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Regular Articles |
From the Department of Pathology,*
Division of
Neuropathology, University of Pittsburgh School of Medicine,
Pittsburgh, Pennsylvania; and the Department of Microbiology and
Immunology,
Oregon Health Sciences University,
Portland, Oregon
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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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The entry of HIV-1-infected monocytes into the CNS is hypothesized to occur through breaches in the blood-brain barrier (BBB). The BBB is composed of a network of continuous capillaries surrounded by a basal lamina and astrocytic foot processes.20 Under physiological conditions, this vascular barrier selectively regulates the intracellular and paracellular exchange of macromolecules and cells between the circulation and CNS through several unique structural and functional attributes. These include specialized endothelial membrane transport systems,21 limited endothelial pinocytosis, and lack of transendothelial fenestrae.22 Most distinct is the presence of high resistance interendothelial zonula occludens, or tight junctions.23,24 These intercellular belts consist of continuous, anastomosing intramembranous strands25 between the outer leaflets of adjacent cerebral endothelial cell membranes and are composed, in part, of a number of proteins, including the integral membrane protein, occludin26,27 and the peripheral membrane protein, zonula occludens-1 (ZO-1).28 Unlike systemic endothelial and epithelial tight junctions, cerebral endothelial tight junctions lack pore-like discontinuities,29 and, hence, create impermeable seals between the cells. In total, these barrier features serve to restrict the exchange between the microvasculature and CNS and, in particular, regulate inflammatory cell egress from the circulation.
During HIV-1 infection, however, multiple studies demonstrate that this strictly regulated exchange is compromised. In clinical studies of HIV-1-infected patients, tomographic analyses show perfusion defects30-32 compatible with cerebral vascular compromise, whereas both computed tomography and magnetic resonance imaging (MRI) studies reveal abnormal white matter signals33-35 that do not correspond to regions of demyelination.36 Elevated cerebrospinal fluid (CSF) markers of BBB damage, including increased CSF-serum albumin ratios,37,38 matrix metalloproteinase levels,39 and nitric oxide metabolites,40 also occur in HIV-1-infected patients. Serum protein extravasation through the BBB occurs in the brains of patients with HIV and HIVE,36,41,42 accompanied by abnormal neuronal and glial immunoreactivity for these proteins. In addition, morphological changes develop in the cerebral endothelium, including endothelial hypertrophy,43 membrane glycoprotein loss,44 and basal lamina thinning.45 Furthermore, functional alterations occur in the levels of the membrane glucose transporter-1,46 a barrier-related protein.
Although these observations clearly demonstrate that both structural and functional BBB perturbations occur during HIV-1 infection, the precise mechanism of these permeability changes and their relationship to the entry of HIV-1-infected monocytes into the CNS remain unclear. To gain insight into this issue, we examined tight junction integrity in autopsy CNS tissue from HIV-1-infected patients, both with and without encephalitis, as well as tissue from HIV-1-seronegative patients. Tight junctions were assessed immunohistochemically for changes in occludin and ZO-1 expression within the frontal cortex and basal ganglia, regions demonstrating histopathological features of HIVE and high HIV-1 burden and associated pathology.47 This expression was compared to that in the cerebellum, a region demonstrating no or minimal HIV-1 burden.47 These changes, in turn, were correlated with BBB permeability, astrocytosis, monocyte accumulation, and viral burden via assessment of fibrinogen, glial fibrillary acid protein (GFAP), CD68, and HIV-1 gp41 immunoreactivity. Our findings demonstrate that significant tight junction disruption, as demonstrated by fragmentation or absence of immunoreactivity for the proteins occludin and ZO-1, is a fundamental feature of HIVE. We further demonstrate that these BBB changes occur in regions of histopathological damage in concert with perivascular accumulation of activated, HIV-1-infected monocytes and microglia, serum protein extravasation, and abundant astrogliosis. We propose that disruption of this critical barrier structure serves as the primary portal of entry whereby activated HIV-1-infected monocytes gain access to the CNS.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six brains from patients with AIDS (mean age 34 ± 6 years; mean HIV-1 seropositivity 6 ± 3 years) and ten brains from patients with HIVE (mean age 40 ± 7 years; mean HIV-1 seropositivity 5 ± 4 years) were selected from our previous study set of 74 consecutive brain-inclusive AIDS autopsies between March, 1981 and July, 1996 at the Presbyterian University Hospital, University of Pittsburgh Medical Center.48 Clinical and pathological data were obtained from the patients’ medical records and autopsy reports, including the date of the first AIDS-defining illness (according to the 1987 Centers for Disease Control criteria). AIDS-dementia complex was defined by HIV-1 seropositivity accompanied by a clinical history and neurological findings of progressive cognitive and/or motor impairment in the absence of opportunistic infection. All selected patients with AIDS and AIDS-dementia complex were male. Risk factors included homosexuality (12), bisexuality (2), intravenous drug abuse (1), and blood transfusions (1). Causes of death in the AIDS group included pneumonia (5) and complications of disseminated Kaposi’s sarcoma (1). Causes of death in the AIDS-dementia complex group included pneumonia (7), multi-organ failure (2), and acute hemorrhage (1).
