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Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA 92037
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. Therefore, the neurological dysfunctions
correlated with increased numbers of CD8+ T cells of an
activated phenotype in the brain, suggesting that virus-host
interactions contributed to the related CNS functional
defects. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The infected cells in the CNS are of the monocytic lineage (6, 8, 9). Virus can be detected in the brain within 2 wk after infection, when acute viremia peaks. In the early stages of experimental SIV infection, viral expression in the brain is localized mainly in perivascular mononuclear cells and within the leptomeninges (4, 10). Later, viral RNA can be detected in the leptomeninges and in brain infiltrating/perivascular macrophages and multinucleated giant cells (3, 9, 10, 11). Of several mechanisms proposed to account for this CNS infection, the "Trojan horse" model, in which infected monocytes and/or T cells migrate into the brain, has received much support (12, 13).
Approximately one-third of HIV-1-infected individuals develop the associated cognitive/motor disorder, also known as the AIDS dementia or neuroAIDS (13, 14, 15). Furthermore, HIV alters sleep patterns, psychomotor activity, and sensory evoked potentials (EPs)3 (16, 17, 18). In humans, progressive motor and cognitive abnormalities are thought to be initiated by the entry of HIV into the CNS (19). Similarly, in SIV-infected rhesus monkeys, functional CNS deficiencies in sensory EPs, behavioral/cognitive ability, and motor skills have been found by us and others (20, 21, 22, 23). In the rhesus/SIV model, the reduction of virus load in the periphery by antiviral treatment restored neurophysiological normalcy, perhaps, in part, because this treatment lowered the number of infected macrophages and T cells entering the CNS (20). However, abnormalities in movement and temperature were unchanged by antiviral treatment. Because cells of the macrophage/microglia lineage (but not neurons) are infected in the CNS, the neurological consequences of the CNS infection are related to indirect mechanisms, such as viral products or molecules produced by the host in response to the virus. However, the triggering events remain unknown.
Tissue damage is frequently associated with host cell-mediated (T cell) responses (24). Virus-specific CD8+ CTLs play a central role in the immune response against viruses, in general, by eliminating virus-infected cells. Macaques additionally have CD8+ cells that lack the CD3 complex and exhibit NK cell activity (25). The importance of CD8+ cells in controlling SIV infection in monkeys was shown when the viral load increased and progression of disease was accelerated in animals whose CD8+ cells were depleted by Ab treatment (26).
Most of the specific effector CTL generated during viral infections
undergo apoptosis, but a fraction of them remains in the circulation,
constituting a pool of memory cells. These memory cells promptly and
efficiently respond to a second specific stimulation. The expression of
CD45RA can be used in rhesus macaques to define whether previously
primed cells are mediating a secondary response. Resting naive
CD8+ cells, not primed by Ag, express high levels
of CD45RA, but memory cells express far less (27).
However, this low expression of CD45RA does not discriminate memory
cells in the resting state from those that are activated. Instead, the
expression of receptors for homing to endothelium of inflamed sites,
such as CD11a (LFA-1), give some clue about the activation status of
these cells (28). The expression of CD95 (Fas) also
increases on activated cells, and its binding to its ligand (FasL) may
induce apoptosis or proliferation of CD8+ cells
(29). CD28 can also be used to define the pattern of
activation. The T cell Ag CD28 is an important provider of
costimulatory signals to TCR-mediated activation, thereby regulating
proliferation, preventing the induction of anergy or apoptosis, and
driving the pattern of cytokine production (30). The
expansion of CD8+CD28-
cells in HIV infection suggests that such cells arise in response to
the virus (31). In fact, the
CD8+CD28- cells can
present characteristics of end-stage effector CTL, such as expression
of perforin, granzymes A and B, and IFN-, and they exhibit cytolytic
activity (32).
We previously identified SIV-specific CD8+ CTL in the cerebrospinal fluid (CSF) of SIV-infected macaques very early after the infection and in the brain at autopsy (33). Yet the contribution of the CD8+ cells and other T cell subpopulations in the progression of CNS functional deficits remains unknown.
