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Blood-Brain Barrier Tight Junction Disruption in
Human Immunodeficiency Virus-1 Encephalitis
Linda M. Dallasta,* Liubomir A. Pisarov,*
James E. Esplen,* Jonette V. Werley,*
Ashlee V. Moses,
†
Jay A. Nelson,
†
and
Cristian L. Achim*
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
The blood-brain barrier (BBB) plays a critical role in
regulating cell trafficking through the central nervous
system (CNS) due to several unique anatomical fea-
tures, including the presence of interendothelial tight
junctions that form impermeable seals between the
cells. Previous studies have demonstrated BBB pertur-
bations during human immunodeficiency virus en-
cephalitis (HIVE); however, the basis of these perme-
ability changes and its relationship to infiltration of
human immunodeficiency virus type 1 (HIV-1)-in-
fected monocytes, a critical event in the pathogenesis
of the disease, remains unclear. In this study, we
examined CNS tissue from HIV-1-seronegative pa-
tients and HIV-1-infected patients, both with and
without encephalitis, for alterations in BBB integrity
via immunohistochemical analysis of the tight junc-
tion membrane proteins, occludin and zonula occlu-
dens-1 (ZO-1). Significant tight junction disruption
(P<0.001), as demonstrated by fragmentation or
absence of immunoreactivity for occludin and ZO-1,
was observed within vessels from subcortical white
matter, basal ganglia, and, to a lesser extent, cortical
gray matter in patients who died with HIVE. These
alterations were also associated with accumulation of
activated, HIV-1-infected brain macrophages, fibrino-
gen leakage, and marked astrocytosis. In contrast, no
significant changes (P>0.05) were observed in cer-
ebellar tissue from patients with HIVE compared to
HIV-seronegative patients or HIV-1-infected patients
without encephalitis. Our findings demonstrate that
tight junction disruption is a key feature of HIVE that
occurs in regions of histopathological alterations in
association with perivascular accumulation of acti-
vated HIV-1-infected macrophages, serum protein ex-
travasation, and marked astrocytosis. We propose
that disruption of this key BBB structure serves as the
main route of HIV-1-infected monocyte entry into the
CNS. (Am J Pathol 1999, 155:1915–1927)
Human immunodeficiency virus type 1 (HIV-1)-associ-
ated dementia complex
1
is a syndrome of progressive
motor, cognitive, and behavioral impairment that occurs
in a significant number of patients with acquired immu-
nodeficiency syndrome (AIDS) late in the course of their
infection.
2,3
The neuropathological correlate, HIV-1 en-
cephalitis (HIVE),
4
is characterized by subcortical and
neocortical damage within the white and gray matter.
5,6
Pathological hallmarks include the formation of multinu-
cleated giant cells and microglial nodules, as well as a
striking perivascular and parenchymal accumulation of
macrophages.
7
In addition, widespread reactive astrocy-
tosis, diffuse white matter pallor, and varying degrees of
neuronal injury and loss are present. These changes are
associated with both increasing CNS viral burden,
8
as
well as the presence of activated, HIV-1-infected infiltrat-
ing macrophages and resident microglia, the primary
central nervous system (CNS) targets of HIV-1.
9–12
Al-
though viral expansion is requisite for the development of
HIVE, recent studies indicate that the number of activated
macrophages and microglia may also serve as a good
indicator for the level of neurological dysfunction.
13
In-
deed, the vast array of secretory products released by
these activated cells is now considered to be the basis of
HIV-1-associated neurotoxicity.
14–19
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 condi-
tions, this vascular barrier selectively regulates the intra-
cellular and paracellular exchange of macromolecules
and cells between the circulation and CNS through sev-
eral unique structural and functional attributes. These
include specialized endothelial membrane transport sys-
tems,
21
limited endothelial pinocytosis, and lack of trans-
endothelial 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 strands
25
between the outer leaflets of adjacent cerebral endothe-
lial cell membranes and are composed, in part, of a
number of proteins, including the integral membrane pro-
Supported by National Institutes of Health grant NS 35419 to C. L. A.
Accepted for publication August 24, 1999.
Address reprint requests to Linda M. Dallasta, M.D., Ph.D., Division of
Neuropathology, Presbyterian University Hospital, A-515, 200 Lothrop
Street, Pittsburgh, PA 15213-2582. E-mail: dallasta@np.awing.upmc.edu.