Nine brains from HIV-1-seronegative patients (mean age 52 ± 15 years) were selected as controls. Causes of death included cardiac arrhythmias or heart failure (3), diffuse alveolar damage (1), alcohol-induced cirrhosis (1), cerebral and pulmonary fat emboli (1), acute supporative meningitis (1), Pick’s disease (1), and complications of chronic multiple sclerosis (1).
Light Microscopy
Autopsies from patients with AIDS and AIDS-dementia complex were performed with a mean postmortem interval of 8.8 ± 6.2 hours and 12.3 ± 6.4 hours, respectively. Autopsies from control patients were performed with a mean postmortem interval of 13.4 ± 8.2 hours. Brains were fixed in 10% formalin for 10 to 14 days before sectioning. Samples from the following regions were paraffin-embedded and stained with hematoxylin and eosin for routine histopathological examination: mid-frontal cortex, caudate nucleus, insular cortex, basal ganglia, thalamus, hippocampus, cerebellum, midbrain, pons, medulla, and spinal cord. Microscopic examination and immunohistochemical stains revealed no evidence of opportunistic infection or neoplasms, including lymphoma. Sections from the mid-frontal cortex, basal ganglia, and cerebellum were then selected for this study.
Tissue sections from the cortical gray matter, subcortical and deep white matter, and deep gray matter were examined for the distribution and abundance of HIV-1 proteins by immunohistochemical staining for gp41, the transmembrane portion of the HIV-1 envelope protein, as previously described.48 In brief, levels of gp41 expression were assessed separately for each region (an average of five fields per region per 20x microscopic objective) and scored on a scale of 0 to 2 as follows: 0 = no cells stained for gp41, 1 = less than 2 cells stained for gp41, 2 = more than 2 cells stained for gp41. A composite score, derived by adding individual scores of the three regions, was scored on a scale of 0 to 6 as follows: 0–1 = absent to minimal HIV-1 burden, 2–3 = moderate HIV-1 burden, 4–6 = abundant HIV-1 burden. In sections from all patients with AIDS-dementia complex, gp41-immunoreactive cells with microglial or macrophage morphology were most prevalent in the deep gray matter (average gp41 score 2.0 ± 0.0), followed by the cortical white matter (average gp41 score 1.7 ± 0.7) and cortical gray matter (average gp41 score 0.9 ± 0.9). The gp41 composite score for all cases was 4.6 ± 1.2, with 90% of the cases demonstrating an abundant HIV-1 burden and 10% a moderate HIV-1 burden.
Immunoperoxidase Staining
Paraffin-embedded tissue sections were deparaffinized in
Histoclear (National Diagnostics, Atlanta, GA), rehydrated, and treated
with 3% hydrogen peroxide (Sigma Chemical Co., St. Louis, MO) for 30
minutes. Sections requiring antigen retrieval were treated with either
pepsin (0.4%; Dako Corporation, Carpinteria, CA) at 37°C for 10
minutes or Citra solution (Biogenex, San Ramon, CA) for 3 to 7 minutes
using the recommended microwave protocol. Sections were rinsed with
phosphate-buffered saline (PBS) and incubated in TNB blocking buffer
(DuPont NEN, Boston, MA) for 30 minutes. After overnight incubation at
4°C with primary antibody (Table 1)
,
the sections were rinsed in PBS and then incubated at room temperature
for 1 hour with either biotinylated goat anti-mouse immunoglobulin G
(IgG) serum (1:100; Caltag Laboratories, Burlingame, CA) or goat
anti-rabbit Ig (1:200; Biogenex). Immunostaining was then performed
using the tyramide signal amplification method according to the
manufacturer’s protocol (biotinylated tyramide, 1:150, 10 minutes;
TSA-Indirect, DuPont NEN). Chromagen reactions were developed with
3-amino-9-ethylcarbazole (AEC; Biogenex) and counterstained with
Mayer’s hematoxylin. Isotype-matched normal mouse serum was
used as a negative reagent control.