Therefore, the present work was designed to evaluate the brain infiltrate and the phenotype of T cells in the CNS compared with those in the periphery, at a relatively early stage of SIV-induced disease, for a possible correlation with neurological dysfunction. The results showed altered EPs and a drastic increase in the number of activated CD8+ T cells expressing predominantly a memory and a cytolytic phenotype in the brains of animals with early SIV infection. These data suggest that SIV infection induces a chronic immune response in the CNS that mediates injury to the brain.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rhesus monkeys, free from SIV, type D simian retrovirus, and herpes B virus were obtained from Covance (Alice, TX) and Charles River Breed Laboratories (Key Lois, FL). All animal experiments were performed with approval from the Institutional Animal Care and Use Committee. Blood samples were drawn from the femoral vein, and plasma was separated by centrifugation from cells in EDTA-anticoagulated blood. CSF was obtained from the cisterna magna. CSF samples containing >0.1% blood contamination (determined by RBC counts) were discarded. Necropsy was performed after terminal anesthesia. During necropsy, animals were perfused intracardially with sterile PBS containing 1 U/ml heparin before tissue samples were taken for cell isolation, virus quantification, and formalin fixation for histology. Animals 350, 352, 353, and 354 were sacrificed at 73, 75, 77, and 80 days postinoculation (p.i.), respectively, and comprised the 11-wk p.i. group. Animals 335, 346, and 347 were sacrificed at 13 days p.i. and comprised the 2-wk p.i. group.
Viral infection
A cell-free stock of SIV (SIVmac182) obtained after serial passage of SIV-infected microglia was the inoculum used (34). Animals received 0.25 ml of SIVmac182 stock, diluted into RPMI 1640 for injection, containing 5 ng/ml p27 (gag) Ag.
Viral quantitation
To measure virus load, SIV RNA in plasma, CSF, and brain tissue was determined by using the quantitative branched DNA (bDNA) signal amplification assay. Bayer Reference Testing Laboratory (Emeryville, CA) performed this assay. Each sample was measured in duplicate and is reported as an average value. Virus was also recovered from the CSF in a coculture assay. Cells were separated from the CSF by centrifugation, and the cells and cell-free supernatant were separately added to cultures of CEMx174 cells; all cultures were maintained for at least 3 wk for examination for syncytia formation.
Electrophysiology
Eletrophysiological analysis of brainstem auditory EP (BSAEP) and auditory EP (AEP) was performed on ketamine (20 mg/kg)-anesthetized animals as described previously (35). Positive waves in BSAEP were denoted as P1, P2a, P2b, P3, P4, and P5. In AEP, two positive (P1 and P2) and two negative (N1 and N2) waves were identified. Averaged peak latencies were calculated and compared individually between animals and as group means using ANOVA.
Peripheral mononuclear cells
Buffy coats obtained from centrifugation of EDTA-anticoagulated blood, and cell suspensions from spleen, deep cervical, and inguinal lymph nodes, passed through a nylon mesh, were submitted to a Ficoll-Isopaque (Pharmacia Biotech, Uppsala, Sweden) gradient centrifugation for isolation of the mononuclear fraction. After washing, cells were enumerated in a Coulter Z2 (Coulter, Miami, FL) and resuspended in complete RPMI 1640, containing 10% FCS at a concentration of 107 cells/ml, for labeling or freezing.
Brain cells
The meninges were carefully removed, and the brain was minced and transferred to a sterile cell sieve with a 40-mesh wire screen. A sterile plunger was used to force this material through the sieve, and the disassociated material was collected by centrifugation at 470 x g for 15 min at 4°C. The supernatants were aspirated, and each pellet brought to a final volume of 30 ml with HBSS, after which 28 U/ml of DNase I and 500 U/ml of collagenase II were added. Digestion was performed at 37°C in a shaking water bath for 1 h. Afterward, the cells were pelleted at 470 x g for 10 min and then washed in HBSS containing 1% FCS. The cell pellet was resuspended in 6.4 ml of a 1.122 g/ml Percoll (Pharmacia Biotech) solution and brought to a final volume of 30 ml with HBSS, resulting in 1.033 g/ml Percoll. Eight milliliters of 1.088 g/ml Percoll was underlayed, and this gradient was centrifuged at 1200 x g for 20 min at 20°C. The cells at the 1.033/1.088 interface were collected, washed, and quantified in a Coulter Z2.
