Download Tumor necrosis factor-alpha in the retina in acquired immune

May 1992 Vol. 33/6
Investigative Ophthalmology
& Visual Science
Articles
Tumor Necrosis Foctor-a in the Retina in Acquired
Immune Deficiency Syndrome
Florence M. Hofman and David R. Hinron
The presence of specific cytokines and the number and distribution of leukocytes were determined in
the retinas of patients with acquired immune deficiency syndrome (AIDS). Using immunohistocytochemical techniques, three retinas from patients with AIDS had focal infiltration by T-lymphocytes and
macrophages. These specimens stained positively for tumor necrosis factor-a (TNF-a) in cells identified morphologically as macrophages and glial cells and showed prominent reactive gliosis. The retinas
from seven other affected patients had minimal leukocytic infiltration and no TNF-a reactivity; gliosis
was present in only one. The retinas from clinically normal patients without human immunodeficiency
virus (HIV) contained no TNF-a-positive cells. Using in situ hybridization for HIV, four of five
patients with AIDS had rare positive cells. No interferon-7 was detected in any of the retinal tissues
tested. These data suggest a role for TNF-a in the development of AIDS-related retinal disease. Invest
Ophthalmol Vis Sci 33:1829-1835,1992
central nervous system (CNS) disease.7 The HlV-infected macrophages appear to be the most likely vehicle for HIV infection of the brain;6 however, once in
the brain, the mechanism of HIV dissemination and
tissue degeneration is not well understood.
Tumor necrosis factor-a (TNF-a) may be critical in
enhancing HIV replication and spread.8 This cytokine has a molecular weight of 17 kD and is produced
most commonly by activated macrophages and monocytes.9 This activation may be induced by mitogens,
antigens, or binding of HIV to the CD4 receptor of
monocytes.8>1° Some affected patients show elevated
TNF-a levels in their sera, which may be a reflection
of the increased levels of TNF-a production by HIVinfected monocytes and macrophages.8 This cytokine
has been shown to mediate cell proliferation, cytokine
production, and cytotoxicity.9 In addition, TNF-a
can enhance HIV production directly by inducing the
synthesis of cellular factors that bind to the nuclear
factor kappa binding site of the HIV long terminal
repeat.8
In the CNS, TNF-a has been detected, and astrocytes can both produce and respond to it. Rat astrocyte cultures manufacture biologically active TNFa,11"13 and the cytokine can induce astrocytic proliferation14-15 and class II antigen expression.16 More
significantly, TNF-a also is cytotoxic to oligodendroglial cells.12 These data suggest that, under specific
conditions, TNF-a may be present in the CNS and
that astrocytes may play a significant role in the pro-
Approximately 58% of patients with acquired immune deficiency syndrome (AIDS) have retinal disease.1 Ischemic lesions are frequent with loss of pericytes, degeneration of endothelial cells, and focal
occlusion of small vessels.2 The substantial abnormalities of the retinal microvasculature in AIDS suggest
that disease progression may involve an alteration in
the blood-retina barrier, leading to leukocytic infiltration and subsequent dissemination of opportunistic pathogens such as cytomegalovirus (CMV).34 The
mechanism of the development of this AIDS-induced
retinal disease is not well understood; however, it may
occur without CMV infection.4 In studies done on
brain tissue from patients with AIDS, the cells most
commonly infected with human immunodeficiency
virus (HIV) are macrophages, microglial cells, multinucleated cells. Endothelial cells and glial cells are
rarely infected.5"7 Although HIV usually does not infect glia, severe gliosis is often apparent, suggesting
indirect pathogenetic mechanisms in the induction of
From the Department of Pathology, University of Southern California School of Medicine, Los Angeles, California.
Supported by National Institutes of Health (Bethesda, Maryland)
grant EYO 8144 (FMH).
Submitted for publication: April 26,1991; accepted December 2,
1991.
Reprint requests: Dr. Florence Hofman, Department of Pathology, USC School of Medicine, 2011 Zonal Avenue, Los Angeles,
CA 90033.
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / May 1992
duction of this cytokine, particularly during disease
progression.