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Double Immunofluorescence Staining
Paraffin-embedded sections were treated with the same protocol as
described for immunoperoxidase staining. Following overnight incubation
at 4°C with the primary antibody (Table 1)
, the sections were rinsed
with PBS and incubated at room temperature for 1 hour with either
biotinylated goat anti-mouse immunoglobulin G (IgG) serum (1:100;
Caltag Laboratories) or goat anti-rabbit Ig (1:200; Biogenex).
Immunofluorescent staining was performed using the tyramide signal
amplification method according to the manufacturer’s protocol
tetramethylrhodamine isothiocyanate (TRITC)-labeled tyramide, 1:100, 10
minutes; excitation peak 570 nm; emission peak 590 nm; TSA-Direct,
DuPont NEN). Sections were then incubated with the second primary
antibody at room temperature for 2 hours. After rinsing in PBS,
sections were incubated at room temperature for 1 hour with either
fluorescein isothiocyanate (FITC)-labeled donkey anti-rabbit Ig (1:100;
Jackson ImmunoResearch Laboratories, West Grove, PA) or FITC-labeled
goat anti-mouse IgG (1:100; Jackson). Immunostaining was then performed
using the tyramide signal amplification method according to the
manufacturer’s protocol (FITC-labeled tyramide, 1:100, 10 minutes;
TSA-Indirect, DuPont NEN). Isotype-matched normal mouse serum
was used as a negative reagent control.
Confocal Microscopy
Double-labeled immunofluorescent sections were analyzed with a Molecular Dynamics laser scanning confocal microscope (Sunnyvale, CA) equipped with an argon/krypton laser, Nikon inverted microscope, and Plan Apo 20x 0.75 NA (air) and Plan Apo 60x 1.40 NA (oil) objective lenses. FITC and TRITC were excited by the laser’s 488-nm and 568-nm lines, respectively, which were delivered to the tissue sections by a 488/568 B/S primary dichroic beamsplitter. Fluorescent light emitted by FITC and TRITC was separated by a 565 B/S secondary dichroic beamsplitter and then passed through a 530DF30 filter and a 600DF40 filter, respectively. Images were collected with a Silicon Graphics Inc. computer (Operating System release 5.3, Farmington, MI) and analyzed using the Image Space software (version 3.2, Molecular Dynamics).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Blood vessels were examined immunohistochemically for changes in tight junction protein expression and distribution using antibodies to the tight junction markers, occludin, an integral membrane protein, and ZO-1, a peripheral membrane protein. In addition, vascular density was assessed by immunostaining for CD34, an endothelial cell marker.
All size blood vessels from all regions in control sections
demonstrated a strong, continuous interendothelial staining pattern
(Figure 1A)
of equal intensity when
stained for either tight junction protein (occludin immunostaining is
shown in Figure 1
; similar reactivity patterns were seen with ZO-1
immunostaining, data not shown). Although regional variations in
vascular density were observed (Figure 2A)
, no statistically significant
differences (P > 0.05) were noted in the mean
number of occludin- or ZO-1-reactive blood vessels (Figure 2, B and C)
compared to the mean number of CD34-reactive blood vessels within the
same region (Figure 2A)
.
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. Similarly, sections from HIV-1 patients without HIVE
demonstrated strong, continuous interendothelial expression of both
occludin and ZO-1 on all cerebellar blood vessels, as well as a
majority of vessels in the cortical gray matter, cortical white matter,
and deep gray matter. Scattered, isolated medium-sized blood vessels in
the cortical white matter, the globus pallidus and, less frequently,
the cortical gray matter, however, showed weak or fragmented expression
of these markers (Figure 1B)
. Single, weakly cross-reactive cells with
astrocytic morphology (Figure 1B)
surrounded occasional altered vessels
in regions of mild, diffuse gliosis. Overall, however, no statistically
significant difference (P > 0.05) was observed
in the mean number of occludin or ZO-1-reactive blood vessels in these
regions compared to the same regions in control sections (Figure 2, B and C)
.
