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Cytokine expression in the brain during the acquired immunodeficiency syndrome.

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ORIGINAL ARTICLES
Cytohne Expression in the Brain during
the Acquired Immunodeficiency Syndrome
William R. Tyor, MD," Jonathan D. Glass, MD,"$ John W. Griffin, MD," P. Scott Becker, MD,"f
Justin C. McArthur, MBBS, MPH,' Lena Bezman, MD," and Diane E. Griffin, MD, PhD't
The pathogenesis of central nervous system (CNS) disease in acquired immunodeficiency syndrome (AIDS) is poorly
understood but may be related to specific effects of the immune system. Cytokines such as tumor necrosis factor and
interleukin-1 may have toxic effects on CNS cells and have been postulated to contribute to the pathogenesis of the
neurological complications of human immunodeficiency virus (HIV) infection. To characterize viral and immunological activity in the CNS, frozen specimens taken at autopsy from the cerebral cortex and white matter of HIVseropositive and -seronegative individuals were stained immunocytochemically for mononuclear cells, major histocompatibility complex (MHC) antigens, HIV, astrocytes, and the cytokines interleukin-1 and -6, tumor necrosis factor-a
and -p, and interferon y . Levels of soluble CD4, CDS, and interleukin-2 receptor, as well as interferon y , tumor necrosis
factor-a, P,-microglobulin, neopterin, and interleukin-6 and - 1f3 were assayed in the cerebrospinal fluid and plasma
of many of these individuals during life. The HIV-seropositive group included individuals without neurological
disease, those with CNS opportunistic infections, and those with HIV encephalopathy. Perivascular cells, consisting
primarily of macrophages with some CD4+ and CDS+ T cells and rare B cells, were consistently MHC class I1 positive.
MHC class I1 antigen was also present on microglial cells, which were frequently positive for tumor necrosis factor-a.
HIV p24 antigen, when present, was found on macrophages and microglia. Endothelial cells were frequently positive
for interleukin-1 and interferon y and less frequently for tumor necrosis factor and interleukin-6. There were gliosis
and significant increases in MHC class I1 antigen, interleukin-1, and tumor necrosis factor-a in HIV-positive patients
compared to HIV-negative brains. Cerebrospinal fluid from most of the patients tested had increased levels of tumor
necrosis factor, &-microglobulin, and neopterin. There was no correlation in HIV-positive individuals between levels
of cytokines and the presence or absence of CNS disease. These data indicate that there is a relative state of "immune
activation" in the brains of HIV-positive compared to HIV-negative individuals, and suggest a potential role for the
immune system in the pathogenesis of HIV encephalopathy.
Tyor WR, Glass JD, GriffinJW, Becker PS, McArthur JC, Bezman L, Griffin DE. Cytokine expression in
the brain during the acquired immunodeficiency syndrome. Ann Neurol 1992;31:349-360
Human immunodeficiency virus type 1 (HIV) infects
the central nervous system (CNS) early 11-31, and cerebrospinal fluid (CSF) abnormalities are common in
the absence of clinically apparent disease [4, 5 ) . At
later stages of infection, symptomatic CNS disorders
are frequent (6-83 and include meningitis, encephalopathy, myelopathy, opportunistic infections, and
neoplasms. The pathology of these disorders has been
delineated (9- 113. Patients with HIV encephalopathy
or dementia may exhibit myelin pallor, microglial nodules, and multinucleated giant cells [9, 10, 12). HIV
antigen 113-181 and RNA [19-21) have been detected in macrophages and microglia. The pathogenesis
of HIV encephalopathy is unknown but is presumed
to be related to the infection of macrophages and microglia within the CNS 122) since oligodendrocytes
and neurons have not consistently been shown to be
infected by HIV (13-211. Partial expression of a portion of the HIV genome or expression of a particular
viral gene may result in the production of viral components that have a cytotoxic effect on neurons or oligodendrocytes, damage myelin in some way, or simulate
a neurotransmitter, resulting in the abnormal stimulation or inhibition of neurons [23). Alternatively, the
immune response to infection may have a role in damaging myelin, neurons, or oligodendrocytes.
One attractive hypothesis is that locally produced
cytokines may be toxic to the nervous system. Activated macrophages and microglia can secrete cytokines
that attract other inflammatory cells to the CNS 1241
and can stimulate endothelial cells, T cells, and astrocytes to produce other soluble factors. Some cytokines
may be able to damage CNS constituents (i.e., myelin,
neurons, and so on) directly or there may be as-yet-
From the Departments of 'Neurology, +Medicine, and $Pathology,
Johns Hopkins University School of Medicine, Baltimore, MD.
Address correspondence to Dr Tyor, Pathology 627 A , The Johns
Hopkins Hospital, 600 N. Wolfe Street, Baltimore, M D 2 1205.
Received Jun 17, 1991. and in revised form Aug 27. Accepted for
publication Sep 1, 1991.