Flow cytometry
Cells isolated as described (2 x 105 to 10 x 105) were stained with 50 µl mixtures of Abs diluted according to a previous titration in staining buffer (HBSS with 2% FCS and 0.01% NaN3). The Abs used for the staining were anti-monkey CD3-biotin (clone FN-18; Biosource International, Camarillo, CA) followed by streptavidin-PerCP or streptavidin-APC (BD PharMingen, San Diego, CA), anti-human CD8-PE (clone DK25; DAKO, Carpinteria, CA), anti-CD11a-FITC (clone 25.3.1; Immunotech, Westbrook, ME), anti-CD28-FITC (clone CD28.2; Immunotech), anti-CD95-FITC (cloneDX2; Caltag, Burlingame, CA), anti-Mac-1-PE (clone M1/70(9) Roche, Indianapolis, IN), and anti-CD4-PE (clone OKT4; hybridoma obtained from the American Type Culture Collection (Manassas, VA), with the secreted Ab purified and PE-conjugated in the laboratory). Isotype controls (BD PharMingen) were also used. The cells were then processed through a FACScan flow cytometer before analysis of data with CellQuest software (BD Immunocytometry Systems, San Jose, CA).
Magnetic columns
Cells isolated from the blood or lymphoid tissues were resuspended to the concentration of 2 x 107 in 200 µl of PBS, and 50 µl of anti-CD8 MACS Microbeads (Miltenyi Biotec, Auburn, CA) were added. The cells were incubated at 4°C for 20 min, washed once, and passed twice through magnetic columns according to the manufacturer’s instructions for the obtention of a CD8-enriched population. The cells were pelleted and frozen for RNA extraction.
RNA extraction and RT-PCR
The RNA was extracted from the cell pellets on a Bead-Beater
(Biospec Products, Bartlesville, OK) with an Ambion Totally RNA kit
(Ambion, Austin, TX), as per the manufacturer’s instructions. A volume
equivalent to 1 µg was subjected to reverse transcription using 125
ng of random primers, 5x first-strand buffer, 0.1 M of DTT, 10 mM of
dNTPs, and 200 U of Superscript II (Life Technologies, Grand Island,
NY), in a final volume of 20 µl. The samples were then incubated at
42°C for 50 min followed by inactivation at 90°C for 5 min. To
design primers for rhesus CD8 
-chain, granzymes A and B, and
perforin, fragments of cDNA derived from activated rhesus monkey
lymphocytes were PCR-amplified using primers derived from human
sequences available in GenBank. These products were molecularly cloned,
and three to seven clones of each product were sequenced using an
Applied Biosystems automated sequencer at the TSRI Protein and Nucleic
Acids Core Facility. Primers were then designed from the monkey cDNA
sequences obtained, as well as the rhesus IFN- sequence available in
GenBank, with the aid of the MacVector program (Accelrys, San Diego,
CA). Next, amounts of cDNA to be used in the PCR were normalized
after determining the relative amounts of 18 S cDNA in the sample by
real-time quantitative PCR (primers and probe from Perkin-Elmer, Foster
City, CA) using a Smart Cycler (Cepheid, Sunnyvale, CA). Finally, the
normalized cDNA was amplified with the following primers: CD8,
5'-CATGG TGAAG AGGTG GCACA GGAGA and 3'-TGGAC TTCTT GGTGG GCTGG GCA
(72°C, 205-bp product); perforin, 5'-ACTTT GCAGC CCAGA AGACC CAC and
3'-CCGTA GTTGG AGATG AGCCT GAGGT (71°C, 193-bp product); granzyme A,
5'-GGGTG GGGGA AGATT CACAA TAGTG C and 3'-CCCTC GGAAA ACACC GTTAC ACAAC
A (60°C, 219-bp product); granzyme B, 5'-GGAAG ATCAA ACGTG CAAAT CCCG
and 3'-GGAGG CTTGC CATTT CTTTG TCCAT (60°C, 177-bp product); and
IFN-, 5'-ACGGG ATGAC TTTGA AAAGC TGACC and 3'-CACTG GGATG CTCTT
CGACC TGC (72°C, 165-bp product).
Histopathology and immunohistochemistry
Tissues were fixed in 10% formalin, embedded in paraffin, and cut into 5-µm sections. After H&E staining, sections from each tissue block were examined microscopically. Representative sections of brain (plus lymph node and spleen controls) were immunohistochemically stained for leukocyte common Ag (LCA) as described (20).