In contrast, despite comparable regional vascular densities (Figure 2A)
, sections from patients with HIVE showed marked alterations in both
the intensity and staining pattern of occludin and ZO-1 when compared
to either control or HIV-1 sections. A majority of small and
medium-sized vessels in the cortical white matter and deep gray matter
showed either weak, fragmented expression or no expression of these
markers (Figure 1, C and E)
. Isolated, similar-sized vessels with
strong, continuous occludin or ZO-1 reactivity were often observed in
the vicinity (Figure 1E)
. Nonetheless, highly statistically significant
differences (P < 0.0001) were observed in the
mean number of occludin- or ZO-1-reactive blood vessels in these
regions compared to the same regions in either control or HIV-1
sections (Figure 2, B and C)
.
Numerous small and medium-sized vessels in the cortical gray matter
from patients with HIVE showed similar alterations in the intensity and
staining pattern of occludin or ZO-1, although to a lesser degree than
that observed in the cortical white matter and deep gray matter (Figure 1D)
. Compared to the mean number of occludin- or ZO-1-reactive blood
vessels in the cortical gray matter in either control or HIV-1
sections, however, very significant decreases (P
< 0.001) in expression of these markers were observed (Figure 2, B and C)
. On the other hand, only focal vascular alterations in occludin or
ZO-1 expression were observed in the cerebellar cortex or cerebellar
white matter in patients with HIVE. Likewise, no statistically
significant difference (P > 0.05) was observed
in the mean number of occludin or ZO-1-reactive blood vessels in these
regions compared to the same regions in either control or HIV-1
sections (Figure 2, B and C)
.
Histopathological features of HIVE were most pronounced in the deep
gray matter and cortical white matter, followed by the cortical gray
matter, and were, likewise, spatially associated with alterations in
the vascular expression of both occludin and ZO-1 (Figure 1, D–F)
.
Mononuclear cell aggregates and multinucleated giant cells were
frequently associated with vessels demonstrating alterations in tight
junction protein expression (Figure 1E)
. Microglial nodules (Figure 1D)
, present in 20% of the cases, were also seen adjacent to vessels
with weak, fragmented, or absent expression of either occludin or ZO-1.
In addition, microglial nodules occasionally engulfed blood vessels
with occludin- or ZO-1-positive vascular remnants (Figure 1F)
.
Tight Junction Protein Disruption Is Associated with Serum Protein Extravasation and Astrocytosis in HIVE
Sections were examined immunohistochemically for structural and functional alterations in BBB integrity using antibodies to occludin and ZO-1, fibrinogen, a serum protein that extravasates during BBB breakdown, and GFAP, an intermediate filament protein that increases in reactive astrocytes.
Blood vessels from control sections demonstrated strong, intravascular
fibrinogen immunoreactivity in association with strong, continuous
interendothelial reactivity for either tight junction protein. (ZO-1
immunostaining is shown in Figure 3A
and
throughout Figure 3
. Similar reactivity patterns were seen with
occludin immunostaining; data are not shown.) Although mild neuronal
cross-reactivity for fibrinogen was observed in the globus pallidus
(Figure 3C)
and cerebellar dentate nucleus, significant perivascular
fibrinogen extravasation was not observed in any region examined
(Figure 2D)
. GFAP-reactive, parenchymal, and perivascular astrocytes
and fibrillar cell processes were identified in the superficial cortex
and cerebellum and, less frequently, in the deep gray matter and
cortical white matter (Figure 3B)
. Alterations in tight
junction proteins or their colocalization with GFAP-reactive cells or
cell processes (Figure 3B)
were not observed in any region.
Furthermore, perivascular fibrinogen extravasation or its
colocalization with GFAP-reactive cells or cell processes was not
identified (Figure 3C)
.
|
. Scattered, isolated medium-sized blood
vessels in the cortical white matter, the deep gray matter, and, less
often, the cortical gray matter, however, showed tight junction protein
fragmentation in association with perivascular astrocytosis (Figure 3D)
. GFAP-reactive, hypertrophic astrocytes surrounding the majority of
these blood vessels colocalized focally with fragmented tight junction
proteins (Figure 3D)
, as well as extravasated fibrinogen (Figure 3F)
.
In addition, some GFAP-reactive, perivascular astrocytes in these
regions colocalized focally with occludin- or ZO-1-positive vessels
showing no apparent alterations in expression of these markers (Figure 3E)
.