Copyright 0 1992 by the American Neurological Association
349
unidentified toxins released by i m m u n e cells that are
harmful to the CNS [251. T u m o r necrosis factor
(TNF)-a and interleukin-1 (IL-1) are known to induce
fever, to mediate inflammation and acute-phase responses, and to be cytotoxic for a n u m b e r of cells
[26-32). Cytokines and o t h e r soluble factors released
by cells have b e e n measured in CSF and plasma during
the course of HIV infection and other inflammatory
diseases 133-361, b u t it is not known whether these
levels accurately reflect their presence in CNS parenchyma. We characterized t h e state of immunological
activation in the brains and CSF of HIV-infected patients using immunological assays and immunocytochemical staining for inflammatory cells, major histocompatibility complex ( M H C ) class I and I1 molecules,
reactive astrocytes, HIV antigens, IL-1, TNF-a and -p,
IL-6, and interferon y (IFN-y).
cellular pathology in the area. The routine neuropathological
diagnoses are listed in Table 1. Neuropathological diagnoses
were based o n studies of formalin-fixed, paraffin-embedded
sections from areas that included frontal, temporal, cirgulate,
parietal, and occipital regions of the cortex with adjacent
white matter, head of the caudate, putamen, globus pallidus,
hippocampus, thalamus, brainstem, cerebellum, spinal cord,
and any area that appeared suspicious clinically or by gross
inspection. The stains used were hematoxylin-eosin, reduced
silver for axons, and L u o l fast blue for myelin. Immunocytochemical study for CMV and papovavirus was performed
where appropriate.
The fresh-frozen specimens were embedded in OCT compound (Ames Company, Division Miles Laboratories, Elkhart, IN) and 5-micron cryosections were cut, attached to
glass slides, and immediately fixed in 95% ethanol for 10
minutes. The slides were then washed and stored in
phosphate-buffered saline solution (PBS) (pH 7.4)at ,4"C for
less than 1 week before staining.
Materials and Methods
Patients
Antisera
Fifteen HIV-infected patients, all with acquired immunodeficiency syndrome (AIDS), and 11 uninfected patients were
studied. Thirteen of the HIV-positive individuals had been
examined by members of the H I V neurology group at one
or more times during their illness, as part of ongoing prospective studies. The clinical profiles of all patients are given
in Table 1. The age range of HIV-positive patients was 6
months to 58 years (mean, 31 years; median, 29 years) and
of controls from 17 to 84 years (mean, 44 years; median, 51
years). Seven of the HIV-positive patients had no clinically
recognized C N S disease, 3 had encephalopathy, and 5 had
opportunistic CNS infections or neoplasm (Zoxoplasma,cytomegalovirus [CMV], progressive multifocal leukoencephalopathy, or lymphoma). The criteria for HIV encephalopathy
were: (a) tIIV type 1 seropositive, (b) progressive cognitive
and behavioral decline, (c) neurological and/or neuropsychological findings consistent with a decline from the premorbid
baseline level, (d) CNS opportunistic processes excluded by
computed tomography (CT) or magnetic resonance imaging
(MRI) and CSF analysis, and (e) no active substance use or
psychiatric disorder. Three control (HIV-negative) subjects
had no CNS disease. The 8 control subjects with C N S disease included patients with C N S sarcoid ( 1 patient), idiopathic basal ganglia disease ( 1 patient), alcoholic liver disease
(2 patients'), and drug abuse (2 patients). Although the controls who were drug abusers had no clinically recognized
CNS disease, we found neuropathological abnormalities (see
below). Clinical information was obtained from the patient's
hospital charts and autopsy reports.
The primary and secondary antibodies are listcd in Table 2,
along with their type, specificity, dilution used, and :source.
Control antibodies included a mouse monoclonal antibody
of irrelevant specificity (antimouse I-Ad, Becton Diclcinson,
Mountain View, CA) at the lowest dilution used (1 : 2 ) and
normal rabbit serum used at a dilution of 1: 25
lmmunoperoxidase and ImmunofEuorescent Staining
Immunoperoxidase staining was performed as previously described [37). Briefly, slides were incubated in 2% normal
horse or goat serum in PBS for 20 minutes, followed by
incubation with primary antibody (see Table 2) diluted in
PBS. Biotinylated secondary antibody (see Table 2) was then
added for 30 minutes. Endogenous peroxidase was blocked
for 30 minutes with 0.1% hydrogen peroxide (H,O,) in
methanol. Slides were then incubated in avidin-DH-biotin
complex (Vector Laboratories, Burlingame, CA) in PBS for
30 minutes, followed by color development with dixninobenzidine (0.5 mg/ml) in PBS with 0.01% H,O, for 20 minutes. After darkening with 0.5% cupric sulfate and 0.15 M
sodium chloride, slides were rinsed in water and counterstained with hematoxylin.
For double immunofluorescent staining, primary antibodies were incubated together (rabbit antihuman and mouse
monoclonal antihuman antibody) for 45 minutes. followed
by fluorescein-conjugated goat antimouse and rhodamineconjugated goat antirabbit immunoglobulin secondary antibodies for 30 minutes (see Table 2). Control slides were
prepared by omitting the primary antibodies.