Statistical analysis
After establishing the normality and homogeneity of variances, the samples were submitted to one- or two-way ANOVA, depending on the number of criteria to be tested, followed by a multiple comparisons t test (Tukey) when appropriate. Only the samples that exhibited a confidence interval of 95% or more were accepted as significant. The tests were performed by SigmaStat version 2.0 for Windows (SPSS Science, Chicago, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A). In the CSF, the temporal
pattern of viral load was similar to that in plasma for two animals
(monkeys 350 and 353), another animal (354) never had virus detectable
by bDNA tests of the CSF at any time (Fig. 1B). Early time
point CSF samples from monkey 352 were too small for quantitation by
bDNA analysis, but CSF from all four animals was positive in coculture
assays on day 14 p.i., suggesting that virus was present in the
CSF simultaneously with the peak load in the plasma of all four
animals.
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A shows that the latency of
the BSAEP P5 increased during the infection in comparison to each
animal’s individual preinoculation baseline, and Fig. 2B
similarly shows the increase in the latency of the AEP N2 auditory time
following viral inoculation. Representative waveforms before and after
infection for the BSAEP are shown in Fig. 2C. These data are
consistent with previous observations of early abnormal sensory EPs
after infection.
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). Because the number of
CD4+ T cells with CD11a phenotype did not change
(Fig. 3), no generalized activation of T cells occurred.
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According to histopathological analysis, SIV encephalitis was not
present, and the CNS had no focal lesions, although infiltrating
lymphoid cells were noted and subsequently verified by reactivity with
LCA. The LCA-positive cells were easily detected on brain sections from
infected animals but were rare in uninfected controls. Positively
stained mononuclear cells formed foci in the perivascular area or were
scattered diffusely within the parenchyma (Fig. 4). No differences were observed in the
intensity or distribution of infiltrating cells among individual
infected animals.
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). Calculation of the number of
CD3+CD8+ cells isolated
from the CNS revealed that an increased number of
CD8+ T cells was found in the brains relative to
controls, but the intensity of the CD8+
infiltrate was not necessarily proportional to the amount of virus
detected in the brain tissue (Table I). Thus, no link was evident
between the amount of virus and the number of
CD8+ T cells at this stage of SIV infection.
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).
The phenotypes of lymphocytes infiltrating the CNS was
determined by flow cytometry and identification of surface markers that
define the subpopulations and their activation status. Fig. 5A depicts the relative
percentage of T cell types in the brains of individual monkeys. The
most dramatic expansion in both absolute and relative counts was found
in the CD3+CD8+ cell
population in the 11 wk p.i. animals; such infected animals had a
significant 5.4-fold increase (F = 38.797;
p < 0.001) relative to the number in controls. Amounts
of other lymphocyte subpopulations also increased somewhat, but not to
a level of statistical significance. That is,
CD3+CD4+ T cells increased
2.3-fold (F = 1.811; p = 0.218) and
CD3-CD8+ cells 2.5-fold
(F = 1.108; p = 0.371) in the brains of
11-wk-infected animals compared with control brains. Although the
number of CD3+CD8+ T cells
in the brain of the 2 wk p.i. group was approximately twice that found
in uninfected control animals, such a change was not statistically
significant (F = 38.797; p =
0.403).
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B). The increase in both memory and naive
CD8+ cells in 11 wk was statistically significant
in comparison to controls (F = 16.669 and
F = 10.334, respectively; p < 0.001).
In contrast, the smaller increase in
CD3+CD8+ cells found at 2
wk p.i. was evenly divided between memory and naive cells. In the CNS
of SIV-infected monkeys, a preferential enrichment of the
CD8+ T cells observed at 11 wk presumably favored
those expressing a memory phenotype.
Furthermore, the foregoing increase of CD8+ T
cells resulting from SIV infection was also observed in peripheral
sites. Fig. 6 presents the CD4:CD8 ratios
in the blood, spleen, and lymph nodes, all of which exhibited lower
values following 2 or 11 wk of infection relative to controls.
Comparison of CD4:CD8 T cell ratios in the brain vs peripheral regions
showed that infected as well as uninfected monkeys had a predominance
of CD8+ T cells in the brain (Fig. 6).
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), suggesting that the infection led to
a systemic enrichment of memory cells. The comparison of
CD8+ T cells from different sites within the same
group of animals revealed that in control monkeys, the percent of
memory CD8+ T cells (expressing
CD45RAlow) in the brain was significantly higher
(F = 10.334; p < 0.01) than in
the periphery (Fig. 7). However, in the SIV-infected monkeys,
the percent of memory CD8+ T cells was comparable
at all sites analyzed. In the CNS, 11 wk-infected and control animals
had the same level of predominance of memory CD8+
T cells, but their absolute number differed greatly.
