| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
,,¶,,¶,||
*
Center for Aging and Developmental Biology,
Program in Neurosciences, and Departments of
Neurology (Child Neurology Division),
§
Microbiology and Immunology,
¶
Pediatrics, and
||
Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY 14642;
#
Center for Neurologic and Neurodegenerative Disease, University of Nebraska Medical Center, Omaha, NE 68198;
**
Department of Pediatrics (Child Neurology Division), Children’s Memorial Hospital, Chicago, IL 60614;
Chemocentryx, San Carlos, CA 94074; and
*
Department of Laboratory Medicine and Pathology (Neuropathology Division), University of Medicine and Dentistry of New Jersey, Newark, NJ 07103
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Several classes of chemokine receptors (CCR5, CXCR4) may function as
coreceptors for HIV-1 in the CNS and have helped to identify
populations of CNS glia that are at risk for infection (9, 10). Macrophages in areas with the histopathologic correlates of
HIVE express ß chemokines, monocyte chemotactic protein-1, macrophage
inflammatory protein-1
, and RANTES, suggesting a role for chemokines
in formation of inflammatory infiltrates and microglial nodules
(9). Deletion of the CXCR4 gene in transgenic mice results
in abnormalities of cerebellar neuronal migration (11).
Thus, chemokine receptor activation may modulate development of the CNS
and mediate immunologic communication between glia and neurons.
Fractalkine (FKN) is a novel chemokine derived from non-hemopoietic cells. It is a large (373 residues) protein that contains the chemokine domain attached to a mucin-like stalk (12). FKN exists in a soluble form with potent chemoattractant activity for T cells and monocytes, and in a membrane-bound form that is induced on activated endothelial cells, and promotes leukocyte adhesion (12). FKN is predominantly expressed in neurons in the CNS. Its receptor, CX3CR1, is expressed in microglia (13). Administration of FKN substantially ameliorates gp120-induced neuronal apoptosis in cultured rat hippocampal neurons (14), which suggests that FKN may play an important role in modulating the neuropathogenesis of HIV-1. We demonstrate for the first time that FKN expression is localized to cytoplasmic structures, that these structures have a vesicular appearance in glutamatergic neurons, and that FKN is up-regulated in neurons adjacent to macrophages and microglia in brain tissue from pediatric patients with HIVE and progressive encephalopathy (PE), compared with pediatric patients with HIV-1 but no HIVE or PE, or to pediatric patients that were HIV-1 seronegative. Furthermore, FKN can function both as a potent mediator of monocyte trafficking across an endothelial cell/astrocyte bilayer, and as a neuroprotective agent when administered with either the HIV-1 neurotoxin platelet activating factor (PAF) or the regulatory protein Tat.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Clinical and neuropathologic data on seven HIV-1-infected children and nine HIV-1-seronegative pediatric controls have been described (2, 15). HIV-1 infection was diagnosed by Centers for Disease Control (CDC) criteria (positive culture <15 mo; presence of HIV-1 Abs >15 mo) (16). One adult case (male, 42, i.v. drug abuser, with symptoms of neurologic disease) with HIVE and one adult case (male, 41, i.v. drug abuser, with no symptoms of neurologic disease) with HIV-1 infection, but without HIVE, were also studied. Formalin-fixed paraffin-embedded tissue blocks were processed as previously described from brain regions used in this study (15). Tissue sections from various brain regions of HIV-1-infected and uninfected patients were incubated with rabbit anti-FKN peptide (Chemocentryx, San Carlos, CA) at 2.5 µg/ml overnight at 4°C. Various concentrations of rabbit anti-FKN peptide were tested to obtain optimal signal to background. Control slides from the same brain regions were incubated with a 22-residue blocking peptide containing the C-terminal sequence of FKN (20 µg/ml) (12), and coincubated with 2.5 µg/ml rabbit anti-FKN peptide, using the Vector ABC avidin-biotin complex with either an alkaline phosphatase or diaminobenzidine (DAB) chromagen. For FKN receptor ICC, tissue sections were incubated with either a human FKN variant that contained a small portion of the mucin stalk and a poly histidine (HIS) tag (Chemocentryx, 10 µg/ml, 1 h, at 22°C) or a chemokine portion of FKN lacking the polyHIS tag to serve as a control condition, followed by incubation with an anti-poly(HIS) mAb (Chemocentryx, 10 µg/ml, 1 h, at 22°C), using the Vector ABC system. Parallel tissue sections were stained with antisera to glutamate (1:500; Research Biochemicals, Natick, MA) to identify glutamatergic neurons. Glial fibrillary acidic protein (GFAP) (1:500 in normal goat serum; Dako, Carpinteria, CA.) was used to identify astrocytes while a mAb (EBM11) to the cell surface Ag CD68 (1:200 in normal horse serum; Dako) was used to identify MPs, and HIV-1 antiserum to p24 (1:500 in normal horse serum; American Bio-Technologies, Cambridge, MA.) was used to identify infected cells, using the Vector ABC system. In some experiments, double immunolabeling with antisera to FKN and antisera to CD68 or glutamate were performed as previously described (15). Ag retrieval was performed to obtain optimal signals from the Abs tested in formalin-fixed brain tissues (15).
