shown). SOD staining was infrequent and faint in
GFAP-positive astrocytes (data not shown). To better define the
iNOS-positive cells, double labeling experiments were performed on the
same cases. By double labeling, we found that both CD68-positive
microglial cells (Fig. 3D) and GFAP-positive astrocytes
(data not shown) expressed iNOS Ags with roughly the same frequency and
intensity in patient 9, whereas iNOS reactivity was detectable but
faint in patient 3 (data not shown). Subsequently, double
immunohistochemical staining was performed on case 9 with SOD and HIV-1
p24-specific mAbs. More than 60% of the p24 Ag-positive cells also
contained SOD Ag (Fig. 3E). Since HIV-1 productively
replicates only in brain macrophages (3, 4, 5), these findings suggest
that SOD expression and possibly superoxide anion production occurred
mostly in HIV-1-infected brain macrophages.
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Since elevated SOD levels were detected in brain macrophages in
tissue obtained from demented AIDS patients, the ability of HIV-1 to
affect SOD expression in macrophages was investigated in vitro.
Therefore SOD expression in HIV-infected macrophages was compared with
replicate uninfected cultures by RT-PCR analysis. As shown in Fig. 4A, the expression of SOD mRNA
in uninfected macrophage cultures decreased in time, whereas the
HIV-infected macrophages continued to express SOD. Importantly, the
time course pattern of HIV mRNA levels was similar to that of SOD mRNA
levels (Fig. 4B). To confirm that HIV RNA expression indeed
led to HIV-1 production, the release of p24 Ag in the culture
supernatants was detected by ELISA (Fig. 4C).
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To investigate whether the relative increased levels of SOD
expression in HIV-infected macrophages could be a consequence of
increases in superoxide anion production, the production of superoxide
anion by HIV-1-infected macrophages was compared with that of
uninfected macrophages using chemiluminescence. Immediately after HIV-1
infection, the ratio of the amount of superoxide anion production
between HIV-infected and uninfected macrophages measured after 30 min
was 1.2. (Fig. 5A). Four days
after HIV-1 infection, the amount of superoxide anion production by
HIV-infected macrophages increased when compared with that of the
control macrophages (Fig. 5B). The 30-min ratio of the
amount of superoxide anion production between the virus-infected and
control cells was 1.8. Eight days after viral inoculation of the
macrophages, the amount of superoxide anion production by HIV-infected
macrophages was substantially increased, when compared with that of the
uninfected cells (Fig. 5C). The ratio of the amount of
superoxide anion production between HIV-infected and uninfected
macrophages measured after 30 min was 2.9. To demonstrate that the
chemiluminescence signal was indeed superoxide anion specific, we added
SOD, resulting in a dose-dependent decrease of the signal (Fig. 6). The chemiluminescence signal was
completely abolished when 100 µg/ml SOD was added (data not shown),
indicating that, in our experiments, lucigenin indeed reacts
specifically with superoxide anion.
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Since the reaction between NO and superoxide anion results in the
formation of peroxynitrite and both these molecules appear to be
present in the brains of demented AIDS patients, elevated levels of
peroxynitrite are to be expected. Therefore, brain sections of patients
4, 9, and 11 were stained for NT, which is observed when large amounts
of peroxynitrite were produced. Whereas the nondemented AIDS patient 4
did not show any substantial reactivity with the NT polyclonal, the
demented AIDS patient 11 showed strong staining for NT parenchymal as
well as perivascular (Fig. 7). In
addition, patient 9 stained heavily for NT (data not shown) although
less than patient 11. Interestingly, patient 9 showed lower expression
of iNOS and SOD than patient 11 (Fig. 2), suggesting a possible
relation between the degree of iNOS/SOD expression and the presence of
nitrosylated proteins. As a control for nonspecific staining, the
primary Ab was omitted. In addition, as a control for NT staining, the
primary Ab was preincubated with NT. Both control experiments resulted
in inhibition of staining (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, it is demonstrated that, although the ability of macrophages to produce superoxide anion in vitro decreases in time, elevated amounts of superoxide anion are produced upon in vitro HIV-1 infection of macrophages compared with control macrophages. Since macrophages and microglia function as a long-term reservoirs for HIV-1 (38), these cells apparently possess a mechanism to protect themselves against the toxic effects of superoxide anion. Indeed, we show here that elevated levels of superoxide anion coincide with elevated levels of cytosolic copper-zinc SOD, an important intracellular scavenger of superoxide anion, in HIV-infected macrophages compared with control macrophages. Since these in vitro data demonstrate that HIV infection of macrophages result in both increased superoxide anion production and in increased SOD expression, changes in SOD mRNA expression in vivo may also be indicative of changes in superoxide anion production. In vivo, SOD mRNA levels were found to be elevated in demented AIDS patients, and immunohistochemical analysis of brain tissue revealed that SOD is localized mostly in HIV-infected brain macrophages. These data suggest that superoxide anion production by HIV-infected macrophages may also be increased in vivo.