Tissue Samples
Analysis of Staining
Cortex and subjacent white matter from the right frontal lobe
of all patients (approximately 2 x 2 x 1 cm) was obtained
at autopsy, frozen in isopentane with dry ice, and stored at
- 70°C. Neighboring regions were fixed in 5 % glutaraldehyde for electron microscopy and in 4% paraformaldehyde
for paraffin embedding and other histological studies. The
49;paraformaldehyde-fixed sections adjacent to the samples
removed for freezing were studied to further characterize the
Cells were considered positively stained by the immunoperoxidase technique if brown reaction product was present
around a nucleus counterstained with hematoxylin. Cells
identified immediately adjacent to a blood vessel were designated as perivascular cells and included endothelial cells,
astrocytes, and microglia as well as infiltrating mononuclear
cells from the blood. For the assessment of perivascular infiltrates, the total number of cells immediately adjacent to a
350 Annals of Neurology Vol 31 No 4
April 1992
Table 1. Patient Profils and Autopsy Diagnoses
-
Patient No.
Clinical Summary
Brain Autopsy Diagnoses
I. HIV-positive
A. No known clinical C N S disease
1
50 years old, F; transfusion + H I V + ; recurrent Nocardzu pneumonia -+ death
2
33 years old, M, homosexual; disseminated TB; sensory neuropathy; ARDS + death
3
4 1 years old, M, homosexual; chronic inflammatory
demyelinating neuropathy; cervical herpes zoster;
PCP + hypoxia; Staphylococcus sepsis .+ death
4
6 months old, M; congenital HIV; persistent fever
and diarrhea; PCP + pneumothorax -+ death
26 years old, M, homosexual; Kaposi's sarcoma;
5
pneumonia + hypoxia
death
6
7 years old, M; congenital HIV; failure to thrive and
chronic diarrhea; disseminated Mycobacterium
a~iumlirrtracellulre;cardiomyopathy ; recurrent
pneumonia and UTIs
7
32 years old, M, homosexual; PCP; disseminated
CMV; chronic renal failure; pneumothorax +
death
B. Clinical encephalopathy
8
58 years old, M; unknown risk factor; recurrent PCP;
mild encephalopathy
28 years old, M, homosexual; history of secondary
9
syphilis; cutaneous neurofibromatosis; encephalopathy; pneumonia --j staphylococcus sepsis + death
-
10
26 years old, M, homosexual; recurrent PCP; CMV
retinitis; disseminated Mycobacterium aiiiuml intracelIulave + MA1 peripheral neuropathy; advanced encephalopathy; myelopathy
C. Other C N S disease (opportunistic infection, neoplasm, etc.)
11
41 years old, M, Haitian; disseminated T'B; C N S
Toxoplasma and CMV retinitis; mental status
changes; sensorimotor neuropathy; PCP + respiratory failure + death
31 years old, M, homosexual; PCP; disseminated TB;
12
PML; encephalopathy
13
34 years old, M, homosexual; recurrent PCP; sensory
neuropath y; presumed C N S ToxopliEswi
14
53 years old, M, homosexual; progressive myelopathy; sensory neuropathy; severe wasting syndrome
15
36 years old, M, homosexual; PCP; hepatitis B; C N S
Toxoplasma: Staphylococcus sepsis + death
11. HIV-negative
62 years old, M; chronic obstructive pulmonary dis16
17
ease and cor pulmonale; bronchogenic carcinoma
45 years old, F; hypertension, depression and Hodgkin's disease, status after chemotherapy; sepsis, disseminated Candida + death
-
Mild diffuse myelin pallor; adjacent section"-mild
gliosis and D M P
Multifocal axonal spheroids in putamen, middle cerebral peduncle, and pyramids; mild thickened meninges and ependymitis; vacuolar myelopathy; ad jacent sectiona-severe gliosis
Many multifocal white matter changes and mulcinucleated giant cells; thickened meninges and ependymitis; adjacent sectiona-moderate gliosis
Normal
Adjacent section"-mild
gliosis
Axonal spheroids in internal capsule; adjacent section"-meningeal thickening, mild-to-moderate gliosis, and mild D M P
Microglial nodules and diffuse gliosis; no CMV in
brain; adjacent section"-moderate gliosis
Slightly thickened meninges; possible white matter
gliosis; adjacent section"-moderate gliosis
Microglial nodules and multinucleated giant cells in
the cortex; severe vacuolar myelopathy; adjacent
section"-microglial nodules and severe gliosis and
DMP
Microglial nodules, scattered small multifocal white
matter lesions and multinucleated giant cells in the
corona radiata; hemosiderin-laden macrophages;
mild ependymitis; ventriculomegaly; vacuolar myelopathy; adjacent section"-normal (not stained
for GFAP)
Multiple Toxoplasma lesions; mild DMP; CMV inclusions-occipital lobe and ventriculitis; adjacent section"-Toxoplasma lesions and perivascular infiltrates
Severe PML-cerebellum and brainstem, but mild
above tentorium; adjacent section"-moderate-tosevere gliosis and moderate D M P
Necrosis right internal capsule, caudate, putamen,
globus pallidus, and thalamus; n o ToxopLasma seen;
thickened meninges
Several calcified blood vessels; ventriculomegaly; central spinal myelinolysis; adjacent sectiona-meningeal thickening, moderate gliosis, and DMP
Primary C N S lymphoma; C N S Toxoplasmu; moderate
amount of microglial nodules in cortex; old contusion in left temporal lobe; vacuolar myelopathy;
adjacent section"-lymphoma, moderate gliosis,
and mild D M P
Normal
Normal
Tyor et al: Cytokine Expression in AIDS Brain
351
7;rMe I .