Quantitative morphometry to measure the relative OD (ROD) of FKN-immunopositive neurons using computerized densitometry (Image Pro Plus for Macintosh, Vs. 3.1) was performed in the following fashion: ROD was corrected for incident light levels in each field. Maximum black levels (shutter closed condition) were the same for each field. FKN-immunopositive neurons in insular cortex were identified by observers blinded to the patient group using standard cytologic criteria. A total of 375 FKN-immunopositive neurons were identified for the HIV- group, 426 FKN immunopositive neurons were identified for the HIV+ group, and 522 FKN immunopositive neurons were identified for the HIVE group. The ROD was integrated from the total surface area of each neuronal soma, and data were expressed as the mean ROD ± SEM. Significance was determined by paired t tests between groups.
Confocal microscopy
ICC for FKN and CD68 or glutamate was performed as described above using secondary Abs conjugated with FITC for either CD68 or glutamate and tetramethylrhodamine isothiocyanate (TRITC) for FKN. Images were recorded at intervals of 11.8 s in increments of 0.2-µm slices for FKN and CD68 and intervals of 9–10.1 s in increments of 0.3-µm slices for FKN and glutamate on an Olympus (Spectra Services, Webster, NY) BX50WI microscope (60 x 1.4 oil-immersion PlanApo objective). Confocal sections were scanned in the center of the tissue section and are represented as a stacked image of all sections (Olympus Fluoview Software). Wavelengths of 488 nm and 568 nm were used to excite fluorescein- and rhodamine-conjugated secondary Abs, respectively. Emission spectra were collected with 510-nm and 560-nm bandpass filters to achieve minimal overlap of fluorophore detection for accurate double labeling. Digitized images from representative fields were prepared from TIFF files (Adobe Photoshop 4.0, Seattle, WA).
Cell culture and preparation of endothelial cell/astrocyte bilayers
Purified human fetal astrocytes (PHFAs) were isolated from
second-trimester human fetal brain tissue obtained from elective
abortions (performed in full compliance with both National Institutes
of Health (NIH) and University of Rochester guidelines), using methods
previously described (17). The purity of the PHFA cultures
was determined by ICC for GFAP. Cultures were used for experiments only
if >97% of cells were GFAP immunopositive. HUVECs (>99% factor
VIII-immunoreactive) were obtained frozen from Cascade Biologics
(Portland, OR). Twenty-four-well transwell inserts were first coated on
the upper chamber side with rat-tail collagen (50 µg/ml;
Collaborative Biomedical Products, Bedford, MA), then coated on both
sides with human fibronectin (500 ng/ml; Life Technologies, Rockville,
MD) (18). PHFAs were plated on the lower chamber side in
an inverted position at 1 x 105 cells/100
µl for 2 h for cell adherence. The transwell was placed upright,
and HUVECs were then plated at a density of 0.5 x
105 cells/200 µl as previously described
(19, 20). Transwell cultures were maintained in Medium-200
(Cascade Biologics) at 37°C, in an atmosphere containing 5%
CO2 for 
5 days before use, with daily changes
of medium. For monocyte migration experiments, human monocytes were
recovered from PBMCs of HIV- and hepatitis B-seronegative donors after
leukapheresis and purified (>98%) by countercurrent centrifugal
elutriation as previously described (4). Monocytes were
adjusted to 1.5 x 105 cells/200 µl, and
200 µl was placed in the upper transwell. Doses of soluble human FKN
(no mucin stalk; Chemocentryx), neutralizing mAbs (IgG1 and IgG2b,
clones 21, 28, 29, 36, 37, and 43; Chemocentryx) to FKN, MCP-1 (R&D
Systems, Minneapolis, MN), or vehicle were placed in the lower chamber
of the transwell, and the chambers were returned to a 37°C, 5%
CO2 incubator for 4 h. Then 400-µl
aliquots from the bottom transwell chamber were combined with FITC CD14
(PharMingen, San Diego, CA). The total number of monocytes migrating
from upper to lower wells through the bilayer was counted for 60 s
on a FACStarPlus (flow cytometry) gating for
monocytes (21). Ten replicates were performed for each
concentration of FKN, and experiments were repeated three times.