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A), especially
in the spleen and lymph nodes, where the differences were statistically
significant (F = 8.733; p < 0.001).
Additionally, both infected and control animals had a significantly
higher (F = 26.521; p < 0.001)
percentage of CD8+ T cells expressing
CD11ahigh in the CNS than in the periphery. Close
to 100% of the cells in the CNS expressed high levels of CD11a, but in
peripheral sites of control as well as infected monkeys, the proportion
of cells was always much below that level. The level of CD95 expression
did not differ statistically between controls and infected monkeys for
each individual site analyzed, but CD8+ T cells
from the CNS tended to have a higher level of CD95 expression than
those in the periphery (Fig. 8B). The expression of CD28 was
also evaluated on CD8+ cells from the CNS and
other sites in SIV-infected monkeys and controls (Fig. 8C)
but the differences were not significant. CD8+ T
cells in the CNS seem to differ phenotypically, then, from those in the
periphery, and the SIV-induced changes in the CNS do not involve
differences on the expression of the surface markers that we analyzed.
Similar to the animals infected for 11 wk, the
CD8+ T cells in the brain of 2-wk-infected
animals did not differ from uninfected controls for the level of
expression of the activation markers analyzed (Fig. 8D).
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represents examples of
fluorescence intensity histograms of the surface markers we
investigated. The differences between controls (Fig. 9A) and
SIV-infected monkeys (Fig. 9B) were strongest for CD11a and
CD95 expression. Clearly, the uninfected controls expressed lower
levels of these markers and fewer cells with an abundance of these
markers than SIV-infected animals. This was true at all sites analyzed,
except the brain. Although control animals varied considerably in CD28
expression, CD8+ T cells from infected animals
had less CD28 than CD8+ cells from three of five
control animals, regardless of site.
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, all agents of tissue
destruction, was identified in the CD8+
cells from the brains of SIV-infected animals at both 2 and 11 wk p.i.
(Fig. 10). In contrast, the mRNA of
CD8+ cells obtained from the brains of control
animals contained heterogeneous and variable expression patterns of the
analyzed molecules.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Periodic evaluation of T cell subpopulations in the circulation of the infected animals over an 11-wk-period showed an enlargement of the CD8 compartment, peaking at 2 wk p.i., although the CD4+ cells remained unaltered. Indeed, a burst of proliferation is found in the CD8+ T cell compartment within the first few weeks of SIV infection (27, 36). Our study as well as those of others show that these CD8+ T cells express high levels of CD11a on the surface (37, 38), indicating that these cells become activated and able toadhere to endothelial cells, possibly followed by transmigration to tissue parenchyma. In parallel with the changes in blood CD8+ T cells at 2 wk p.i., a corresponding mild increase in the number of cells is found in the brain at 2 wk p.i. However, at 11 wk p.i., while the number of CD8+ T cells in the circulation has declined from the 2 wk p.i. peak, the CD8+ T cell presence in the brain has increased to over 5-fold in number compared with controls.
In peripheral sites such as the blood, spleen, and lymph nodes, the CD4:CD8 ratio of the infected animals decreased in comparison to control animals. This finding suggests that the virus infection induced an expansion of the CD8 compartment relative to CD4 that was not restricted to the brain, but was a systemic phenomenon, confirming previous observations in the SIV model and also in HIV (39). However, the CD4:CD8 ratio in the brains of SIV-infected monkeys and their uninfected counterparts was not statistically different and, in both, was lower than in the periphery. This evidence points to the predominance of CD8+ T cells in the CNS even under nonpathological conditions, as has been found in the CSF of monkeys (40) and brains of mice (41).
Naive CD8 cells in monkeys express high levels of CD45RA, but memory
cells lose these molecules from the surface (27). However,
an effector CD8+ T cell does not necessarily
present low levels of CD45RA, because cells with naive phenotype can
have effector activity, with expression of high levels of CD11a.
Effector CD8+ T cells initiate transcription of
cytokines such as IFN- early in the activation process. Other
effector molecules such as granzymes A and B are expressed upon
acquisition of cytotoxic function, and their expression is a useful
marker of CTL activation in vivo (32, 42). Here, we found
that brain-infiltrating CD8+ cells expressed mRNA
for the cytotoxic granule proteins perforin, granzyme A and granzyme B,
as well as for IFN-, indicating an effector phenotype at 2 and 11 wk
after infection. CD8+ T cells can regulate
IFN- expression in an Ag-specific manner, expressing it when in
contact with their cognate Ag (42). The expression pattern
we found here is consistent with our previous immunohistochemical and
in situ hybridization studies identifying granzyme B and IFN-
expression in the brains of SIV-infected monkeys (20, 43),
and our finding of infiltrating SIV-specific cells with CTL activity in
the brains of infected monkeys (33).