American Journal of Pathology, Vol. 155, No. 6, December 1999
Copyright © American Society for Investigative Pathology
1915
tein, occludin
26,27
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 be-
tween the microvasculature and CNS and, in particular,
regulate inflammatory cell egress from the circulation.
During HIV-1 infection, however, multiple studies dem-
onstrate that this strictly regulated exchange is compro-
mised. In clinical studies of HIV-1-infected patients, to-
mographic analyses show perfusion defects
30–32
compatible with cerebral vascular compromise, whereas
both computed tomography and magnetic resonance
imaging (MRI) studies reveal abnormal white matter sig-
nals
33–35
that do not correspond to regions of demyeli-
nation.
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 pa-
tients. Serum protein extravasation through the BBB oc-
curs in the brains of patients with HIV and HIVE,
36,41,42
accompanied by abnormal neuronal and glial immunore-
activity 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, func-
tional 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 pa-
tients, 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 histopatholog-
ical features of HIVE and high HIV-1 burden and associ-
ated 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 accumu-
lation, and viral burden via assessment of fibrinogen, glial
fibrillary acid protein (GFAP), CD68, and HIV-1 gp41
immunoreactivity. Our findings demonstrate that signifi-
cant tight junction disruption, as demonstrated by frag-
mentation 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.
Materials and Methods
Tissues
Six brains from patients with AIDS (mean age 34 66
years; mean HIV-1 seropositivity 6 63 years) and ten
brains from patients with HIVE (mean age 40 67 years;
mean HIV-1 seropositivity 5 64 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 Dis-
ease Control criteria). AIDS-dementia complex was de-
fined 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-de-
mentia complex were male. Risk factors included homo-
sexuality (12), bisexuality (2), intravenous drug abuse (1),
and blood transfusions (1). Causes of death in the AIDS
group included pneumonia (5) and complications of dis-
seminated 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 52 615 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 suppo-
rative meningitis (1), Pick’s disease (1), and complica-
tions of chronic multiple sclerosis (1).
Light Microscopy
Autopsies from patients with AIDS and AIDS-dementia
complex were performed with a mean postmortem inter-
val of 8.8 66.2 hours and 12.3 66.4 hours, respectively.
Autopsies from control patients were performed with a
mean postmortem interval of 13.4 68.2 hours. Brains
were fixed in 10% formalin for 10 to 14 days before
sectioning. Samples from the following regions were par-
affin-embedded and stained with hematoxylin and eosin
for routine histopathological examination: mid-frontal cor-
tex, caudate nucleus, insular cortex, basal ganglia, thal-
amus, hippocampus, cerebellum, midbrain, pons, me-
dulla, and spinal cord. Microscopic examination and
immunohistochemical stains revealed no evidence of op-
portunistic 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, subcor-
tical 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 expres-
sion were assessed separately for each region (an aver-
age of five fields per region per 203microscopic objec-
1916 Dallasta et al
AJP December 1999, Vol. 155, No. 6
tive) and scored on a scale of 0 to 2 as follows: 0 5no
cells stained for gp41, 1 5less than 2 cells stained for
gp41, 2 5more than 2 cells stained for gp41. A com-
posite score, derived by adding individual scores of the
three regions, was scored on a scale of 0 to 6 as follows:
0–1 5absent to minimal HIV-1 burden, 2–3 5moderate
HIV-1 burden, 4 – 6 5abundant HIV-1 burden. In sections
from all patients with AIDS-dementia complex, gp41-im-
munoreactive cells with microglial or macrophage mor-
phology were most prevalent in the deep gray matter
(average gp41 score 2.0 60.0), followed by the cortical
white matter (average gp41 score 1.7 60.7) and cortical
gray matter (average gp41 score 0.9 60.9). The gp41
composite score for all cases was 4.6 61.2, 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), rehy-
drated, 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 pro-
tocol. Sections were rinsed with phosphate-buffered sa-
line (PBS) and incubated in TNB blocking buffer (DuPont
NEN, Boston, MA) for 30 minutes. After overnight incu-
bation at 4°C with primary antibody (Table 1), the sec-
tions 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 us-
ing the tyramide signal amplification method according to
the manufacturer’s protocol (biotinylated tyramide, 1:150,
10 minutes; TSA-Indirect, DuPont NEN). Chromagen re-
actions were developed with 3-amino-9-ethylcarbazole
(AEC; Biogenex) and counterstained with Mayer’s hema-
toxylin. Isotype-matched normal mouse serum was used
as a negative reagent control.