In contrast, a majority of small and medium-sized blood vessels in the
cortical white matter and deep gray matter from HIVE sections showed
marked alterations in both the intensity and staining pattern of
occludin and ZO-1 in association with abundant, perivascular fibrinogen
extravasation, diffuse parenchymal fibrinogen immunoreactivity, and
diffuse astrocytosis. Vessels with minimal to absent tight junction
protein expression showed marked perivascular fibrinogen extravasation
in these regions (Figure 3G)
. Similarly, vessels with disrupted tight
junction proteins were accompanied by marked, diffuse perivascular and
parenchymal astrocytosis (Figure 3H)
. GFAP-reactive hypertrophic
astrocytes surrounding permeable blood vessels colocalized extensively
with fibrinogen (Figure 3I)
, but not with occludin- or ZO-1-positive
remnants (Figure 3H)
. The cortical gray matter showed similar, but less
extensive, changes. All of these regions, however, demonstrated highly
statistically significant decreases (P 
0.0001) in the mean number of blood vessels containing intravascular
fibrinogen compared to the same regions in control or HIV-1 sections
(Figure 2D)
.
Blood vessels in the cerebellar cortex and white matter from HIVE
sections, on the other hand, showed only focal changes in tight
junction protein integrity, accompanied by focal vascular fibrinogen
permeability and perivascular astrocytosis. The extent of these
alterations was similar to that seen in the cortical gray matter in
HIV-1 sections. Likewise, no statistically significant difference
(P > 0.05) was observed in the mean number of
cerebellar blood vessels containing intravascular fibrinogen compared
to the same regions in control or HIV-1 sections (Figure 2D)
.
Tight Junction Protein Disruption and BBB Permeability Are Associated with Mononuclear Cell Infiltrates and Microglial Nodules in HIVE
Sections were examined immunohistochemically for BBB alterations and their relationship to mononuclear cell and microglial aggregates using antibody to CD68, a marker for peripheral blood monocytes and tissue macrophages, including microglia. In addition, sections were analyzed for the expression of HLA-DR, a Class II major histocompatibility complex (MHC) activation marker, as well as gp41, an HIV-1 envelope protein expressed by a percentage of circulating viral-infected monocytes during HIV-1 infection.
All regions in control sections contained a small number of round
CD68-reactive cells and short, CD68-reactive cell processes scattered
throughout the parenchyma (data not shown). Infrequent blood vessels
walls contained large CD68-positive infiltrating mononuclear cells that
colocalized with tight junction proteins (ZO-1 immunostaining is shown
in Figure 4A
and throughout Figures 4 and 5
; similar reactivity patterns were seen
with occludin immunostaining, data not shown) and expressed Class II
MHC molecules (Figure 5A)
. Less often, flattened periadventitial cells
that did not colocalize with tight junction proteins were identified
adjacent to the abluminal aspect of blood vessel walls. No alterations
in tight junction protein expression (Figure 4A)
or fibrinogen
permeability (data not shown) were observed in any vessel wall. In
addition, no viral protein expression was detected in any parenchymal
or intravascular cell (data not shown).
|
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within
both the cortex and cerebellum. Furthermore, scattered gp41-positive
cells were observed within the blood vessels walls and parenchyma of
all sections examined (Figure 5D)
. Blood vessels that showed no
alterations in tight junction protein expression or fibrinogen
permeability were variably associated with CD68-positive (Figure 4B)
or
gp41-positive cells (Figure 5D)
. On the other hand, CD68-positive
cells, both isolated and in small clusters, were consistently
associated with scattered cerebral vessels showing tight junction
protein fragmentation (Figure 4C)
or fibrinogen permeability (Figure 4G)
. Despite expression by these cells of Class II MHC molecules,
however, no overall increase in HLA-DR expression was observed in any
region compared to the same region in control sections (Figure 5B)
.
Compared to both HIV-1 and control sections, HIVE sections demonstrated
a marked increase in parenchymal and perivascular CD68-positive cells,
including CD68-reactive microglial nodules, within the cortical white
matter, the deep gray matter, and, to a lesser extent, the cortical
gray matter (Figure 4D)
. Cerebellar sections, on the other hand,
contained only a slightly greater number of immunoreactive cells than
that observed in all HIV-1 sections (data not shown). In all regions,
blood vessels with no alterations in tight junction protein expression
or fibrinogen permeability were variably associated with CD68-positive
cells (data not shown), similar to that observed in HIV-1 sections. In
contrast, tight junction-disrupted and fibrinogen-permeable blood
vessels were consistently associated with CD68-positive mononuclear
cell aggregates or microglial nodules (Figure 4, D and H)
. Many of
these disrupted blood vessels were surrounded, often asymmetrically, by
clusters of hypertrophic, periadventitial CD68-reactive cells that
variably colocalized with tight junction protein remnants (Figure 4E)
.