Despite having a similar pattern of surface marker expression, CD8+ cells in the brains of uninfected animals have an inconsistent or mixed pattern of mRNA expression for the proinflammatory and cytolytic molecules. This suggests that the few CD8 cells in the brain of uninfected animals are circulating cells not carrying out an effector function, but may represent cells in what is termed a surveillance mode (44). Highly activated cells, silent in terms of effector molecule production, can circulate in the blood and tissues, switching to an effector mode upon Ag stimulation. At the second week after infection, although only a limited increase in the number of CD8+ T cells in the brain was observed, the cells that got into the brain were indeed qualitatively different, because the expression of these molecules was consistent and homogeneous. At 11 wk, a highly significant increase in the CD8+ T cell population occurred with maintenance of a consistent and homogeneous level of production of effector molecules. These findings suggest that the CD8 cells that are present in the brain of SIV-infected animals are qualitatively as well as quantitatively distinct from controls.
Certainly, the increased number of CD3+CD8+ T cells at 11 wk, a relatively early stage of infection, was a most remarkable finding in terms of brain cell populations. However, the fact that we identified 1) far greater expression of activation markers on brain-derived CD8+ T cells than on those in the periphery and 2) expression of RNA to cytolytic and proinflammatory molecules adds a functional dimension to the quantitative changes. Immunohistochemical and FACS studies by others have indeed shown that CD8+ T cells are present in the brains of SIV-infected macaques both at the acute, and later stages of the disease (3, 45, 46). The CD8+ T cells that migrate into the brain can have a protective antiviral role (45), as suggested by the lowering of brain viral load in the 11 wk vs 2 wk p.i. animals and as described for other CNS viral infections such as influenza encephalitis (47). However, a pathogenic role of CD8+ T cells in virus-induced CNS disease has been suggested not only by us for SIV-infected monkeys (33), but for HTLV-I in humans (48), as well as mouse hepatitis virus, Semliki, Borna, and Theiler’s virus infections in rodents (49, 50, 51, 52).
It is notable that there was a temporal coincidence between peripheral and CNS events related to the peak of virus load, but not to the increase of CD8+ T cells. There is a time lag between the expansion of CD11ahigh CD8+ T cells in the periphery and their accumulation into the brain. This suggests that the increase of CD8+ T cells in the brain is not exclusively due to a systemic circumstance of activation and expansion of the CD8 subset, although such a change is potentially reflected in the mild increase in brain CD8+ T cells found at 2 wk p.i. It is recognized that activated T cells can cross the unaltered blood-brain barrier into the brain, independent of Ag specificity, as part of general immune surveillance of the CNS (53). Naive T cells can also enter an inflamed CNS (54). However, the retention and accumulation of T cells in the CNS requires Ag recognition (55, 56), as does effector molecule production (54). Thus, following infection and the consequent Ag stimulation, the CD8+ T cell population observed in the brain has changed from one mainly composed of cells in the surveillance mode to one of effector activity, capable of producing molecules that can target infected cells, but also with the capacity of inducing tissue damage. The CNS is a specialized tissue with a very low degree of functional redundancy and is thus quite sensitive to cellular injury that can compromise the ability of the brain to carry out its critical role.
Overall, our data support the concept that activated cells are present in the CNS. With respect to the expression of CD45RA in our experiments, the percentage of naive and memory CD8+ T cells in the brain was not statistically different between SIV-infected and control animals. This was due to the fact that uninfected controls presented a higher percentage of CD8+ T cells with memory phenotype in the brain in comparison to the periphery. Interestingly, recent data indicate that specific CD8+ memory and effector T cells migrate to nonlymphoid tissues (57). In other experimental models, particularly in rodents, specificity and even MHC restriction were not crucial factors for T cells to infiltrate the CNS, suggesting that SIV-specific cells are not the only ones to enter the brain (53, 54). However, the CD8+ lymphocytes we found in the brain of either infected or control monkeys were close to 100% CD11ahigh and CD95high, indicating an activation status independent of the expression of memory markers (28).