Sections were analyzed semiquantitatively with an
Olympus BX40F-3 microscope (Tokyo, Japan) and pho-
tographed with an Olympus SC35 camera. Vessels dem-
onstrating strong, continuous interendothelial reactivity
for either occludin or ZO-1 were considered positive, and
vessels demonstrating weak, fragmented, or no expres-
sion of either occludin or ZO-1 were considered negative.
An average of five fields of blood vessels was counted
separately for each region with a micrometer and a 203
microscopic objective. Sections from a patient with
chronic multiple sclerosis showed no tight junction pro-
tein expression on blood vessels within burned-out
plaques, and, thus, served as a negative control. Strong
expression of both markers was present on vessels in the
surrounding, uninvolved parenchyma, however, and
these areas were used for quantification. Blood vessels
showing CD34 reactivity or intravascular fibrinogen reac-
tivity without extravasation were counted in a similar man-
ner. Statistical analysis was performed using the un-
paired Student’s t-test, where two-tailed Pvalues ,0.05
were considered significant, Pvalues ,0.01 were con-
sidered very significant, and Pvalues ,0.0001 were
considered highly significant.
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
Table 1. Panel of Antibodies Used for Immunohistochemistry
Antibody Specificity Manufacturer Host/class Dilution
Occludin Integral membrane protein of
epithelial and endothelial
tight junctions (26)*
Zymed, San Francisco, CA Rabbit Ig 1;250
ZO-1 Peripheral membrane protein of
epithelial and endothelial
tight junctions (28, 49)* and
adherens junctions (50)*
Zymed Rabbit Ig 1;250
Fibrinogen Plasma protein Dako, Carpinteria, CA Rabbit Ig 1;10,000
Human Glial Fibrillary
Acidic Protein
(GFAP)
Astrocytes, some ependymal
cells
Dako (Clone 6F2) Mouse IgG
1
1;50
CD68 Peripheral blood mononuclear
cells and tissue
macrophages, including
microglia
Dako (Clone PG-M1) Mouse IgG
3
1;100
Human CD34 Endothelial cells, hematopoietic
cells, collagen IV
Biogenex, San Ramon, CA
(Clone QBEnd/10)
Mouse IgG
1
Neat
HLA-DR Major histocompatibility Class II
antigens
Pharmingen, San Diego, CA
(Clone 4U39)
Mouse IgG
2a
1;50
gp41 Transmembrane portion of HIV-
1 envelope protein
Genetic Systems, Seattle,
WA
Mouse IgG
1
1;750
*Numbers in parentheses correspond to references.
Tight Junction Disruption in HIV Encephalitis 1917
AJP December 1999, Vol. 155, No. 6
biotinylated goat anti-mouse immunoglobulin G (IgG) se-
rum (1:100; Caltag Laboratories) or goat anti-rabbit Ig
(1:200; Biogenex). Immunofluorescent staining was per-
formed using the tyramide signal amplification method
according to the manufacturer’s protocol tetramethylrho-
damine isothiocyanate (TRITC)-labeled tyramide, 1:100,
10 minutes; excitation peak 570 nm; emission peak 590
nm; TSA-Direct, DuPont NEN). Sections were then incu-
bated with the second primary antibody at room temper-
ature 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 ana-
lyzed with a Molecular Dynamics laser scanning confocal
microscope (Sunnyvale, CA) equipped with an argon/
krypton laser, Nikon inverted microscope, and Plan Apo
2030.75 NA (air) and Plan Apo 6031.40 NA (oil) ob-
jective lenses. FITC and TRITC were excited by the la-
ser’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 di-
chroic beamsplitter and then passed through a 530DF30
filter and a 600DF40 filter, respectively. Images were
collected with a Silicon Graphics Inc. computer (Operat-
ing System release 5.3, Farmington, MI) and analyzed
using the Image Space software (version 3.2, Molecular
Dynamics).
