H.G. Saadati et al.264

Immunolocalization of IL-1βββββ and IL-6 in optic nerves of patients

with AIDS

Hossein G. Saadati, Iftikhar A. Khan, Xiang-Hong Lin, Anish Kadakia, Keith B. Heller, Alfredo A. Sadun

Department of Ophthalmology, Doheny Eye Institute, University of Southern California School of Medicine, LosAngeles, CA, USA

Current Eye Research 0271-3683/99/1903-0264 $15.001999, Vol. 19, No. 3, pp. 264–268 © Æolus Press

Correspondence: Alfredo A. Sadun, MD, PhD, Department ofOphthalmology, Doheny Eye Institute, University of Southern Cali-fornia, Los Angeles, CA 90033, USA, Tel: (323) 442-6417, Fax:(323) 442-6688, E-mail: asadun@hsc.usc.edu

Received on February 3, 1999; revised and accepted on May 18, 1999

Abstract

Purpose. Immunohistochemical procedures were employedto test the hypothesis that cytokines such as interleukin-1β(IL-1β) and interleukin-6 (IL-6) are involved in AIDS-re-lated optic neuropathy and to determine the primary celltypes involved.

Methods. Fixed sections of six human HIV-1 infected opticnerves were immunostained for the presence of IL-1β andIL-6, using horseradish peroxidase and diaminobenzidineas markers.

Results. IL-1β and IL-6 were found in astrocytes, macro-phages, microglia, and endothelial cells. The great majorityof astrocytes demonstrated strong immunoreactivity.

Conclusion. Our findings support the premise that IL-1βand IL-6 are significant pro-inflammatory mediators in AIDS-related optic neuropathy. This finding supports the theorythat HIV infection stimulates the release of IL-1β and IL-6 in astrocytes, macrophages, and endothelial cells in theoptic nerve which ultimately leads to demyelination, astro-gliosis, and neuronal destruction.

Keywords: AIDS; immunohistochemistry; interleukin-1β;interleukin-6; optic nerve

Introduction

In AIDS-related optic neuropathy, the pathology of axons,myelinating oligodendrocytes, and other glial cells of theoptic nerve are not well explained by direct infection ofthese cells by the HIV-1 virus. In HIV encephalopathy,neither oligodendrocytes nor neurons have been shown tobe infected with the virus.1 However, in AIDS it has beenshown that infiltrating macrophages and resident microgliaare directly infected with the HIV-1 virus and allow viralreplication.2 It is probable that the HIV-1 virus inducesthese cells to release pro-inflammatory mediators such ascytokines which can be toxic to the neurons and glialcells.1,3 We have previously published on tumor necrosisfactor alpha (TNF-α) and its potential for mediating anAIDS-associated optic neuropathy.4 Interleukin–1beta (IL-1β) and interleukin-6 (IL-6) are two other cytokines whichhave been shown to play an important role in the immuneresponse and pro-inflammatory process in the central ner-vous system (CNS).2,3 Because of related effects and mecha-nisms of production, IL-1, IL-6, and TNF-α have been groupedtogether as similar immune factors.5

Human IL-1 consists of two forms, IL-1α and IL-1β,which are 26% homologous in amino acid sequence andbind to the same receptor; however it is IL-1β that is pre-dominantly found in most human tissues.5 Elevated levelsof IL-1β have been measured in the cerebro spinal fluid(CSF) in patients with AIDS Dementia Complex (ADC) aswell as HIV-infected patients without neurological signs.6

IL-1β has been shown to stimulate HIV-1 LTR reportergene expression in glioblastoma, astrocytoma, and primaryastrocytes suggesting enhanced replication of the virus inglial cells.7

IL-6 is produced by many activated cell types in theCNS including astrocytes, microglia, endothelial cells, andmacrophages.1,2 IL-6 is often found in association with IL-1β, which in addition to TNF-α may induce IL-6 geneexpression. IL-6 may also increase the production of theHIV-1 virus, as HIV-1 infected monocyte clones have dem-

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Immunolocalization of IL-1β and IL-6 in optic nerves of patients with AIDS 265

onstrated increased production of the virus in response toIL-6 from human astrocytes.2 Other effects of IL-6 may alsoinclude astrogliosis and B-cell activation and differentia-tion.3 In addition, increased CSF levels of IL-6 have alsobeen measured in 42% of 38 patients with ADC6 as well asin cases of acute viral meningitis.8

We proposed that cytokines may be a major contributingfactor in the demyelination, axonal damage, and astrogliosisseen in AIDS-related optic neuropathy. The presence of IL-1 and IL-6 may thus be important in the pathogenesis ofAIDS-related optic neuropathy. In addition, with the recentadvents in combination protease inhibitor and nucleotidetherapy, patients are living longer and will experience in-creasing morbidity such as ADC and AIDS-related opticneuropathy suggesting a growing relevance to understand-ing the pathogenesis of AIDS related optic neuropathy.

Materials and methods

Tissue preparation

Six optic nerves from HIV-positive patients were obtainedat necropsy from the Shiley Eye Institute, University ofCalifornia at San Diego and delivered in 50% ethanol.Among the six nerves, four were from patients who hadcytomegalovirus (CMV) or other types of retinitis; the othertwo did not have abnormal findings. Three normal HIVnegative eyes were obtained from the Lion’s eye bank atthe Doheny Eye Institute processed in the same fashion andprepared as controls. All the HIV-positive patients had grosslynormal psychometric evaluations.