Although studies have indicated that low levels of CD28 expression
correlate with effector function and infection (31), we
saw no such differences, at least not to the level of significance, for
the expression of this marker in the infected monkeys. In part, this
may have resulted from the uneven expression of CD28 in the uninfected
animals. However, not all effector cells exhibit low levels of CD28, as
SIV-specific CTL, identified by tetramer binding studies, are divided
for the expression of this marker (38). Others have shown
a population of CD8+CD28+
cells that expanded during viral infection, produced IFN- and
perforin, and had effector function. This cell subset, as opposed to
the CD28- population, retained the ability to
proliferate (58). The nature of these cells in the
periphery as well as the brain has yet to be examined in SIV-infected
subjects. Our results emphasize that alterations in CNS T cell
subpopulations soon after infection result in an abnormally large
number of activated CD8+ T cells in the brain;
this may increase the concentrations of cytolytic and proinflammatory
cytokines to a pathological level.
The purified brain CD8+ cells obtained from
SIV-infected animals expressed the mRNA for perforin, granzymes A and
B, important molecules involved in the apoptotic process and cell
lysis, as well as for IFN-. Proinflammatory cytokines, among them
IFN-, are known to induce an increase in the expression of adhesion
molecules like ICAM-1 on endothelial cells (59). ICAM-1 is
the ligand for CD11a (LFA-1) expressed on the leukocyte cell surface.
The up-regulation of this molecule may play a role in the adhesion of
leukocytes to the endothelial cell, a step that precedes the
transmigration to the tissue, especially if LFA-1 expression is also
increased on the cell surface, as we observed here on
CD8+ T lymphocytes. Thus, IFN- can contribute
to the migration of CD11ahigh
CD8+ T cells to the parenchyma. Additionally,
IFN- can regulate cell traffic into the brain by inducing chemokine
synthesis (60) and can function as a potent
macrophage/microglia activator that induces the production of
potentially neurotoxic molecules.
Our results suggest that the SIV infection promotes a numerical increase of CD8+ T lymphocytes with the capacity to enter the CNS. The accumulation of such cells in the brain, despite having a protective role, can potentially induce cellular damage in the brains of SIV-infected monkeys and incite the functional CNS abnormalities characteristic of this and similar infections.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Howard S. Fox, Department of Neuropharmacology, The Scripps Research Institute, 10550 North Torrey Pines Road, CVN 8, La Jolla, CA 92037. E-mail address: hsfox{at}scripps.edu
3 Abbreviations used in this paper: EP, evoked potential; CSF, cerebrospinal fluid; p.i., postinoculation; bDNA, branched DNA; BSAEP, brainstem auditory EP; AEP, auditory EP; LCA, leukocyte common Ag.
Received for publication May 10, 2001. Accepted for publication August 31, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and human immunodeficiency virus infection. Proc. Natl. Acad. Sci. USA 92:12080. shapes immune invasion of the central nervous system via regulation of chemokines. J. Immunol. 164:2759.This article has been cited by other articles:
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 S. Sadagopal, S. L. Lorey, L. Barnett, R. Basham, L. Lebo, H. Erdem, K. Haman, M. Avison, K. Waddell, D. W. Haas, et al. Enhancement of Human Immunodeficiency Virus (HIV)-Specific CD8+ T Cells in Cerebrospinal Fluid Compared to Those in Blood among Antiretroviral Therapy-Naive HIV-Positive Subjects J. Virol., November 1, 2008; 82(21): 10418 - 10428. [Abstract] [Full Text] [PDF]
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 M. Eilers, U. Roy, and D. Mondal MRP (ABCC) Transporters-Mediated Efflux of Anti-HIV Drugs, Saquinavir and Zidovudine, from Human Endothelial Cells Experimental Biology and Medicine, September 1, 2008; 233(9): 1149 - 1160. [Abstract] [Full Text] [PDF]