In this study, we determined (1) whether or not
TNF-a was present in the retinas of patients with
AIDS and (2) how the presence of this cytokine correlated with retinal disease, particularly leukocytic infiltration. To accomplish this, we examined retinas
from ten HIV-seropositive patients with AIDS and
five HIV-negative neurologically normal control subjects to detect the presence and distribution of leukocyte subpopulations and the cytokines TNF-a and interferon-7 (IFN-7). In three affected patients, intense
TNF-a staining was found in association with macrophages and glial cells in the nerve fiber and ganglion
cell layers; these three had retinal infiltration by Tlymphocytes and macrophages. In the remaining
seven patients and five normal control subjects, the
absence of T-cells and relatively low numbers of macrophages correlated with little or no TNF-a positivity.
These data suggest a role for TNF-a in the development of AIDS-induced retinal disease.
Materials and Methods
Tissue
Fresh retinas from patients with AIDS and neurologically normal control subjects were obtained at autopsy within 24 hours postmortem. The globes were
bisected horizontally, and the superior half was snap
frozen in liquid nitrogen-cooled isopentane for immunoperoxidase analyses. The inferior half was fixed
in buffered formalin 10% for in situ hybridization
studies.
Reagents
Mouse monoclonal antibodies used in this study
were the following: CD4, CD8, CDl 1C, and HLA-Dr
class II (Becton Dickinson, San Jose, CA); anti-human TNF-a (Genentech, South San Francisco, CA);
anti-human IFN-7 (ICN, Costa Mesa, CA); and antibovine glial fibrillary acidic protein (GFAP; Dako,
Carpenteria, CA). The secondary linking reagents
(biotinylated horse anti-mouse and avidin-biotinperoxidase complex) were obtained from Vector
(Burlingame, CA). The HIV probe (Dupont, Boston,
MA) and in situ hybridization reagents were available
commercially.
Immunocytochemical Analysis
The frozen tissue was cut at 5-7 nm, air dried, and
fixed in reagent-grade acetone for 10 min. After drying, the slides were incubated with phosphate-buffered saline (PBS, pH 7.4) for 5 min. The tissues then
were blocked with normal horse serum (20 min) and
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Vol. 33
treated sequentially with the primary monoclonal antibody (30 min), the secondary biotinylated horse
anti-mouse (30 min), and avidin-biotin-peroxidase
complex (20 min) with 10-min PBS washes after each
step. Amino-ethyl carbazole solution, which produces
a red precipitate in the presence of peroxidase, was
added to the slides for 10 min. They were rinsed and
counterstained with Mayer's hematoxylin for 3 min,
followed by a rinse in tap water for 10 min. Subsequently, the slides were mounted in a solution of glycerol and PBS. Primary antibodies were titrated initially on known positive cell preparations. Controls
for the staining procedure included both the use of an
irrelevant antibody or PBS in place of the primary
antibody. The frequency of positive cells was determined by counting the number of stained cells in
three selected 1-mm2 fields in which preliminary
staining indicated leukocytic infiltrates.
In Situ Hybridization
The alkaline phosphatase-linked HIV-specific
RNA probe used was a cocktail of three probes specific for the two GAG and one envelope regions. The
procedure, as recommended by the manufacturer
(Dupont), required the tissue to be rehydrated in 100
nmol/1 MgCl2 for 10 min at room temperature. Then
the slides were prehybridized for 10 min at 70 °C in
formamide and rinsed in 5X SSC. The samples were
heated to 55°C, incubated with probe solution for 60
min at 55°C, and then treated with detection buffer
for 4 hr in the dark at 37°C. The development was
stopped by rinsing the slides in distilled water. The
slides were counterstained with either methyl green or
nuclear fast red for 25 sec, rinsed with distilled water,
and allowed to air dry in the dark.
Results
Tissues from ten HIV-seropositive patients were
analyzed in this study; their clinical findings and
treatments are summarized in Table 1. The retinal
tissues were examined for the presence and distribution of infiltrating T-cells and macrophages using immunocytochemical methods. In three of ten retinal
tissues from patients with AIDS, increased numbers
(>5 cells/mm2) of T-cells were present (Table 2A).
One of these retinas had retinal toxoplasmosis (from
patient 2); however, the other two did not have opportunistic infection. These T-cells were localized within
the nervefiberand ganglion cell layers both in perivascular and intraparenchymal areas. Approximately
equal numbers of CD4-positive (Fig. 1) and CD8-positive cells were identified. In the other seven specimens
(Table 2B), no extravascular T-cells were found in the
retinal tissues, and the retinal structure and cell distri-
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TNF-a IN THE RETINA IN AIDS / Hofman and Hinron
No. 6
Table 1. Clinical findings of HIV seropositive patients
No. Age Sex
1
33
M
2
57
M
Clinical
ophthalmologic findings
Systemicfindings,major
Eye pathology—P.M.