Results
The Distribution of Tight Junction Proteins Is
Disrupted in HIVE
Blood vessels were examined immunohistochemically for
changes in tight junction protein expression and distribu-
tion using antibodies to the tight junction markers, occlu-
din, an integral membrane protein, and ZO-1, a periph-
eral 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 sec-
tions demonstrated a strong, continuous interendothelial
staining pattern (Figure 1A) of equal intensity when
stained for either tight junction protein (occludin immu-
nostaining is shown in Figure 1; similar reactivity patterns
were seen with ZO-1 immunostaining, data not shown).
Although regional variations in vascular density were ob-
served (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) com-
pared to the mean number of CD34-reactive blood ves-
sels within the same region (Figure 2A).
Sections from HIV-1 patients without HIVE showed no
differences in vascular density when compared to the
same regions in control sections (Figure 2A). 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 cor-
tical 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 sig-
nificant 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 den-
sities (Figure 2A), sections from patients with HIVE
showed marked alterations in both the intensity and stain-
ing 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 ex-
pression or no expression of these markers (Figure 1, C
and E). Isolated, similar-sized vessels with strong, con-
tinuous 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 ves-
sels 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 cor-
tical 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 expres-
sion were observed in the cerebellar cortex or cerebellar
white matter in patients with HIVE. Likewise, no statisti-
cally 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 pro-
nounced in the deep gray matter and cortical white mat-
ter, followed by the cortical gray matter, and were, like-
wise, spatially associated with alterations in the vascular
expression of both occludin and ZO-1 (Figure 1, D–F).
Mononuclear cell aggregates and multinucleated giant
1918 Dallasta et al
AJP December 1999, Vol. 155, No. 6
Figure 1. The distribution of tight junction proteins is disrupted in HIVE. A: In control sections, blood vessels show strong immunoreactivity for the tight
junction-associated protein, occludin, in a continuous, interendothelial staining pattern. B: In HIV sections, isolated medium-sized vessels are weakly immuno-
reactive for occludin (arrowhead). C–F: In HIVE sections, numerous small and medium-sized vessels exhibit decreased to absent occludin immunoreactivity (C)
in association with microglial nodules (Dand F), mononuclear cell infiltrates (Dand E), and multinucleated giant cells (E). Similar changes were observed in
ZO-1-immunostained sections. Dand E: Original magnification, 3200. A–C and F: Original magnification, 3400. CGM, cortical gray matter; CWM, cortical white
matter; DGM, deep gray matter.
Tight Junction Disruption in HIV Encephalitis 1919
AJP December 1999, Vol. 155, No. 6
cells were frequently associated with vessels demon-
strating 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 occa-
sionally 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 asso-
ciation 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 immunostain-
ing; data are not shown.) Although mild neuronal cross-
reactivity for fibrinogen was observed in the globus pal-
lidus (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). Alter-
ations in tight junction proteins or their colocalization
with GFAP-reactive cells or cell processes (Figure 3B)
were not observed in any region. Furthermore, perivas-
cular fibrinogen extravasation or its colocalization with
GFAP-reactive cells or cell processes was not identi-
fied (Figure 3C).
Similarly, a majority of blood vessels from all HIV-1
sections showed strong, continuous interendothelial re-
activity for the tight junction markers, occludin or ZO-1, in
association with strong, intravascular immunoreactivity
for fibrinogen. No statistically significant difference (P.
0.05) in vascular fibrinogen permeability was observed in
any region compared to the same regions in control
sections (Figure 2D). Scattered, isolated medium-sized
Figure 2. Semiquantitative analysis of blood vessel immunoreactivity for the endothelial cell marker, CD34 (A), the tight junction proteins, occludin (B) and ZO-1
(C), and the intravascular serum protein, fibrinogen (D), in sections from control, HIV, and HIVE brains (average and standard error of five fields of blood vessels
per 203objective). Two-tailed Pvalues of ,0.01 (*) and ,0.0001 (**) are noted.