Production of Tat
Recombinant HIV-1 Tat1–86 was expressed and purified as a thrombin cleaved GST fusion protein (22) and stored at -70°C. Tat1–86 was characterized and quantified using the Lowry method, SDS-PAGE, and by immunoblot analysis, using a polyclonal Ab (AIDS Research and Reference Reagent Program, National Institute of Allergy and Infectious Diseases (NIAID), NIH) (22). To control for contaminating bacterial proteins that might elicit neurotoxicity, an Escherichia coli strain containing a GST expression plasmid without the Tat1–86 sequence was used to prepare extracts, which were purified in the same manner as the Tat fusion protein and used as vehicle control.
Neuronal cell culture and quantitative analyses of cell death
Cerebellar brain tissue from 7 day Sprague Dawley rats was
harvested according to the guidelines of the Animal Welfare Act (1987)
and NIH policies. Briefly, cerebellum was trypsinized in 0.25 mg/ml and
0.1% DNase (
10 ml volume/cerebellum), minced into 2
mm3 pieces, triturated, and incubated for 20 min
at 37°C (23). The tissue was filtered through nylon
mesh, and the cell suspension was loaded over a two-step percoll
gradient and centrifuged at 1000 x g for 15 min at
4°C to remove glia. Neurons were collected, washed twice in
serum-free medium, then resuspended in fresh DMEM:F12 + 10% horse
serum. Cells were triturated and plated at 2 x
105 cells/12 mm, on glass coverslips precoated
with poly(L-lysine) (Sigma, St. Louis, MO) in
24-well culture dishes. After 1.5 days in culture, fUDR
(5-fluoro-deoxyuridine) was added to the cultures at 20 mg/ml and
uridine at 50 mg/ml, to eliminate astrocytes. The purity of the
neuronal population was verified by ICC for neuronal markers. Under
these conditions, neuronal cultures have <3–5% glial contamination.
Neurons were cultured for 
7 days at 37°C in 5%
CO2. Serum-free DMEM:F12 was replaced every
3 days.
In situ detection of apoptotic neurons by TUNEL stain
Neurons cultured on 12mm poly(L-lysine)-coated coverslips were treated with vehicle or reactions, and apoptotic cells were stained using an in situ TdT-mediated digoxigenin-TUNEL assay method (Oncor, Gaithersburg, MD) as previously described (22). Cell cultures were fixed in 4% paraformaldehyde, rinsed with PBS, postfixed with a 100% ethanol:acetic acid solution (2:1), and rinsed with PBS. Neurons were pretreated with 2% H2O2 to quench endogenous peroxidase, before the addition of TdT. Anti-digoxigenin-peroxidase was added and catalytically reacted with 0.05% DAB in PBS. TUNEL-stained neurons were counted from 15 randomly selected fields. Each field of at least 100 neurons was counted for immunoreactive cells. Data are expressed as the percentage of TUNEL-positive neurons per high power field. For dose-response studies, data were analyzed using Sigmaplot (Vs. 3.0) to obtain the resulting sigmoidal curves using the following equation: Y = (Vmax x (1 - (x/(x + Ki))) + os. Inhibitor values (Ki) for soluble FKN (sFKN) and the C22 analogue of FKN (FKN22), as well as Hill coefficients (nH) were then determined from the resulting dose-response curves.