Bilateral CMV retinitis
CN VI palsy, left
Retinal atrophy and gliosis in
posterior pole consistent with
treated CMV retinitis
Not determined (terminally Retinal toxoplasmosis (Focal)
in hospice)
3
43
M
Normal
No observable pathology
4
5
58
38
M
M
Normal
Normal
No observable pathology
No observable pathology
6
34
M
Normal
No observable pathology
7
24
F
Normal
No observable pathology
8
51
M
Normal
No observable pathology
9
40
M
Normal
No observable pathology
10
32
M
Normal
No observable pathology
bution appeared to be normal. Macrophages, identified as CDllC-positive cells, were present in all 10
affected patients, although they generally were present in greater numbers in those with T-cell infiltration
(Table 2). In all instances, macrophages were localized to the nerve fiber and ganglion cell layers and
were adjacent to the blood vessels (Fig. 2).
The retinas from patients with AIDS were analyzed
for the presence of HIV using in situ hybridization. In
four offivepatients (Table 1), rare HIV-positive cells
were detected in the retinal tissue (1-5 cells/mm2).
Antiviral/fungal therapy
CMV encephalitis
Cerebral gliosis
Azidothymidine
Ganciclovir
Primary HIV encephalitis
Amphotericin B
Disseminated cryptococcosis
Cerebral gliosis, perivasculitis
Pneumocystis pneumonia
CMV-adrenal
Cerebral gliosis, perivasculitis
Pneumocystis pneumonia
Bronchopneumonia
Tuberculosis
Cerebral gliosis
Pneumocystis pneumonia
Kaposi's sarcoma
Cryptococcal meningitis
Cerebral toxoplasmosis
Cerebral gliosis
Pulmonary Candida
Aspergillus infections
Pneumocystis pneumonia
Cerebral gliosis
Pneumocystis pneumonia
CMV encephalitis
Azidothymidine
None
None
None
None
Amphotericin B
None
Azidothymidine
Azidothymidine
These positive cells were identified morphologically
as macrophages and were located in the ganglion and
inner plexiform layers (Fig. 3A). Positive cells located
in the inner nuclear layer (Fig. 3B) had a morphology
suggesting Miiller cells.
The expression of HLA-Dr class II antigen was increased in retinas of affected patients with increased
leukocytic infiltration (Table 2); reactivity was localized to the nerve fiber and ganglion cell layers and
around the blood vessels. Class II-positive cells appeared to be morphologically identifiable macro-
Table 2. AIDS retina tissue
T
lymphocytes
M0
TNF-i
(2)t
(3)
+2
+1
+1
+1
+1
+1
+ 1*
+2§
+2§
(4)
(5)
(6)
(7)
(8)
(9)
(10)
_
-
+
(1)
±
±
±
+
+1
+1
HLA-Dr
HIV
GFAP*
+1
+1
+1"
ND
-
- = no positive cell present; ± = rare positive cell (<5 cells/mm 2 ); + 1 = ^ 5
< 20 positive cells/mm 2 ; +2 = S;20 positive cells/mm 2 ; ND = not done; M 0
= macrophage; TNF-a = tumor necrosis factor-alpha; IFN-7 = interferongamma; HLA-Dr = class II; HIV = human immunodeficiency virus; GFAP
= glial fibrillary acidic protein.
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IFN-y
±
+
+1"
+1"
ND
ND
ND
ND
* Number of plusses indicates relative intensity of staining.
f Primary HIV encephalitis with retinal toxoplasmosis.
t Predominant staining of astrocytes.
§ Both astrocytes and Mueller cells staining.
11
Predominant staining of blood vessels.
ND
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / May 1992
Vol. 33
V.
Fig. 1. Cryostat sections of AIDS retina were examined for the
presence of CD4-positive T lymphocytes. Low numbers of cells
(arrows) were present perivascularly in the ganglion and nerve fiber
layers (X400).
Fig. 2. Frozen sections of AIDS retina demonstrated CD1 lC-positive macrophages (arrows) associated with blood vessels, and in
the ganglion and nerve fiber layers (X400).
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3*3
Fig. 3. HIV in situ hybridization was performed on AIDS retinas.
(A) HIV-positive cells were associated with perivascular macrophages (X1000). (B) Positive cells were also located in the inner
nuclear cell layer (arrows) (X400).
phages, lymphocytes, and endothelial cells (Fig, 4),
with focal staining consistent with astrocyte end feet.