1920 Dallasta et al
AJP December 1999, Vol. 155, No. 6
Figure 3. Tight junction protein disruption is associated with marked serum protein extravasation and astrocytosis in HIVE. A–C: In control sections, blood vessels
display intact, ZO-1-positive tight junctions (red fluorochrome, Aand B) and retain the intravascular serum protein, fibrinogen (green fluorochrome, A). Weakly
staining, GFAP-reactive cell processes surround the intact BBB (green fluorochrome, Band C), however, do not overlap with either ZO-1-positive tight junctions
(red fluorochrome, B) or intravascular fibrinogen (red fluorochrome, C). D–F: In HIV sections, scattered blood vessels with ZO-1-fragmented tight junctions (red
fluorochrome, D) and, less often, intact ZO-1-positive tight junctions (red fluorochrome, E) overlap (yellow) with GFAP-reactive cell processes (green
fluorochrome, Dand E). In addition, GFAP-reactive astrocytes (green fluorochrome, F) colocalize focally (yellow) with extravasated fibrinogen (red fluoro-
chrome, F) from the focally disrupted BBB. G–I: In HIVE sections, abundantly extravasated fibrinogen (green fluorochrome, G) and numerous hypertrophic,
GFAP-reactive astrocytes (green fluorochrome, H), surround ZO-1-disrupted blood vessels (red fluorochrome, Gand H). Colocalization of the two markers with
ZO-1 is not observed due to extensive disruption of the tight junction protein (red fluorochrome, Gand H). GFAP-reactive astrocytes (green fluorochrome, I),
on the other hand, colocalize extensively (yellow) with extravasated fibrinogen (red fluorochrome, I) from the markedly disrupted BBB. Similar changes were
observed in occludin immunostained sections. Double-label immunofluorescence laser confocal microscopy; original magnification, 3600. CGM, cortical gray
matter; CWM, cortical white matter, DGM, deep gray matter.
Tight Junction Disruption in HIV Encephalitis 1921
AJP December 1999, Vol. 155, No. 6
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 associa-
tion with perivascular astrocytosis (Figure 3D). GFAP-
reactive, hypertrophic astrocytes surrounding the major-
ity 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 ves-
sels 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 fibrino-
gen extravasation, diffuse parenchymal fibrinogen immu-
noreactivity, and diffuse astrocytosis. Vessels with mini-
mal 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 per-
meable blood vessels colocalized extensively with fibrin-
ogen (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 de-
creases (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 perivascu-
lar astrocytosis. The extent of these alterations was sim-
ilar to that seen in the cortical gray matter in HIV-1 sec-
tions. Likewise, no statistically significant difference (P.
0.05) was observed in the mean number of cerebellar
blood vessels containing intravascular fibrinogen com-
pared 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 histocom-
patibility complex (MHC) activation marker, as well as
gp41, an HIV-1 envelope protein expressed by a per-
centage of circulating viral-infected monocytes during
HIV-1 infection.
All regions in control sections contained a small num-
ber of round CD68-reactive cells and short, CD68-reac-
tive cell processes scattered throughout the parenchyma
(data not shown). Infrequent blood vessels walls con-
tained large CD68-positive infiltrating mononuclear cells
that colocalized with tight junction proteins (ZO-1 immu-
nostaining is shown in Figure 4A and throughout Figures
4 and 5; similar reactivity patterns were seen with occlu-
din immunostaining, data not shown) and expressed
Class II MHC molecules (Figure 5A). Less often, flattened
periadventitial cells that did not colocalize with tight junc-
tion proteins were identified adjacent to the abluminal
aspect of blood vessel walls. No alterations in tight junc-
tion protein expression (Figure 4A) or fibrinogen perme-
ability (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).
Compared to control sections, HIV-1 sections demon-
strated a mild increase in CD68-positive cells and cell
processes (Figure 4B) within both the cortex and cere-
bellum. 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 iso-
lated and in small clusters, were consistently associated
with scattered cerebral vessels showing tight junction
protein fragmentation (Figure 4C) or fibrinogen perme-
ability (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 com-
pared to the same region in control sections (Figure 5B).