Measurement of TNF-, NO, and superoxide anion
TNF- was measured in primary human monocyte or
monocyte-derived macrophage cultures exposed to FKN species by ELISA
(R&D Systems) as previously described (24). NO production
was estimated by Griess reaction as previously described
(25). Superoxide anion was measured in cultures of human
monocytes or monocyte-derived macrophages (4, 15) using
the Lumimax superoxide anion detection kit (Stratagene, La Jolla, CA).
Briefly, cultures exposed to either soluble or immobilized FKN were
then reacted with PMA, a bioactive peptide (fMLP), or opsonized zymosan
capable of inducing superoxide anion. Luminol and the proprietary
enhancer were added to a final concentration of 100 mM and 125 mM,
respectively. Results (compared with a standard curve) were read on a
luminometer.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
A.), a patient with HIV-1
without either encephalitis or PE (Fig. 1B.), and a patient
with HIVE (Fig. 1C.) are shown in Fig. 1. Little or no FKN
was detected in astrocytes, macrophages, microglia, or
oligodendrocytes. In all patient groups, neurons were the predominant
cell type to express FKN, which was localized to the cytoplasm. In the
patient with HIVE, immunopositive neurons were present in the insula of
the temporal lobe (Fig. 1C), caudate, putamen, claustrum,
and globus pallidus. In the caudate and putamen of the HIVE case, small
neurons were immunopositive for FKN, but large neurons did not express
FKN (Fig. 1, E and F). A similar pattern of FKN
staining was seen in the insular cortex, globus pallidus, and claustrum
of the patient with HIV-1, but not HIVE (Fig. 1B). To
quantitate the relative difference in FKN immunostaining between
patient groups, we performed computerized morphometry of the ROD of
immunostaining in neurons expressing FKN. The mean ROD of FKN
immunostaining in neurons from HIVE brain tissue was 0.709 ±
0.011 vs 0.590 ± 0.005 for neurons in HIV-1 brain tissue vs
0.347 ± 0.004 for neurons in HIV- brain
tissue (all comparisons between groups significant at p
< 0.001). Thus, mean ROD of FKN immunostaining was 2.04 times greater
than the mean ROD of FKN immunostaining in neurons from
HIV- brain tissue, but only 1.2 times greater
than the mean ROD of FKN immunostaining in neurons from HIV-1 brain
tissue (Fig. 1).
|
E). Brain tissues from patients with HIV-1 and
HIV-1-seronegative patients were devoid of axonal staining for FKN
(data not shown). Specificity of FKN immunostaining in neuronal
cytoplasm and neuropil in HIVE tissue was shown by preincubation with a
blocking peptide for FKN (Fig. 1F). With these conditions,
FKN expression was reduced to near background levels.
Confocal microscopy of brain tissue sections stained for the macrophage
and microglial marker CD68 and FKN revealed focal inflammatory
infiltrates in cortex and basal ganglia where perivascular macrophages
and activated microglia appeared to be in cell-to-cell contact with
neurons expressing FKN in brains with HIVE (Fig. 2). Neuronal expression of FKN was
localized to punctate structures with a vesicular appearance in the
cytoplasm of the soma (Fig. 2) and in the neuropil, in agreement with
the results obtained using conventional immunohistochemistry (Fig. 1)
on HIVE brain tissue. Because Fig. 2 is a digital summation of stacked
images obtained by successive scans in the z direction
(i.e., through the entire thickness of the section), it does not depict
that, in some sections, neuronal FKN immunostaining is discrete, but
contiguous with CD68-positive microglia. Identical results were
obtained in brain tissue from an adult patient with HIVE (data not
shown). In contrast, CD68-positive macrophages and microglia were
infrequently observed in proximity to FKN-expressing neurons in
patients with either HIV-1, but not HIVE and PE, or control patients
without HIV infection (data not shown).