More often, fragmented and permeable blood vessel walls were pavemented
or infiltrated by colocalizing CD68-positive mononuclear cells, often
in aggregates (Figure 4, F and I)
. A marked increase in Class II MHC
expression was observed in these vascular- and
perivascular-associated cells, as well as short arborizing cell
processes within the surrounding parenchyma (Figure 5C)
.
In contrast to the regional differences in CD68 reactivity, a similar
difference was not observed in the number of gp41-positive cells within
the cortex and cerebellum of HIVE sections (Figure 5, F and G)
.
Although both areas contained a greater number of gp41-positive cells
compared to HIV-1 sections, significant tight junction protein
alterations were not observed in vessels within the cerebellum, despite
focal vascular infiltration by gp41-positive cells (Figure 5G)
.
Furthermore, viral proteins were not consistently observed among
fragmented (Figure 5F)
or permeable vessels of the cerebrum (Figure 5, H and I)
, despite focal colocalization with CD68-reactive mononuclear
cells within blood vessel walls (Figure 5E)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results clearly demonstrate that tight junction alterations correlate with serum protein extravasation, underscoring the importance of this critical structure in maintaining the relative impermeability of the BBB. The demonstration of widespread serum protein extravasation in HIVE is in agreement with previous reports36 but in contrast to others, which did not detect a difference in BBB permeability between brain tissue from HIV-1-infected patients with and without encephalitis.42 This disparity may be a reflection of the more sensitive immunohistochemical techniques employed in this study. The focal tight junction disruption and serum protein extravasation observed in the frontal cortex and basal ganglia from HIV-infected patients contrasts sharply with the extensive changes seen in these regions from HIV-infected patients with encephalitis. These findings support the idea that BBB permeability changes develop slowly and precede the onset of HIVE,36 which occurs late in the course of infection. These findings also suggest that permeability changes may contribute directly to the CNS injury observed in HIVE. Distention of the extracellular space by vasogenic edema may contribute to alterations in the ionic milieu and, in turn, cellular membrane function.36
In addition, the development of astrocytosis, a prominent feature that
accompanied tight junction and permeability alterations in this study,
may play a dual role in both protecting the CNS from BBB-induced
alterations and augmenting loss of BBB integrity. Astrocytosis is
consistently reported as the earliest neuropathologic change in the CNS
of HIV-1-infected patients.53
The relationship between
focal BBB compromise and perivascular astrocytosis in HIV-1 tissue in
this report suggests that this reactive change is triggered by loss of
BBB integrity and serves, initially, to wall off impending damage to
the CNS microenvironment. Reactive astrocytes, however, are potent
mediators of vascular permeability-inducing cytokines, including tumor
necrosis factor-
(TNF-) and interleukin-6 (IL-6), which may
augment permeability changes with time.54,55
Furthermore,
under physiological conditions, astrocytes play a critical role in
maintaining tight junction integrity,56
a role that may be
compromised during gliosis. These possibilities are intriguing in light
of the observation that focal overlap of GFAP-reactive processes and
tight junction proteins was observed in vessels without light
microscopic evidence of BBB perturbations in tissue from HIV-1-infected
patients without encephalitis.
On the other hand, the presence of comparable numbers of gp41-infected monocytes within both affected and unaffected cerebral and cerebellar vessels in HIVE strongly suggests that the presence of this HIV-1 envelope protein, by itself, is not sufficient for tight junction modulation or permeability alterations during this disease. Other HIV-1 proteins, however, may play a role in compromising tight junction integrity, as supported by the demonstration that the HIV-1 envelope protein, gp120, can induce intercellular gaps in rat brain endothelium through a mechanism that may involve substance P.57 Although the direct transmission of HIV-1 through endothelial cells of the BBB remains controversial,51,58-61 reports of gp120 adsorptive endocytosis across cerebral endothelial cells in vitro62,63 suggest that this pathway may also constitute a significant route of viral entry into the CNS.