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S. J. Bissel, G. Wang, D. Bonneh-Barkay, A. Starkey, A. M. Trichel, M. Murphey-Corb, and C. A. Wiley Systemic and Brain Macrophage Infections in Relation to the Development of Simian Immunodeficiency Virus Encephalitis J. Virol., May 15, 2008; 82(10): 5031 - 5042. [Abstract] [Full Text] [PDF]
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C. C. Clay, D. S. Rodrigues, Y. S. Ho, B. A. Fallert, K. Janatpour, T. A. Reinhart, and U. Esser Neuroinvasion of Fluorescein-Positive Monocytes in Acute Simian Immunodeficiency Virus Infection J. Virol., November 1, 2007; 81(21): 12040 - 12048. [Abstract] [Full Text] [PDF]
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 S. Gorantla, J. Liu, H. Sneller, H. Dou, A. Holguin, L. Smith, T. Ikezu, D. J. Volsky, L. Poluektova, and H. E. Gendelman Copolymer-1 Induces Adaptive Immune Anti-inflammatory Glial and Neuroprotective Responses in a Murine Model of HIV-1 Encephalitis J. Immunol., October 1, 2007; 179(7): 4345 - 4356. [Abstract] [Full Text] [PDF]
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M. C. G. Marcondes, T. H. Burdo, S. Sopper, S. Huitron-Resendiz, C. Lanigan, D. Watry, C. Flynn, M. Zandonatti, and H. S. Fox Enrichment and Persistence of Virus-Specific CTL in the Brain of Simian Immunodeficiency Virus-Infected Monkeys Is Associated with a Unique Cytokine Environment J. Immunol., May 1, 2007; 178(9): 5812 - 5819. [Abstract] [Full Text] [PDF]
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 E. S. Roberts, S. Huitron-Resendiz, M. A. Taffe, M. C. G. Marcondes, C. T. Flynn, C. M. Lanigan, J. A. Hammond, S. R. Head, S. J. Henriksen, and H. S. Fox Host response and dysfunction in the CNS during chronic simian immunodeficiency virus infection. J. Neurosci., April 26, 2006; 26(17): 4577 - 4585. [Abstract] [Full Text] [PDF]
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Y. Zhu, J. Antony, S. Liu, J. A. Martinez, F. Giuliani, D. Zochodne, and C. Power CD8+ lymphocyte-mediated injury of dorsal root ganglion neurons during lentivirus infection: CD154-dependent cell contact neurotoxicity. J. Neurosci., March 29, 2006; 26(13): 3396 - 3403. [Abstract] [Full Text] [PDF]
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R. Sancho, L. de la Vega, A. Macho, G. Appendino, V. Di Marzo, and E. Munoz Mechanisms of HIV-1 Inhibition by the Lipid Mediator N-Arachidonoyldopamine J. Immunol., September 15, 2005; 175(6): 3990 - 3999. [Abstract] [Full Text] [PDF]
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 M. C. G. Marcondes, G. C. Furtado, A. Wensky, M. A. Curotto de Lafaille, H. S. Fox, and J. J. Lafaille Immune Regulatory Mechanisms Influence Early Pathology in Spinal Cord Injury and in Spontaneous Autoimmune Encephalomyelitis Am. J. Pathol., June 1, 2005; 166(6): 1749 - 1760. [Abstract] [Full Text] [PDF]
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M. Moniuszko, C. Brown, R. Pal, E. Tryniszewska, W.-P. Tsai, V. M. Hirsch, and G. Franchini High Frequency of Virus-Specific CD8+ T Cells in the Central Nervous System of Macaques Chronically Infected with Simian Immunodeficiency Virus SIVmac251 J. Virol., November 15, 2003; 77(22): 12346 - 12351. [Abstract] [Full Text] [PDF]
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B. O. Kim, Y. Liu, Y. Ruan, Z. C. Xu, L. Schantz, and J. J. He Neuropathologies in Transgenic Mice Expressing Human Immunodeficiency Virus Type 1 Tat Protein under the Regulation of the Astrocyte-Specific Glial Fibrillary Acidic Protein Promoter and Doxycycline Am. J. Pathol., May 1, 2003; 162(5): 1693 - 1707. [Abstract] [Full Text] [PDF]
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 S. Sanchez-Ramon, J. Ma Bellon, S. Resino, C. Canto-Nogues, D. Gurbindo, J.-T. Ramos, and M. Munoz-Fernandez Low Blood CD8+ T-Lymphocytes and High Circulating Monocytes Are Predictors of HIV-1-Associated Progressive Encephalopathy in Children Pediatrics, February 1, 2003; 111(2): e168 - 175. [Abstract] [Full Text] [PDF]
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E. M. E. Burudi, M. C. G. Marcondes, D. D. Watry, M. Zandonatti, M. A. Taffe, and H. S. Fox Regulation of Indoleamine 2,3-Dioxygenase Expression in Simian Immunodeficiency Virus-Infected Monkey Brains J. Virol., October 25, 2002; 76(23): 12233 - 12241. [Abstract] [Full Text] [PDF]
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