|
). A
representative field is shown from the cerebral cortex of an adult
patient with HIVE. FKN expression was localized to punctate cytoplasmic
structures in the neuronal soma. There was colocalization with
cytoplasmic glutamate in FKN-expressing neurons. Superimposition of the
summated confocal images revealed that FKN and glutamate may be
localized to different cytoplasmic compartments in some of the neurons
examined. Similar results were observed in pediatric patients with HIVE
and PE (data not shown).
|
. In pediatric brain tissue with the
neuropathologic hallmarks of HIVE, cytoplasmic staining for the FKN
receptor was seen in neurons and microglia. A similar pattern of
neuronal staining was observed in the HIV-1-infected pediatric brain
tissue without HIVE, whereas limited neuronal staining of the FKN
receptor was seen in pediatric brain tissue from HIV-seronegative
children (data not shown). Brain tissue reacted with the chemokine-only
portion of FKN lacking the poly(HIS) tag (control condition) was devoid
of immunostaining when probed with the anti-poly(His) Ab (data not
shown).
|
). FKN
had a monocyte migration index of 2.85 (1–100 ng/ml). The interaction
between FKN and monocytes was specific. Coincubation of FKN (10 ng/ml)
with a neutralizing dose of a mixture of 5 mAbs to FKN (final
concentration 1 µg/ml) completely abrogated FKN's ability to act as a
chemoattractant (Fig. 5).
|
, PAF,
eicosanoids, free radical species derived from NO, and agonists for
excitatory amino acid receptor subtypes (4, 5, 6, 7, 27, 28).
Since FKN served as a potent chemoattractant for monocytes across the
endothelial cell/astrocyte bilayer, we investigated whether FKN could
also induce production of HIV-1-induced neurotoxic substances from
monocytes. Both soluble and immobilized FKN, at doses ranging from
1–1000 ng/ml, failed to induce production of TNF-, NO, or
superoxide in cultures of primary human monocytes or monocyte-derived
macrophages (data not shown).
These data, and data from a recent report by Meucci et al.
(14) demonstrating that coadministration of FKN
ameliorated gp120-induced toxicity to hippocampal neuronal cultures,
suggested that increased expression of FKN in neurons from patients
with HIVE and PE may represent a compensatory neuroprotective mechanism
against HIV-1-induced neurotoxins. We tested the ability of soluble FKN
or a 22-residue peptide analogue to the C-terminal region of FKN to
ameliorate the neurotoxicity of two HIV-1 neurotoxins, Tat and PAF,
that play key roles in initiating neuronal apoptosis (23, 29). Coincubation of soluble FKN at 100 ng/ml (the dose that
induced maximal numbers of monocytes to migrate across an in vitro
endothelial cell/astrocyte bilayer) was able to significantly decrease
the amount of neuronal apoptosis induced by carbamyl PAF (cPAF) (48%
reduction) or Tat (35% reduction) in highly purified cultures of rat
cerebellar granule neurons with less than 3–5% glial contamination
(Fig. 6A). Coincubation of the
22-residue peptide (100 ng/ml) was able to significantly decrease the
amount of neuronal apoptosis induced by cPAF (37% reduction) or Tat
(50% reduction)(Fig. 6B). These findings suggest that the C
terminus of FKN plays a significant role in neuroprotection. Here cPAF
was used because it is resistant to inactivating acetylhydrolases
present in brain (30). Administration of either soluble
FKN or the 22-residue FKN peptide alone were not significantly
neurotoxic relative to control conditions. Dose-response analyses
confirm that soluble FKN and the C22 analogue of FKN (FKN22) have
similar effective doses (Ki for soluble FKN
= 14.45 ± 5.5 ng/ml vs 55.71 ± 16.7 for the C22 FKN
peptide) for ameliorating cPAF-mediated neurotoxicity in rat cerebellar
granule neurons (Fig. 6C). Transformation of the data to
Hill plots reveals that the nH for both soluble FKN and FKN22 is 1.0,
suggesting that positive or negative cooperativity is not involved in
this biologic effect.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(macrophage inflammatory protein 1) and RANTES from
macrophages are present in inflammatory infiltrates in brain tissue
with the histopathologic correlates of HIVE (9). Recently,
Conant et al., (31) have shown that the HIV-1 regulatory
protein Tat can stimulate astrocytes to release MCP-1 in vitro. Levels
of MCP-1 are elevated in brain and cerebrospinal fluid (CSF) of
patients with HIV-1-associated dementia. These data lend credence to
the hypothesis that HIV-1 gene products can initiate a cycle of
inflammation through chemokine production and subsequent signaling.