Compared to both HIV-1 and control sections, HIVE
sections demonstrated a marked increase in parenchy-
mal 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 ob-
served in all HIV-1 sections (data not shown). In all re-
gions, blood vessels with no alterations in tight junction
protein expression or fibrinogen permeability were vari-
ably associated with CD68-positive cells (data not
shown), similar to that observed in HIV-1 sections. In
contrast, tight junction-disrupted and fibrinogen-perme-
able 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 per-
meable 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 ar-
1922 Dallasta et al
AJP December 1999, Vol. 155, No. 6
Figure 4. Tight junction protein disruption and BBB permeability are associated with mononuclear cell infiltrates and microglial nodules in HIVE. A: In control
sections, infrequent blood vessels are infiltrated (yellow) by CD68-positive mononuclear cells (green fluorochrome) with no apparent disruption of ZO-1-positive
tight junctions (red fluorochrome). B, C, and G: In HIV sections, increased CD68-positive mononuclear cells and microglial processes (green fluorochrome, B)
are present among and colocalize focally (arrowheads, yellow) with ZO-1-positive blood vessels (red fluorochrome, B). In addition, scattered ZO-1-fragmented
(red fluorochrome, C) and fibrinogen-permeable blood vessels (red fluorochrome, G) are infiltrated (arrowheads, yellow) by CD68-positive mononuclear cells
(green fluorochrome, Cand G). D–F, H, and I: In HIVE sections, numerous ZO-1-disrupted (red fluorochrome, D) and fibrinogen-permeable blood vessels (red
fluorochrome, H) are associated with CD68-positive mononuclear cell aggregates and microglial nodules (green fluorochrome, Dand H) and microglial nodules.
CD68-reactive cells (green fluorochrome, E, F, and I) both surround (E) and infiltrate (yellow, Fand I) the disrupted blood vessels (red fluorochrome: ZO-1, E
and F; fibrinogen, I). Similar changes were observed in occludin-immunostained sections. B, D, G, and H: Double-label immunofluorescence laser confocal
microscopy; original magnification, 3200. Aand C: Double-label immunofluorescence laser confocal microscopy; original magnification, 3400. E, F, and I:
Double-label immunofluorescence laser confocal microscopy, original magnification, 3600. CGM, cortical gray matter; DGM, deep gray matter.
Tight Junction Disruption in HIV Encephalitis 1923
AJP December 1999, Vol. 155, No. 6
Figure 5. BBB disruption in HIVE is associated with mononuclear cell expression of Class II MHC activation markers but not HIV envelope protein. In control
(A) and HIV (B) sections labeled for endothelial cells (green fluorochrome), infrequent infiltrating (yellow) mononuclear cells express the MHC Class II activation
marker, HLA-DR (red fluorochrome). C: In HIVE sections, perivascular and parenchymal expression of the MHC Class II activation marker (red fluorochrome)
is pronounced. D: In HIV sections, gp41-positive cells (green fluorochrome) infiltrate ( yellow) and surround blood vessels with intact ZO-1-positive tight junctions
(red fluorochrome). E: In HIVE sections, infiltrating CD68-positive mononuclear cells (green fluorochrome) colocalize focally (yellow) with the HIV envelope
protein, gp41 (red fluorochrome). Fand G: In HIVE sections, gp41-positive cells (green fluorochrome) associate with both intact and fragmented (large
arrowhead) ZO-1-positive tight junctions (red fluorochrome). Likewise, some fragmented tight junctions show no association with gp41-positive cells (small
arrowhead, F). H–I: In HIVE sections, fibrinogen-permeable blood vessels (red fluorochrome) are also variably associated with gp41-positive cells (green
fluorochrome). A–D, F, and G: Double-label immunofluorescence microscopy; original magnification, 3200. E: Double-label immunofluorescence laser confocal
microscopy, original magnification, 3600. Hand I: Double-label immunofluorescence laser confocal microscopy, original magnification, 3200. CGM, cortical gray
matter; DGM, deep gray matter.
1924 Dallasta et al
AJP December 1999, Vol. 155, No. 6
borizing cell processes within the surrounding paren-
chyma (Figure 5C).
In contrast to the regional differences in CD68 reactiv-
ity, 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 com-
pared to HIV-1 sections, significant tight junction protein
alterations were not observed in vessels within the cere-
bellum, despite focal vascular infiltration by gp41-posi-
tive 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 mononu-
clear cells within blood vessel walls (Figure 5E).
Discussion
HIV-1-associated dementia complex is characterized his-
tologically by an invasion of large numbers of infected
monocytes into the CNS late in the course of the dis-
ease.