SOD is known to be involved in several other neurodegenerative diseases, like amyotrophic lateral sclerosis, Down’s syndrome, and Alzheimer’s disease (39, 40, 41, 42, 43). Although SOD can scavenge superoxide anion, this reaction will not take place in the presence of NO. When produced in large amounts, iNOS is the only molecule that can effectively out-compete SOD for superoxide anion by generating NO, a highly diffusible molecule that is able to react with superoxide anion to form peroxynitrite (20, 21, 22, 23, 24). Recently, iNOS has been shown to be involved in the pathogenesis of AIDS dementia complex (16, 19) and to be directly or indirectly responsible for neuronal damage (9, 44, 45). In addition to the elevated mRNA levels of SOD, we here also show that iNOS mRNA levels are significantly elevated in brain tissue of demented AIDS patients compared with nondemented AIDS patients. This suggests that, besides superoxide anion, levels of NO may be elevated as well in brains of demented AIDS patients.
In general, cellular interactions between astrocytes and
immune-activated macrophages/microglia are believed to be responsible
for the production of neurotoxic as well as neurotrophic factors
(46, 47, 48). Although we show that in vivo microglia are able to express
iNOS, the ability of human macrophages to produce NO remains highly
controversial. Despite the presence of iNOS in human macrophages, the
production of NO by these cells in vitro is presumably very low (49) or
even absent (13, 15). Indeed, we were also not able to demonstrate any
NO production or expression of iNOS by HIV-infected macrophages in
vitro (data not shown). However, recently it was demonstrated that
macrophages isolated from active multiple sclerosis lesions showed
immunoreactivity for iNOS and were able to produce NO (50). In
addition, it has been reported that IFN- can induce iNOS and NO in
human monocytes (51). This implicates that there might be a trigger
involved in vivo that is not present in vitro. NOS has been detected in
primary human astrocytes, and, for these cells, IL-1ß is the key
proinflammatory cytokine involved in the induction of this molecule
(13, 14). Interestingly, elevated levels of macrophage-derived IL-1ß
have been detected in brain tissue of demented AIDS patients (11, 12),
suggesting that, in AIDS dementia complex, immune-activated macrophages
are able to evoke the release of NO from astrocytes (Fig. 8). Together with the macrophage-derived
superoxide anion, this astrocyte-derived NO may result in the formation
of the highly neurotoxic peroxynitrite. Importantly, we show that NT,
the footprint of peroxynitrite, is detected more frequently and more
intensely in brain sections of demented AIDS patients compared with
nondemented AIDS patients, indicating that peroxynitrite was indeed
present in the brains of these patients. In conclusion, neuronal damage
and death may be the result of interactions between both
immune-activated microglia and astrocytes and the subsequent production
of combined toxic reactive oxygen intermediates like peroxynitrite
(Fig. 8). Thus, although HIV-1 replicates in macrophages, astrocytes
might also participate in the neuropathogenesis of HIV-1 infection.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Leonie A. Boven, Eijkman-Winkler Institute, Section of Neuroimmunology, AZU, hp G04.614, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. E-mail address:
3 H.S.L.M.N. is a fellow of the Royal Netherlands Academy of Sciences and Arts.
4 Abbreviations used in this paper: NO, nitric oxide; iNOS, inducible NO synthase; GFAP, glial fibrillary acidic protein; RFU, relative fluorescence unit; SOD, superoxide dismutase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NT, nitrotyrosine.
Received for publication June 30, 1998. Accepted for publication January 11, 1999.
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References |
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B. Balabanli, Y. Kamisaki, E. Martin, and F. Murad Requirements for heme and thiols for the nonenzymatic modification of nitrotyrosine PNAS, November 9, 1999; 96(23): 13136 - 13141. [Abstract] [Full Text] [PDF]
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