Since a possible functional interaction exists between neuronally
expressed FKN and its receptor, CX3CR1 in
activated microglia (13), we investigated whether FKN was
up-regulated in brain tissue from patients with HIVE, and whether it
functioned in monocyte recruitment into the CNS. Figs. 1 and 2 show
that FKN expression is up-regulated in neurons and in the neuropil of
brain tissue from patients with HIVE, compared with brain tissue from
patients seronegative for HIV-1 or patients with HIV-1 but not HIVE. It
is technically difficult to quantify FKN expression in HIVE tissue
because it is unknown at the time of dissection whether focal
inflammatory infiltrates are present in the brain regions of interest
(blocks of fresh-frozen tissue corresponding to the formalin-fixed
tissue blocks used in this study were not available for these studies),
which precludes Western blot analyses. In all cases, FKN immunostaining
was abolished upon addition of a blocking peptide (based on the C
terminus of FKN) to the anti-FKN Ab used in these studies. Thus,
the staining results were specific. The explanation for the presence of
extracellular FKN is unknown. Since data from confocal studies show
that FKN is present in punctate structures with a vesicular appearance
in neuronal soma and the neuropil, FKN may be released into the
neuropil as a neural-immune modulator to signal brain-resident
macrophage and microglia. These data show that CD68-expressing
perivascular macrophages and activated microglia are in close proximity
to, and may actually be in cell-to-cell contact with, neurons
expressing FKN (Fig. 2), in agreement with another model of neuronal
FKN signaling to microglia after neuronal injury (13).
Unlike HIVE, facial nerve axotomy is a model of microglial activation without a strong inflammatory response. Here FKN mRNA is decreased, and perineuronal microglia expressing the FKN receptor (CX3CR1) are increased (13). In this model it is unclear whether the total protein changes, but lower m.w. species (presumably secreted FKN) increase when measured by Western blot. Our data suggest that levels of FKN expression in neurons correlate with inflammation in patients with HIVE. To further explore the relationship between FKN mRNA and protein levels in areas of focal brain inflammation, it will be necessary to use animal models for lentiviral infection such as simian immunodeficiency virus (SIV) or the SCID mouse model of HIVE (28).
Our confocal data demonstrate that increased expression of FKN in
vesiculoid cytoplasmic structures in neurons in brain tissue with HIVE
are consonant with the idea that FKN, in addition to other chemokines
such as MCP-1 (31), may be another signal for monocyte
recruitment into the CNS. This hypothesis is confirmed by the data in
Fig. 5, which show that FKN is a potent chemoattractant in an in vitro
model of an endothelial cell/astrocyte bilayer. FKN's role as a
chemoattractant is confirmed by coincubation of FKN with neutralizing
mAbs to FKN with no subsequent monocyte migration across
endothelial/astrocyte transwell membranes (Fig. 5). Further studies are
necessary to resolve the relative contribution between soluble and
membrane-bound FKN in initiating monocyte recruitment and microglial
activation into the CNS.
The neuroprotective role of FKN is shown by the marked reduction in
neuronal apoptosis after coincubation of FKN with cPAF or Tat (Fig. 6).
Excessive production of PAF in the CNS is regulated by TNF- and
correlates with neurologic disease and immunosuppression (7, 29) and PAF may play a pivotal role in induction of neuronal
apoptosis via glutamatergic mechanisms (7). The HIV-1
regulatory protein Tat also induces neuronal apoptosis, in part through
a mechanism that involves production of TNF- and activation of
glutamatergic mechanisms (22, 24). These data, combined
with the ability to ameliorate gp120-induced neuronal apoptosis in rat
hippocampal cultures with and without glia (14), suggest
that FKN-mediated activation of CX3CR1 receptors
results in amelioration of neuronal apoptosis from structurally diverse
HIV-1 neurotoxins that signal through different pathways. Reduction of
neuronal apoptosis induced by either cPAF or Tat resulted from
coincubation with either soluble FKN or the C-terminal containing
peptide, suggesting that this region is important in neuroprotection.