13
Previous studies have demonstrated that the
BBB, which normally regulates cell trafficking into the
CNS parenchyma, is compromised in HIVE
36,41,42
and
that this alteration may occur as a result of interactions
with activated monocytes and their soluble products.
51,52
The actual basis of the disruption, however, has not been
elucidated. We now report that highly significant disrup-
tions in BBB tight junctions, as demonstrated by immu-
nohistochemical changes in the expression and distribu-
tion of the proteins, occludin and ZO-1, occur during
HIVE. We further demonstrate that these alterations occur
in CNS regions susceptible to histopathological damage
in association with serum protein extravasation, astrocy-
tosis, and the accumulation of activated, HIV-1-infected
monocytes and microglia. Together, these data suggest
that intercellular conduits created by the disruption of
BBB tight junctions may serve as the pathological mech-
anism whereby HIV-1-infected monocytes gain signifi-
cant access to the CNS.
Our results clearly demonstrate that tight junction al-
terations correlate with serum protein extravasation, un-
derscoring the importance of this critical structure in
maintaining the relative impermeability of the BBB. The
demonstration of widespread serum protein extravasa-
tion in HIVE is in agreement with previous reports
36
but in
contrast to others, which did not detect a difference in
BBB permeability between brain tissue from HIV-1-in-
fected patients with and without encephalitis.
42
This dis-
parity may be a reflection of the more sensitive immuno-
histochemical techniques employed in this study. The
focal tight junction disruption and serum protein extrava-
sation observed in the frontal cortex and basal ganglia
from HIV-infected patients contrasts sharply with the ex-
tensive 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 per-
meability 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 func-
tion.
36
In addition, the development of astrocytosis, a promi-
nent feature that accompanied tight junction and perme-
ability 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 re-
lationship between focal BBB compromise and perivas-
cular 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 dam-
age to the CNS microenvironment. Reactive astrocytes,
however, are potent mediators of vascular permeability-
inducing cytokines, including tumor necrosis factor-
a
(TNF-
a
) and interleukin-6 (IL-6), which may augment per-
meability 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 in-
triguing 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 pa-
tients without encephalitis.
On the other hand, the presence of comparable num-
bers of gp41-infected monocytes within both affected
and unaffected cerebral and cerebellar vessels in HIVE
strongly suggests that the presence of this HIV-1 enve-
lope protein, by itself, is not sufficient for tight junction
modulation or permeability alterations during this dis-
ease. 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 endo-
thelium through a mechanism that may involve substance
P.
57
Although the direct transmission of HIV-1 through
endothelial cells of the BBB remains controver-
sial,
51,58– 61
reports of gp120 adsorptive endocytosis
across cerebral endothelial cells in vitro
62,63
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 essen-
tial role in breaching the BBB,
51,52,64
thus, facilitating
both neurotoxicity
14–18
and viral transmission into the
CNS.
51,65
Several lines of evidence indicate that the local
production of monocyte-generated cytokines, in particu-
lar TNF-
a
, are the most likely candidates for mediating
tight junction disruption after the adherence of activated
monocytes to the cerebral vascular lining. TNF-
a
and
interferon-
g
induce a striking fragmentation of ZO-1 via F
actin rearrangement in cultures of microvascular endo-
thelial 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 produc-
Tight Junction Disruption in HIV Encephalitis 1925
AJP December 1999, Vol. 155, No. 6
tion of high levels of pro-inflammatory cytokines, includ-
ing TNF-
a
.
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 differ-
ent 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 prod-
ucts promotes their avid adherence to cerebral endothe-
lial cells in vitro.
51
Furthermore, capillary up-regulation of
these molecules occurs in HIVE in association with infil-
trating mononuclear cells, in contrast to ubiquitous up-
regulation of ICAM-1 by both endothelial and parenchy-
mal cells.
51
In summary, the results of this study demonstrate that
significant, site-specific alterations in tight junction integ-
rity occur during HIVE and are associated with marked
BBB permeability, activated HIV-1-infected monocyte ac-
cumulation, and astrocytosis. Further investigation of the
precise molecules and mechanisms that disrupt this crit-
ical 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 patho-
genesis of HIV-1-associated dementia.
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