/~~ot/tit//~id/
Patient N o .
-
Clinic:tl Summary
Brain Autopsy Diagnoses
84 years old, M: pancreatic carcinoma and chronic
White matter gliosis
obstructive pulmonary disease o n steroids; movement disorder with idiopathic basal ganglia degeneration
4 3 years old. M; alcohol abuse + cirrhosis + ascites,
jaundice. peritonitis, uremia; testicular carcinoma;
hypotension + death
30 years old, M; suicide-gunshot wound to lower
abdcimen
20 years old. M; drug abuse; drug overdose + death
,i9 years old, M; acute myelogenous leukemia, status
past chemotherapy; pulmonary aspcr,gilrosis; fever,
sepsis -+ hypotension + death
32 y e u s old, M ; CNS sarcoid, left cerebellar lesion;
hycirocephalus * shunt; CSF pleocytosis; chronic
steroid therapy; seizure + death
years old, F; cardiac arrest
56 y e x s old, F; alcohol abuse -+ cirrhosis
ascites,
esophageal varices; recurrent pleural effusions;
myocardial infarction; sepsis + death
1 7 y e . m old, F; myocarditis with cardiomyopathy;
Jrug abuse; pneumonia + death
-j
Normal
Normal
Normal
Basal ganglia calcifications; thickened meninges and
granular epentiymitis
Diffuse sarcoidosis
White matter gliosis
Mild white matter gliosis
Normal
-
.'These par3tormdldehvdc-~xcL{s e c t i ~ n swere adpccnr to the frozen white matter section anci were typically stained with hematoxvlin-eosin,
t o r :ha1 tibrillairy acidit: protein (GFAP], and Lux01 fdsr blue.
DhlP = cliftusc*rnyclin pallor, PCP = Pxezmm).rtii cuuvzt?ii pneumonia; UTI = urinary tract mfec tion; CMV = cyron~cgalovirus;TH
A u l t rcspir;in>ry
tuhcrculosis; M A I = mycobacrcriuni avium inrracellulare, PML = progess~vcmulrifocal Icukoencephal~,pathy.ARDS
~
~
distress svntlronie.
1 was thc denominator, anci the total number o f
stained cclls arounci the same vessel was the numerator for
the antibody analyz.ed. For each antibody. 10 periLascrular
a r c s were counted and averaged t o obtain the percentage
of perivascular cells staineci. T w o observers (W. R. T. and
L. 13.) counteJ the ci:lls and their results were averaged Independent results of these observers generally were in close
agreement. For graded staining of various antibodies (see
a blinded observer (W. R. T.) rated each antibody
Fig i),
separately, for each case. W e observed no differencei in the
amount of staining of any of the antibodies in the cortex
cornparecl with the white matter.
A.r.wy.<f.t, Solidde Pt*odui.t.\ oj- Immzrrie Cells
Eight CSF samples and four plasma samples from
-7 HIVpositive patients hall been stored frozen and were available
for analysis. Soluble CD4, soluble CD8, soluble IL-2 receptor. TNF-ol (T Cell Sciences, Cambridge, MA), IL-0 (Genzyme, Boston. MA,, and IL- 10 (R & D Systems, Mirineapolis. M N ) were me;isured by enzyme immunoassay. IFN-y
(Centocor. Malvern, PA), neopterin (Henning Berlin, Berlin,
Germany 1, and (3-microglobulin (Pharmacia Diagnostics,
Uppsala, Sweden) were measured by radioimmurioassay.
Assays were performed according to the manufacturers' instructions. Normal values for serum and CSF were obtained
from the literature [ i'l,
3 - , 3 8 ) or the manufacturer, or were
based on v'ilues established within o u r laboratory [391.
352
Annals o f Neurology
Vol 3 1 No
4
April 1992
Stuticticul AnaIysiJ
Groups were compared using Student's t test with a twotailed analysis.
Results
Periifumdar Cells
In general, AIDS brains had a similar number of perivascular cells compared to HIV-negative specimens
(mean of 2 1.6 cells per vessel, with a srandard error o f
2.1 for HIV-positive samples; and 17.5 cells per vessel,
with a standard error of 2.3 for HIV-negative samples).