Data from in vitro studies, and our data demonstrating colocalization
of FKN and glutamate in neurons in brain tissue of patients with HIVE,
suggest that FKN may function as a neuroprotective chemokine via a
paracrine mechanism. FKN may activate CX3CR1
receptors located directly on neurons as opposed to microglia, since
the rodent CGN cultures used in our experiments lack significant
numbers of astrocytes or microglia. One intriguing area for further
investigation is whether FKN regulates the release of presynaptic
glutamate and thus controls the amount of glutamate available in the
synapse to induce excitotoxic stimulation of vulnerable neurons.
These data suggest that, in HIVE, FKN may function as a neuroimmune modulator that is released from cytoplasmic neuronal structures with a vesicular appearance into the neuropil, and may participate in recruiting peripheral monocytes into the CNS, and possibly mediate cell-to-cell contact between FKN and neurons and microglia adjacent to focal inflammatory infiltrates. Despite the fact that macrophage recruitment into the CNS may increase the potential for productive HIV-1 infection, our data suggest that FKN may serve a neuroprotective role by interfering with HIV-1 neurotoxin induction of neuronal apoptosis. However, it is unlikely that FKN blocks neuronal apoptosis in HIVE by direct antagonism of HIV-1-induced neurotoxins, since PAF, Tat, and gp120 (14) can initiate neuronal apoptosis by distinct signaling pathways that may involve both glia and neurons. A more likely possibility is that FKN blocks apoptosis at a level that is downstream from neuronal or glial signaling by these neurotoxins or related intermediaries, possibly by interfering with glutamate release. Equally intriguing, FKN may promote production of glial protective factors that influence neuronal survival in neurons exposed to HIV-1 neurotoxins. Thus strategies to modulate or stimulate FKN signaling may have important therapeutic implications in the treatment of HIV-1 associated neurologic disease.
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Harris A. Gelbard, University of Rochester Medical Center, Box 631, 601 Elmwood Avenue, Rochester, NY 14642. E-mail address:
3 Abbreviations used in this paper: HIVE, HIV-1 encephalitis; ABC, avidin-biotin complex; PAF, platelet-activating factor; cPAF, carbamyl PAF; FKN, fractalkine; GFAP, glial fibrillary acidic protein; HIS, histidine; MP, mononuclear phagocytes; PE, progressive encephalopathy; PHFA, purified human fetal astrocytes; TRITC, tetramethylrhodamine isothiocyanate; ICC, immunocytochemistry; DAB, diaminobenzidine; ROD, relative OD.
Received for publication July 9, 1999. Accepted for publication November 11, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
inhibits glutamate uptake by primary human astrocytes: implications for pathogenesis of HIV-1 dementia. J. Biol. Chem. 271:15303. and MIP-1ß in human fetal microglia. J. Immunol 160:1449. and activation of non-NMDA receptors by a NF-
B-independent mechanism. J. Biol. Chem. 273:17852. release by lipopolysaccharide stimulated macrophages and microglia: implications for HIV-1 dementia. J. Neuroimmunol. 95:55.[Medline]
This article has been cited by other articles:
|
|
 |
|