Our findings, on the other hand, lend credence to the hypothesis that
HIV-1-infected monocytes play an essential role in breaching the
BBB,51,52,64
thus, facilitating both
neurotoxicity14-18
and viral transmission into the
CNS.51,65
Several lines of evidence indicate that the local
production of monocyte-generated cytokines, in particular TNF-, are
the most likely candidates for mediating tight junction disruption
after the adherence of activated monocytes to the cerebral vascular
lining. TNF- and interferon-
induce a striking fragmentation of
ZO-1 via F actin rearrangement in cultures of microvascular endothelial
cells.66
Indeed, high levels of these cytokines are present
in the CNS of patients with HIV-1-associated dementia
complex.67
In addition, activated monocytes adhere and
migrate readily through interendothelial gaps in artificial BBB
systems, in association with their production of high levels of
pro-inflammatory cytokines, including TNF-.52,68
In addition, the cerebral endothelial lining itself may play an important role in determining sites of inflammation through its phenotypic and antigenic diversity. This is supported by our finding that tight junction alterations were confined to small and medium-sized vessels within the subcortical white matter and basal ganglia in HIVE. An accumulating body of evidence strongly indicates that the selective expression of adhesion molecules by different caliber vessels within different regions of the CNS may contribute to site-specific inflammation observed in various encephalitides. In studies of HIV-1-infected monocytes, up-regulation of endothelial E-selectin and VCAM-1 by these activated cells and their soluble products promotes their avid adherence to cerebral endothelial cells in vitro.51 Furthermore, capillary up-regulation of these molecules occurs in HIVE in association with infiltrating mononuclear cells, in contrast to ubiquitous up-regulation of ICAM-1 by both endothelial and parenchymal cells.51
In summary, the results of this study demonstrate that significant, site-specific alterations in tight junction integrity occur during HIVE and are associated with marked BBB permeability, activated HIV-1-infected monocyte accumulation, and astrocytosis. Further investigation of the precise molecules and mechanisms that disrupt this critical barrier structure may be of value in abrogating CNS injury, as well as the paracellular migration of activated HIV-1 monocytes in the CNS, critical events in the pathogenesis of HIV-1-associated dementia.
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Footnotes |
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Supported by National Institutes of Health grant NS 35419 to C. L. A.
Accepted for publication August 24, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 J. D. Morrey, A. L. Olsen, V. Siddharthan, N. E. Motter, H. Wang, B. S. Taro, D. Chen, D. Ruffner, and J. O. Hall Increased blood brain barrier permeability is not a primary determinant for lethality of West Nile virus infection in rodents J. Gen. Virol., February 1, 2008; 89(2): 467 - 473. [Abstract] [Full Text] [PDF]
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G. Schreibelt, G. Kooij, A. Reijerkerk, R. van Doorn, S. I. Gringhuis, S. van der Pol, B. B. Weksler, I. A. Romero, P.-O. Couraud, J. Piontek, et al. Reactive oxygen species alter brain endothelial tight junction dynamics via RhoA, PI3 kinase, and PKB signaling FASEB J, November 1, 2007; 21(13): 3666 - 3676. [Abstract] [Full Text] [PDF]
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 E. Behling-Kelly, D. McClenahan, K. S. Kim, and C. J. Czuprynski Viable "Haemophilus somnus" Induces Myosin Light-Chain Kinase-Dependent Decrease in Brain Endothelial Cell Monolayer Resistance Infect. Immun., September 1, 2007; 75(9): 4572 - 4581. [Abstract] [Full Text] [PDF]
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A. Reijerkerk, G. Kooij, S. M. A. van der Pol, S. Khazen, C. D. Dijkstra, and H. E. de Vries Diapedesis of monocytes is associated with MMP-mediated occludin disappearance in brain endothelial cells FASEB J, December 1, 2006; 20(14): 2550 - 2552. [Abstract] [Full Text] [PDF]
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M. Toborek Do not Rho tight junctions (or they become leaky) Blood, June 15, 2006; 107(12): 4581 - 4581. [Full Text] [PDF]