Little or no class II expression was found in most of
those retinas without significant leukocytic infiltration (Table 2). Staining for GFAP revealed positive
Miiller cells and astrocytes in all instances; however,
the intensity and extent of staining varied markedly
among patients (Table 2). In control eyes and in six of
seven TNF-a-negative eyes from patients, the Miiller
cells were positive in the inner nuclear layer, with only
minimal staining of processes in the outer plexiform
and outer nuclear layers. The astrocytes in the nerve
fiber layer showed diffuse reactivity without identifiable processes. In the TNF-a-positive retinas from patients with AIDS, Miiller cells showed more prominent GFAP reactivity extending from inner to outer
limiting membranes, and thick immunoreactive astrocyte processes were found in the nerve fiber layer
(Fig. 5).
Retinal tissues from the ten patients were examined
for the presence of specific cytokines. We found TNFa-positive cells localized to the nerve fiber and ganglion cell layers (Fig. 6A). There was prominent labeling of end feet adjacent to blood vessels consistent
TNF-a IN THE RETINA IN AiDS / Hofman and Hinron
No. 6
1833
population of TNF-a-positive cells had processes terminating at endothelial cells, suggesting the end-feet
processes of astrocytes (Fig. 6B). In two patients (2
and 3), the population of TNF-a-reactive cells consisted of parallel elongated cells, spanning the inner
retina, and suggesting Miiller cell morphology (Fig.
6C). Although TNF-a originally was isolated from activated macrophages,9 this cytokine has been shown
to be synthesized by resident cells of the CNS. Rat
astrocyte cell cultures, exposed to IFN-y, interleukinla, or lipopolysaccharide produced TNF-a, measured by the release of functional protein and induction of specific mRNA.12'13 Viral infections of astrocyte cultures also can induce TNF-a production.12
These data suggest that, at least in vitro, astrocytes can
manufacture TNF-a in the presence of immunoregulatory molecules or after direct viral infection. Studies
using tissue specimens of patients with multiple sclerosis, an immune-mediated demyelinating disease with
prominent leukocyte infiltration,17 or those with subacute sclerosing panencephalitis, a virally induced demyelinating disease, found TNF-a-positive cells with
the morphologic characteristics of astrocytes.18 Using
Fig. 4. Class II-positive cells were identified as leukocytes, astro
eytes, and endothelial cells (X400).
with astroglial cell morphology (Fig. 6B). In two of
four instances, these processes extended through the
thickness of the retina, well into the inner nuclear and
plexiform layers, indicating TNF-a reactivity in
Miiller cells (Fig. 6C). No TNF-a staining was detected in specimens without lymphocytic infiltration
(Table 2B). No IFN-7 was detected in any of the retinal specimens tested, although activated peripheral
blood leukocyte specimen control cells were IFN-Y
positive.
Five neurologically normal HIV-seronegative retinas also were analyzed (Table 3). No T-cells were
present in any of these five specimens, and the number of macrophages varied from none (two offive)to
rare (three of five). In all instances, no TNF-a was
detected. A few cells were HLA-Dr positive, but most
-of the reactivity was confined to endothelial cells. No
HIV was detected in these tissues.
Discussion
We showed that TNF-a is present in the retinas of
patients with AIDS. The presence of this cytokine is
associated with infiltration of T-lymphocytes and
macrophages and gliosis. Based on immunocytochemical and morphologic methods, the cellular source of
TNF-a in the retina is associated with astrocytes and
Miiller cells. In all three TNF-a-positive cases, the
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B
Fig. 5. Retinas from AIDS patients stained for glial fibrillary
acidic protein (GFAP). (A) In a TNF-a-positive retina (case #1),
gliosis is present, with enlarged Miiller cell processes extending
through the thickness of the retina (arrows) (X400). (B) In a TNF-«negative retina (case #10), normal appearing Miiller cells are present whose processes are thinner, and in which GFAP-positive staining predominates in the inner half (X400),
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INVESTIGATIVE OPHTHALMOLOGY & VI5UAL 5CIENCE / May 1992
Fig. 6. TNF-a-positive cells were localized in the nervefiberand
ganglion cell layer, with prominent staining of morphologically
identifiable astrocytes (arrows) (A) and end feet adjacent to blood
vessels (arrow) suggestive of astrocytes (B). TNF-«-positive cells
exhibited stained, vertically oriented processes suggestive of glial
morphology (arrows) (C) (X400).
double-staining techniques, TNF-a staining colocalized with GFAP reactivity, indicating that TNF-a was
associated with astrocytes.17 Additional studies are
needed to determine whether the astrocytes themselves produce TNF-a or bind the released TNF-a
produced by activated infiltrating macrophages.