Monocytesimacrophages (EBMIII) were the most
prevalent mononuclear cell type identihed in perivascular cuffs (Fig lA), followed by T cells (Leu-4). Slightly
more CD4+ (Leu-3a) cells were found than C 1 X i
(Leu-2a) cells. B cells ( B l ) were rare. Most of the inliltracing perivascular cells were class 11-positive (Iig
IS). In HIV-positive specimens there was no apparent
correlation between the presence or absence of CNS
disease, and the percentages of class 11-positive cells,
macrophages, or T cells. There was a mean of 27.3
class 11-positive perivascular cells counted per HIVpositive patient (for each, 10 blood vessels were
counted), compared to 18.0 class I I-positive cells per
HIV-negative patient ( p = 0.031). There was a mean
Table 2. Desrviption of-Antibodies U s ~ d
Name (Clone Number)
Type
Specificity
Source
Dilution
Mouse MAb (IgG1)
Macrophages (CD68)
Dako, Santa Barbara, CA
1:80
Mouse MAb (IgG1)
T cells (CD3)
1:50
4. Leu-2a (SK1)
5 . B1 (H299)
6. (L243)
Mouse MAb (IgG1)
Mouse MAb (IgG1)
Mouse MAb (IgG2a)
Mouse MAb (IgG1)
7. HLA-ABC (W6I32)
Mouse MAb (IgG2a)
CD4+ cells
CD8+ cells
B cells (CD20)
Class I1 molecules (common
determinant on D R and
DQ)
Class I molecules (HLA-ABC
45-kDa glycoprotein)
&-Microglobulin
Bovine GFAP
Becton Dickinson,
Mountain View, CA
Becton Dickinson
Becton Dickinson
Coulter, Hialeah, FL
American Type Culture
Collection, Rockville,
MD
Dako
1. Dakomacrophage
(EBMI 11)
2. Leu-4 (SK 7)
3. Leu-3a (GK 1.5)
8. P,-Microglobulin
9. Glial fibrillary acidic protein (GFAP)
10. 9284 or 7054
11. 9283
12. 9282
13. HIV gp41 (C41)
14. HIV gp120, 160
(2E12.1)
15. HIV p24, 5 5 (2G12.1)
16. Interleukin-1 (IL- 1)
17. Interferon y (IFN)
18. Interleukin-6 (IL-6)
19. Tumor necrosis factor-a
(TNF-a)
20. TNF-P
21. Biotinylated horse antimouse immunoglobulin (Ig)
22. Biotinylated goat antirabbit Ig
23. FITC goat anti-mouse
IgG
24. Rhodamine goat antirabbit IgG
Rabbit
Rabbit
Mouse
Mouse
Mouse
Mouse
MAb
MAb
MAb
MAb
(IgG1)
(IgG1)
(IgG1)
(IgG1)
HIV gp120
HIV p24
H I V p17
HIV gp41
Mouse MAb (IgG1)
H I V gp120, 160
Mouse MAb (IgG1)
Rabbit
Rabbit IgG
Rabbit
Rabbit
HIV p24, 55
Human IL-1, a and p
Human IFN-y
Human IL-6
Human TNF-a
Rabbit
Horse
Dako
Biomedical Technologies,
Stoughton, MA
Dupont, Wilmington, DE
Dupont
Dupont
Olympus, Lake Success,
NY
Olympus
1:lO
1:2
1:2
1 :40
1:100
1: 200
1 : 1,000
1:lO
1: 10
1:lO
1:50
1:50
1:50
1:40
1 : 1,000
1:25
1 :40
Human TNF-P
Mouse Ig
Olympus
Endogen, Boston, MA
Genzyme, Boston, MA
Genzyme
Genentech, South San
Francisco, CA
Genzyme
Vector, Burlingame, CA
Goat
Rabbit Ig
Vector
1: 100
Goat
Mouse IgG
Tago, Burlingame, CA
1 : 100
Goat
Rabbit IgG
Tago
1: 100
of 26.0 perivascular macrophages counted per HIVpositive patient, compared to 18.7 macrophages per
HIV-negative patient (p = 0.056).
Soluble CD8 and CD4, but not IL-2 receptor, were
occasionally detected in the CSF, while soluble CD8
and IL-2 receptor, but nor CD4, were frequently increased in plasma (Table 3).
MHC Class 1 and Class 11 Molecules
Class I molecules (see Table 2) were found on numerous vascular endothelial cells in HIV-positive (Fig 1C)
as well as HIV-negative specimens, and also on brain
parenchymal cells, some of which were stellate (Fig
ID), resembling microglia. Other positive parenchymal
1:10
1: 100
cells were not easily identified by their morphological features but could be non-process-bearing microglia and macrophages, astrocytes, infiltrating mononuclear cells from the blood, or oligodendrocytes. The
patterns of staining with antibodies to HLA-ABC (p
chain) and to P,-microglobulin (achain) were identical.
&Microglobulin was elevated in all available samples
of CSF of all HIV-positive patients (see Table 3).
Class 11-positive cells were found in all cases. In
general, class 11-positive stellate parenchymal cells
were found only in HIV-positive patients (Fig lE),
while in HIV-negative individuals most positive cells
were perivascular. The class 11-positive parenchymal
cells resembled microglia and, with double immuno-
Tyor et al: Cytokine Expression in AIDS Brain
353
Fig I . Reprtcentatiiv. 5-micron-thick sectiom. sluiized immunorytochemicaliy..from frontal hbe white matter of HI V-po.ritive
patients. The brou,n reaction product indicutes positiue staining.