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Y. Persidsky, D. Heilman, J. Haorah, M. Zelivyanskaya, R. Persidsky, G. A. Weber, H. Shimokawa, K. Kaibuchi, and T. Ikezu Rho-mediated regulation of tight junctions during monocyte migration across the blood-brain barrier in HIV-1 encephalitis (HIVE) Blood, June 15, 2006; 107(12): 4770 - 4780. [Abstract] [Full Text] [PDF]
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 E. A. Eugenin, R. Gamss, C. Buckner, D. Buono, R. S. Klein, E. E. Schoenbaum, T. M. Calderon, and J. W. Berman Shedding of PECAM-1 during HIV infection: a potential role for soluble PECAM-1 in the pathogenesis of NeuroAIDS. J. Leukoc. Biol., March 1, 2006; 79(3): 444 - 452. [Abstract] [Full Text] [PDF]
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E. A. Eugenin, K. Osiecki, L. Lopez, H. Goldstein, T. M. Calderon, and J. W. Berman CCL2/Monocyte Chemoattractant Protein-1 Mediates Enhanced Transmigration of Human Immunodeficiency Virus (HIV)-Infected Leukocytes across the Blood-Brain Barrier: A Potential Mechanism of HIV-CNS Invasion and NeuroAIDS J. Neurosci., January 25, 2006; 26(4): 1098 - 1106. [Abstract] [Full Text] [PDF]
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J. Haorah, B. Knipe, J. Leibhart, A. Ghorpade, and Y. Persidsky Alcohol-induced oxidative stress in brain endothelial cells causes blood-brain barrier dysfunction J. Leukoc. Biol., December 1, 2005; 78(6): 1223 - 1232. [Abstract] [Full Text] [PDF]
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 B. T. Hawkins and T. P. Davis The Blood-Brain Barrier/Neurovascular Unit in Health and Disease Pharmacol. Rev., June 1, 2005; 57(2): 173 - 185. [Abstract] [Full Text] [PDF]
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 H. Wen, D. D. Watry, M. C. G. Marcondes, and H. S. Fox Selective Decrease in Paracellular Conductance of Tight Junctions: Role of the First Extracellular Domain of Claudin-5 Mol. Cell. Biol., October 1, 2004; 24(19): 8408 - 8417. [Abstract] [Full Text] [PDF]
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Y. C. Chang, M. F. Stins, M. J. McCaffery, G. F. Miller, D. R. Pare, T. Dam, M. Paul-Satyasee, K. S. Kim, and K. J. Kwon-Chung Cryptococcal Yeast Cells Invade the Central Nervous System via Transcellular Penetration of the Blood-Brain Barrier Infect. Immun., September 1, 2004; 72(9): 4985 - 4995. [Abstract] [Full Text] [PDF]
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Y. Persidsky and H. E. Gendelman Mononuclear phagocyte immunity and the neuropathogenesis of HIV-1 infection J. Leukoc. Biol., November 1, 2003; 74(5): 691 - 701. [Abstract] [Full Text] [PDF]
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 D. Zenker, D. Begley, H. Bratzke, H. Rubsamen-Waigmann, and H. von Briesen Human blood-derived macrophages enhance barrier function of cultured primary bovine and human brain capillary endothelial cells J. Physiol., September 15, 2003; 551(3): 1023 - 1032. [Abstract] [Full Text] [PDF]
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 X. Luo, K. A. Carlson, V. Wojna, R. Mayo, T. M. Biskup, J. Stoner, J. Anderson, H. E. Gendelman, and L. M. Melendez Macrophage proteomic fingerprinting predicts HIV-1-associated cognitive impairment Neurology, June 24, 2003; 60(12): 1931 - 1937. [Abstract] [Full Text] [PDF]
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 J. D. Huber, V. S. Hau, L. Borg, C. R. Campos, R. D. Egleton, and T. P. Davis Blood-brain barrier tight junctions are altered during a 72-h exposure to lambda -carrageenan-induced inflammatory pain Am J Physiol Heart Circ Physiol, October 1, 2002; 283(4): H1531 - H1537. [Abstract] [Full Text] [PDF]
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J. D. Huber, K. A. Witt, S. Hom, R. D. Egleton, K. S. Mark, and T. P. Davis Inflammatory pain alters blood-brain barrier permeability and tight junctional protein expression Am J Physiol Heart Circ Physiol, March 1, 2001; 280(3): H1241 - H1248. [Abstract] [Full Text] [PDF]
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Y. Persidsky, J. Zheng, D. Miller, and H. E. Gendelman Mononuclear phagocytes mediate blood-brain barrier compromise and neuronal injury during HIV-1-associated dementia J. Leukoc. Biol., September 1, 2000; 68(3): 413 - 422. [Abstract] [Full Text] [PDF]
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