  C. Lauro, S. Di Angelantonio, R. Cipriani, F. Sobrero, L. Antonilli, V. Brusadin, D. Ragozzino, and C. Limatola Activity of Adenosine Receptors Type 1 Is Required for CX3CL1-Mediated Neuroprotection and Neuromodulation in Hippocampal Neurons J. Immunol., June 1, 2008; 180(11): 7590 - 7596. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Zhang, G. J. Harry, P. J. Blackshear, and D. C. Zeldin G-protein Pathway Suppressor 2 (GPS2) Interacts with the Regulatory Factor X4 Variant 3 (RFX4_v3) and Functions as a Transcriptional Co-activator J. Biol. Chem., March 28, 2008; 283(13): 8580 - 8590. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Ishida, J.-L. Gao, and P. M. Murphy Chemokine Receptor CX3CR1 Mediates Skin Wound Healing by Promoting Macrophage and Fibroblast Accumulation and Function J. Immunol., January 1, 2008; 180(1): 569 - 579. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Lauro, M. Catalano, F. Trettel, F. Mainiero, M. T. Ciotti, F. Eusebi, and C. Limatola The Chemokine CX3CL1 Reduces Migration and Increases Adhesion of Neurons with Mechanisms Dependent on the beta1 Integrin Subunit J. Immunol., December 1, 2006; 177(11): 7599 - 7606. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Ancuta, J. Wang, and D. Gabuzda CD16+ monocytes produce IL-6, CCL2, and matrix metalloproteinase-9 upon interaction with CX3CL1-expressing endothelial cells. J. Leukoc. Biol., November 1, 2006; 80(5): 1156 - 1164. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Le Priol, D. Puthier, C. Lecureuil, C. Combadiere, P. Debre, C. Nguyen, and B. Combadiere High Cytotoxic and Specific Migratory Potencies of Senescent CD8+CD57+ Cells in HIV-Infected and Uninfected Individuals J. Immunol., October 15, 2006; 177(8): 5145 - 5154. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. R. Wallace, R. W. Vaughan, E. Kondeatis, R. Mathew, Y. Chen, E. M. Graham, and M. R. Stanford A CX3CR1 Genotype Associated with Retinal Vasculitis in Patients in the United Kingdom. Invest. Ophthalmol. Vis. Sci., July 1, 2006; 47(7): 2966 - 2970. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Yoshida, H. Hanawa, K. Toba, H. Watanabe, R. Watanabe, K. Yoshida, S. Abe, K. Kato, M. Kodama, and Y. Aizawa Expression of immunological molecules by cardiomyocytes and inflammatory and interstitial cells in rat autoimmune myocarditis Cardiovasc Res, November 1, 2005; 68(2): 278 - 288. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-C. Gevrey, B. M. Isaac, and D. Cox Syk Is Required for Monocyte/Macrophage Chemotaxis to CX3CL1 (Fractalkine) J. Immunol., September 15, 2005; 175(6): 3737 - 3745. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. H. Ahmed, R. Lutjens, L. D. van der Stap, D. Lekic, V. Romano-Spica, M. Morales, G. F. Koob, V. Repunte-Canonigo, and P. P. Sanna Gene expression evidence for remodeling of lateral hypothalamic circuitry in cocaine addiction PNAS, August 9, 2005; 102(32): 11533 - 11538. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. M. Mitrasinovic, A. Grattan, C. C. Robinson, N. B. Lapustea, C. Poon, H. Ryan, C. Phong, and G. M. Murphy Jr Microglia Overexpressing the Macrophage Colony-Stimulating Factor Receptor Are Neuroprotective in a Microglial-Hippocampal Organotypic Coculture System J. Neurosci., April 27, 2005; 25(17): 4442 - 4451. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Hoshino, D. B. Tse, G. Rochford, S. Prabhakar, S. Hoshino, N. Chitkara, K. Kuwabara, E. Ching, B. Raju, J. A. Gold, et al. Mycobacterium tuberculosis-Induced CXCR4 and Chemokine Expression Leads to Preferential X4 HIV-1 Replication in Human Macrophages J. Immunol., May 15, 2004; 172(10): 6251 - 6258. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. R. Latchney, M. A. Fallon, D. J. Culp, H. A. Gelbard, and S. Dewhurst Immunohistochemical Assessment of Fractalkine, Inflammatory Cells, and Human Herpesvirus 7 in Human Salivary Glands J. Histochem. Cytochem., May 1, 2004; 52(5): 671 - 682. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. O. Kim, Y. Liu, B. Y. Zhou, and J. J. He Induction of C Chemokine XCL1 (Lymphotactin/Single C Motif-1{alpha}/Activation-Induced, T Cell-Derived and Chemokine-Related Cytokine) Expression by HIV-1 Tat Protein J. Immunol., February 1, 2004; 172(3): 1888 - 1895. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