The nuclei zc 'ere iounterstuined with hemutoxylin. (A,Perivasrulur mucrophuge (arrowhead). (Bi ClusJ II-poritiz,e periwzscufur ~ l(arrowhead
l
). iCi Class I-positiw uuscu:ur endothelial
1-eNs. ( D i Clurs I-positive stellate parenchjmul cdl. Arrowhead
354 Annals of Neurology
Vol 31
N o 4 April 1992
indicutes the heniutox~~liii-rozi~zter.rtuI~~e~
nuc1eii.r and hroicw
perikuqm. und the urro'0?4indiinlo L i houm prom I , ( E i C1u.i.i
11-positii'e stellate parenchj mill c.rll (nrrowhead 1. i F i Iwrtrleukin-1 iIL-I)-po.ritiw ra.it.uIur endothelzlnl telli. i C , I IL-1posititme .ctellilte parenchyma/ c-ell (arrowhead). i H , p2.i
Antigen-positif e ccll (arrowheaci ). V -- blood wrsd
Tuble 3. L e d s of Soluble Products of Imniune Cells in Pla.smu
and CerebroJpinal Fluid ICSFI of HlV-Po~ztzz~e
Puttexts Studied PoJtmovtem
~~
T-cell Products
Patient
No.
Plasma
3
9
14
15
CSF
3
9
10
11
13
14
Time
before
Death
Soluble
CD4
(unitsiml)
Soluble
CD8
(unitsiml)
2 yr
9mo
2mo
5 mo
-
3,400"
678
4,300"
877"
4 yr
-
2 yr
9mo
2mo
2mo
1 wk
1 mo
2mo
-
-
16
nd
22
40
150"
5
nd
IFN--y
(unitsiml)
110
7 78"
950"
nd
-
0.5
-
nd
0.1
0.1
0.2
1.8"
-
10
420"
"Values above the normal range.
- _ - amounts were below detectable levels; nd
Soluble
IL-2
Receptor
(unitsiml)
=
20
nd
80
80
40
Macrophage Products
=
-
TNF-a
(pgiml)
30
-
38
42
39"
-
nd
4 0"
7 1"
12a
55"
P2-Microglobulin
(mgiliter)
Neoprerin
(nMiliter)
IL-6
(pgiml)
IL-1P
(pgiml)
4.4d
2.4
5.4"
4.4"
34"
20"
24"
28a
-
-
2.9
nd
2.2"
3.0"
8.0"
6.2"
2.2"
3.0"
3.6"
4.2"
23"
17.5"
47a
89"
27"
29"
32"
11.5"
not determined.
fluorescent staining, were negative for glial fibrillary
acidic protein (GFAP), a marker for astrocytes (data
not shown). GFAP-positive cells were class II-negative by double immunofluorescent staining (data not
shown), further suggesting that the class 11-positive
parenchymal cells were microglia. Class I1 antigen was
more abundant in the brains of HIV-positive patients
than HIV-negative patients (see Fig 3, p = 0.004).
Cytokines
IL-1 staining was very similar in all HIV-positive specimens (Fig 1F). Virtually all endothelial cells were positive and antigen was detected a short distance into the
parenchyma. Stellate parenchymal cells were also occasionally positive (Fig 1G), as were some non-processbearing parenchymal cells. The IL-1-positive stellate
parenchymal cells were GFAP negative by double immunofluorescent labeling (data not shown). In HIVnegative specimens, IL- 1-positive endothelial cells
were much less common and IL-1-positive parenchymal cells were only rarely seen (see Figure 3, p =
0.001). IL-1(3 was not detectable in plasma or CSF,
suggesting that most of the IL-1 remained cell associated (see Table 3).
TNF-a staining was present on some endothelial
cells, but primarily on stellate parenchymal cells (Fig
2A,B). Although most of these cells were GFAP negative by double immunofluorescent labeling (data not
shown), a few TNF-a-positive cells were also GFAP
positive. HIV-positive specimens showed more TNF-a
staining than did HIV-negative specimens (Fig 3, p =
0.03). TNF-a was increased in the CSF in 5 of 7 patients studied (see Table 3).
IFN-y staining was similar in distribution to IL-1
staining (Fig 2C). Endothelial cells and stellate parenchymal cells resembling microglia were most frequently positive. In general, IFN-y staining was more
common in HIV-positive than HIV-negative specimens (see Fig 3) although this difference did not reach
significance (p = 0.12). Although IFN-y was elevated
in only one CSF sample and no plasma samples, neopterin, an indirect measure of IFN-y-induced activation of macrophages [40], was elevated in all CSF and
plasma samples tested (see Table 3).
IL-6 was present on endothelial cells, although staining was less intense than that observed for IL-1 and
IFN-y (Fig 2D). Occasionally stellate parenchymal
cells stained positively for IL-6 and these were probably microglia. Generally, HIV-positive specimens displayed more IL-6 staining than did control samples (see
Fig 3). The pattern for TNF-(3 was similar, although
fewer endothelial cells and more stellate parenchymal
cells were positive for TNF-/3 than IL-6 (see Fig 3).