As shown in our study, the presence of TNF-a was
correlated positively with increased class II expression
on glial cells. This cytokine acts synergisticaliy with
IFN-7 and viral infection to induce class II expres-
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sion.16-19 Class II expression on glial cells enhances
their ability to act as antigen-presenting cells and
thereby mediate immune interaction with activated
T-cells. The presence of HIV did not appear to correlate with either TNF-a production or class II expression because class II expression appeared to be independent of detectable virus. In addition, TNF-a induced astrocytic proliferation in several in vitro
systems,1415 including the human astrocytoma cell
U373.14 The strong correlation between the presence
of TNF-a and retinal gliosis, as determined by the
intensity and extent of staining for GFAP, suggests an
altered microenvironment leading to reactive structural changes in the retina. The cytokine TNF-a can
cause cytotoxicity of oligodendroglial cell cultures"
and degeneration of myelin sheaths,20 thus implying
that TNF-a directly damages CNS cells. This damage
may be mediated by TNF-a produced by activated
leukocytes which have migrated into the CNS21 or by
endogenously stimulated astrocytes.
The role of the T-lymphocyte in triggering TNF-a
production in the retina is unclear. T-lymphocytes
produce a series of cytokines, including IFN-7 (a potent inducer of TNF-a and upregulator of TNF-a receptors on target cells).22 However, no IFN-7 was detected in the tissue we examined, suggesting that this
cytokine is not present at this stage of disease. Other
studies have shown that IFN-7 can be found in the
vitreous of patients with AIDS who have accompanying opportunistic infections.23 As the authors suggest,
the source of this IFN-7 may have been the activated
T-lymphocytes in the vitreous or systemic IFN-y
gaining access to the vitreous after breakdown of the
blood-ocular barrier.23 Based on our findings, IFN-7
is not likely to be derived from retinal tissue per se.
The high level of TNF-a in the three affected retinas
(Table 2A) may be associated with the high numbers
of macrophages present in those tissues. Not only are
macrophages an important source of TNF-a, this cytokine has been shown to activate macrophages, recruit additional macrophages, and increase their cytotoxic potential.24'25 Thus, in the presence of both
TNF-a and macrophages, an amplification circuit for
Table 3. Normal retina tissue
lymphocytes
M0
TNF-a
(1)
(2)
(3)
(4)
(5)
Abbreviations and symbols as in Table 2.
* Predominant staining of blood vessels.
IFN-y
HLA-Dr
+*
HIV
TNF-a IN THE RETINA IN AIDS / Hofman and Hinron
No. 6
this cytokine can be initiated. Macrophages were present in all affected retinas studied, providing a potential source of HIV-infected activated macrophages
(Table 2). It has been suggested that' HIV-infected
macrophages may be the vehicle for HIV infection in
the CNS.7 Thus, HIV-infected macrophages entering
the retina may be the initial source of TNF-a, which
then alters the microenvironment of the retina and,
ultimately, the functions of glial, neuronal, and endothelial cells.
The major pathologic process in HIV infection of
the retina may not be direct HIV infection of neuronal cells because few HIV-positive cells were detected
in the tissue from patients with AIDS, as shown by us
and others.26 Furthermore, TNF-a does not appear to
be acting as an amplification signal for HIV replication because the presence of HIV was independent of
the absence or presence of TNF-a. The pathologic
findings observed in AIDS may, however, be caused
by changes in the composition of the cytokine microenvironment in the retina, thereby increasing the
probability of blood vessel occlusions, cytotoxicity,
and opportunistic infections. We suggest that TNF-a
may play a central role in this cytokine microenvironment and be responsible in part for the initiation, progression, and dissemination of disease in the retina.
8.
9.
10.
11.
12.
13.
14.
15.
16.
Key words: AIDS, retina, tumor necrosis factor (TNF),
Muller cells, astrocytes
17.
Acknowledgments
18.
The authors thank Drs. Larry Nichols and Edward Klatt
for providing tissue; Ms. Robin Bonner, Mr. Albion D.
Wright, and Mr. Marcellino Pantangco for their expert
technical assistance; and Mrs. Florence Miyagawa for help
in preparing the manuscript.
19.
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