IL-6 was detected in only one of four plasma samples
and was nor detected in any CSF samples tested (see
Table 3).
HIV Antigen
A number of monoclonal antibodies were used to detect HIV antigens (see Table 2), but only 2 patients
(Patients 2 and 9) exhibited p24 antigen positivity (see
Fig 1H). This antigen was present on cells that closely
Tyor et al: Cytokine Expression in AIDS Brain
355
356 Annd5 of Neurologv
Vol 3 1
No 'I April 1092
4.0
1
I
T T
H
+
ClaSSII
GFAP
30
0
IL-I
IL-6
c
0
.c
d
2.0
TNF-a
TNF-P
IFN-Y
0
2
(3
10
HIV+
HIV~~~
Fig 3. Auerage gradations for the presence of major histocompat-
ibility complex (MHCI class I1 antigen. astrocytes Iglialjibrillaty acidic protein [GFAP]), ctnd the cytokines interleukin-1
(1L-1) and -6 (IL-6), trmor necrosis factor-a (TNF-a) and -p
(TNF-P), and interferon y (IFN-y).
resembled macrophages or microglia. Serial sections
from these areas stained positively for EBM/11 (macrophages) and class 11, suggesting that the p24-positive
cells were class 11-positive macrophages. No other
HIV antigens (i.e., p17, gp41, or gp120) were detected.
Reactive Astrocytes
Gliosis (increased numbers of astrocytes with abnormally abundant cytoplasm and vesiculated nuclei or
>2 + staining for GFAP) was observed in 13 of
15 HIV-positive and 6 of 11 HIV-negative individuals
(see Fig 3, p = 0.002). Adjacent paraformaldehydefixed, paraffin-embedded sections from 12 of the 15
HIV-positive patients were stained for GFAP and gliosis was found (more in the white matter than in the
gray matter) in all specimens that were examined (see
Table 1).
Discussion
We have shown evidence of T-cell and macrophage
activation in the C N S during H I V infection. Soluble
products of immune cells were assessed in the CSF by
immunoassay and in the frontal cortex and white matter by immunocytochemical staining. Localization of
the staining suggested that infiltrating T cells and macrophages, and local microglia and endothelial cells participated in the immunological activation within the
CNS during the late stages of H I V infection.
Of the perivascular cells identified immunocytochemically, most were macrophages. However, the T
cells present were activated, as indicated by their expression of class I1 M H C antigen and frequent production or induction of soluble factors found in the CSF.
Infiltrating macrophages cannot be differentiated from
ameboid macrophages derived from resident microglia
and are likely to be of similar lineage [41,42]. Macrophages and microglia appear to be major participants
in HIV infection of the CNS. Both macrophages and
microglia in vitro express CD4 and are susceptible to
HIV infection 143, 443, and it is these cells in the brain
that are most often found to be infected with HIV in
vivo C14-18, 20, 21f. In our study we found p24positive cells that resembled macrophages or microglia
in 2 patients (Patients 2 and 9). One might expect more
patients to have H I V antigen-positive cells but this
was probably related to the relatively small area of
brain that was stained for H I V antigen. However, our
data indicate that there is a relative state of immune
activation in the brain, regardless of the presence or
absence of H I V antigen in the sections examined. Serial sections revealed cells appearing similar to the
HIV-positive cells that were class I1 positive, suggesting that H I V is harbored primarily in activated
macrophages or microglia that are theoretically capable
of antigen presentation to C D 4 + T cells, which are also
present in the tissue. Macrophages and microglia also
frequently expressed class I M H C antigen and therefore are potentially capable of presenting antigen to
CD8+ T cells as well. The presence of activated macrophages and T cells in the C N S of HIV-infected individuals creates the potential for a number of cytokines to
be stimulated and expressed by these and other cells
in the CNS 124, 27).
Endothelial cells stained positively for IL-6 and occasionally for TNF, but were most remarkable for their
expression of IL-1. IL-1 is secreted by both microglia
and endothelial cells in vitro 1451 and has many activities, including stimulation of T cells, activation of endothelial cells and macrophages, induction of fever, and
mediation of inflammation and acute-phase responses
[26, 27). IL-1 also induces astrocyte proliferation in
vitro [46].
Proliferation of glial cells can occur at a distance
from a CNS lesion {47), suggesting that a diffusible
factor such as IL-1 may play a role. Gliosis has also
been described by others as a feature of AIDS CNS
pathology [9-11f and our findings are consistent with
this. IL-1 also is cytotoxic for some cells either in its
membrane-bound or in its secreted forms [ 2 8 , 31, 32)
and therefore could play a role in cytokine-induced
CNS disease.
Although sonie macrophage and microglial cells in
patients with AIDS expressed IL-1 and IL-6, which can
upregulate H I V expression [48), these cells were most
notable for their expression of TNF-a. This suggests
that macrophages and microglia, which can secrete
T N F in culture {27,49, SO}, also produce T N F in vivo.
Moreover, we found elevated levels of T N F in the
CSF from 5 of 7 individuals tested. Since T N F induces
fever, inflammation, acute-phase responses, and septic
shock [26, 27) and is cytotoxic for certain cell lines
either in its membrane-bound or in its secreted forms
[28, 291, it could play a role in the development of
encephalopathy, and indeed a number of investigators
Tyor et al: Cytokine Expression in AIDS Brain
357
have suggested this possibility { 5 l , 52). ’TNF is toxic
to oligodendrocytes in culture [53] and is increased in
the serum and CSF in HIV-induced neurological disease 135, 36), It has been found on astrocytes and
macrophages in the brains of patients with multiple
sclerosis, an immunologically mediated disease, and
subacute sclerosing panencephalitis 154). Lastly, monocytes from symptomatic H I V type 1-infected patients
produce larger amounts of T N F than do monocytes
from control or asymptomatic H I V type I-infected
persons { 5 5 , 561, and T N F enhances H I V transcription
in lymphocytes E48, 5 7 , 581. It is not clear whether
HIV infection of macrophages per se induces T N F
production { S l , 59, 6 0 ) o r whether activated T cells
induce the macrophage and microglial T N F production, even though T cells are reduced in number late
in infection.
Evidence for T-cell activation within the C N S during
AIDS comes from increases in soluble CD8, neopterin, and P?-microglobulin. Neopterin is produced
by macrophages, and possibly microglia, in response to
IFN-y stimulation [6 11 and is progressively increased
in the CSF during symptomatic phases of HIV infection and particuiarly in HIV encephalopathy {33, 62).
IFN-y stimulates MHC class I and I1 antigen expression, and class 11 antigen was significantly increased in
HIV-positive cornpared to HIV-negative brains in our
study. Production of IFN-y by C D 8 and CD4 T cells
is well documented, but endothelial cells, which were
the primary cell type found to be positive in this study,
have not been reported to produce IFN-.y. It is possible that these endothelial cells may have bound IFN-y
produced by T cells. Most of the IFN-y produced is
bound in situ, since serum and CSF levels of IFN-y
are not generally elevated 1331. Notably, consistent
increases in IFN-y, IL-6, and IL-16 were not detected
in the CSF and blood, despite the increased staining
of these cytokines in tissue, suggesting that CSF and
blood cytokine levels do not necessarily correlate with
the presence of these factors in brain tissue.
Both microglia and endothelial cells expressed
M H C class I. The source of M H C class 1 antigen is of
interest since Pz-rnicroglobulin has been found to be
elevated in the CSF from patients with AIDS [63},
especially those with encephalopathy, and was elevated
in all CSF available for study in our patients. W e found
P1-microglobulin staining on vascular endothelial cells
and some stellate parenchymal cells (probably microglia) of all ,4IDS patients and on endothelial cells, but
not parenchymal cells, in many of the control subjects
as well.
Parenchymal cells, especially microglia, may therefore be a primary source of increased CSP P,-microglobulin in H I V encephalopathy. Although we and
others have found increased levels of @,-microglobulin
and neopterin in HIV-positive patients with encepha-
358 Annals of- Neurology Vol 31 No /t
April 199.2
lopathy, compared with those without C N S disease
[ 3 3 , 62, 631, we found no significant differences between the two groups in the presence of immune cells,
cytokines, and M H C antigens in the brain. This suggests that some other factor is necessary for the development of encephalopathy in addition to the “immune
activation” observed in AIDS brains.
O u r data suggest that compared with HIV-negative
individuals, a relative state of “immune activation” occurs in the brains of patients with AIDS. Macrophages
and microglia, a few of which are infected with HIV,
are generally activated. Many T cells and macrophages
present in the tissue, as well as resident microglia, endothelial cells, and astrocytes, appear to be involved in
the response to and generation of cytokines. These
cytokines can modulate the immune response, and may
also have toxic effects on the CNS. A large proportion
of patients with AIDS develop encephalopathy. The
pathogenesis of encephalopathy and the myelin pallor
often associated with it are not well understood, but
the immune system may play a role [23, 641. Involvement of the C N S with H I V infection occurs early
[l-31 and, therefore, activated macrophages, T cells,
and cytokines such as IFN-y, TNF-a, and IL-1 may be
present in the brains of HIV-infected patients for many
years. This long period of exposure to “immune activity” should be distinguished from relatively short periods of exposure that would be expected to occur in
acute inflammatory diseases such as meningitis o r encephalitis. These cytokines may be directly involved in
the pathogenesis of myelin pallor and encephalopathy
by damaging oligodendrocytes, myelin, or neurons.
_ _ _ _ _ ~ ~ ~
~~
This study was supported by grants from the National Institutes of
Health (>POI-NS-26643,N01-A1-72034, and NS-071’9).
We rhank Drs Nikki Baumrind and Richard Johnson tor helpful
discussions; Mark Shagogue for preparation of the figures, Carol
Schlough, RN, BSN, for clinical assistance; Linda Kelly and Kingsley
Brooks for prepararion of rhe tnanuscript; Bonita Nsah and Suzanne
Amerr for technical assistance wlth rhe autopsy specimens; and Dr
Donald Price, Department of Pathologv. Johns Hopkiris School of
Medicine.
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