All nine nerves were refixed in 4% paraformaldehydewith 0.1 M sodium cocodylate for 1 hour, and rinsed in a10% sucrose solution in 0.1 M phosphate buffer. They werethen placed in a 30% sucrose solution for 72 hours at 4°C.The fixed nerves were frozen in liquid nitrogen, imbeddedin Tissue Tek O.C.T. Compound, frozen in liquid nitrogenand stored at –20°C. The nerves were then sectioned into7 micron slices on a cryostat. The sections were air driedfor 2 hours and rehydrated in 0.01 M sodium phosphatebuffered 0.9% saline (PBS), pH 7.4 for 5 min.

Immunohistochemistry

Immunohistochemical staining was conducted on 7 micronsections. Aliquots of 100 µl from each of the antibodiesand block solutions were used to incubate individual opticnerve sections. Three washes for 10 min each in PBS weredone between each individual incubation step in block orantibody solution. Block containing 2% bovine serumalbumin and 2% goat serum in PBS was applied for 30minutes to reduce non-specific binding. The primarypolyclonal goat anti-human IL-1β (R&D Research, Catalog# ab201na) and IL-6 (R&D Research, Catalog # ab-206na)antibodies were purchased from R&D Systems and run at

optimum dilutions of 1:100 for ∝ IL-1β and 1:50 for ∝ IL-6. The sections were incubated in primary antibody over-night at 4°C. Incubation for 2 hours at room temperaturewas performed with biotinylated rabbit anti-goat secondaryantibody (Jackson Immunoresearch catalog # 305065003)and run at an optimum dilution of 1:100. Endogenous pe-roxidase activity was quenched with application of a methanolhydrogen peroxide mixture in a ratio of 5:1 for 20 minutes.An avidin:biotinylated horseradish peroxidase complex (ABCkit) obtained from Vector Laboratories (Catalog #: PK-400)and was then added for 30 minutes. The substrate used asthe chromagen was 3′3 diaminobenzidine (DAB) also pre-pared from a kit (Catalog # SK-4100) purchased from Vec-tor Laboratories. The reaction was stopped in running tapwater when any background staining became evident (1–5min). The sections were then counterstained with Hema-toxylin to illustrate contrast and also washed in tap water.Finally, Glycergel mounting media was applied and theslides were coverslipped for examination by light micros-copy. A grading scale of (0–4) for staining intensity wasemployed. Tissue controls were developed on all tissuesections cut from the six different patients in the followingmanner: (1) omitting the primary antibody sections, (2)omitting the secondary, (3) utilizing goat serum as theprimary antibodies, and (4) three normal eyes were pro-cessed in the same fashion.

Results

Control sections were negative for IL-1β (Fig 1A) and IL-6 (Fig. 1B) in all cell types. Normal whole human eyes werealso stained for IL-1β and IL-6 with negative results (datanot shown).

Immunoreactivity was prevalent in astroctyes, endothe-lial cells, and macrophages in both IL-1β and IL-6 incu-bated sections in all HIV-infected optic nerves. The astro-cytes exhibited strong immunoreactivity (+3 on at 0–4scale) for IL-1β, while macrophages exhibited lighter stain-ing (+1)(Fig 2A, 2B). Endothelial cells exhibited displayedweak immunoreactivity (+1 to +2).

Astrocytes (+4) with large extending processes also stainedpositive in immunoreactivity for IL-6 (Fig. 3A, 3B). How-ever, macrophages were barely stained for IL-6 (0 to +1)(Fig. 3B). Some macrophages contained lipid droplets inabundant cytoplasm and this helped us to differentiate themfrom microglia.

In summary, oligodendrocytes and macrophages in thefield exhibited some immunoreactivity for both IL-1β andIL-6 (0 to +1), but less than the reactive astrocytes (+3 to+4). An estimated 85–95% of the astrocytes stained positivelyfor IL-1β and IL-6 (+3 to+4). Endothelial cells were alsomoderately positive for IL-6 (+2 to +3). There was no de-tectable difference in the immunoreactivity of IL-1β andIL-6 in the HIV-positive optic nerve from the patients who

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Figure 1. Normal control optic nerves. Negative immunohistochemical staining for IL-1β (A) and IL-6 (B). 200 × (1 cm = 50 µm).Figure 2. A) Optic nerve of AIDS patient showing astrocytes with strong immunohistochemical staining for IL-1β (arrow). Macrophages(m) exhibited lighter staining. B) Optic nerve of AIDS patient without abnormal ocular findings. Endothelial cells (arrow) displayed weakimmunoreactivity for IL-1β. 550 × (1 cm = 18 mm). Macrophage (m).Figure 3. (A+B): Immunohistochemical staining for IL-6 in optic nerves of AIDS patients. Well stained astrocytes with some large extend-ing processes (3A). However, macrophages were barely stained (3B). 550 × (1 cm = 18 µm). Astrocyte (a), Macrophage (m).

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Immunolocalization of IL-1β and IL-6 in optic nerves of patients with AIDS 267

had CMV-retinitis. Further, the histopathology of the HIV-positive optic nerves did not qualitatively vary. Table 1summarizes the results.

Discussion

Previous histopathologic studies have shown losses in therange of 18–25% of optic nerves axons (without retinitis)in addition to dropout of cortical neurons in AIDSpatients.9,10,11,12 In vitro, it has been observed that IL-1 andTNF-α can cause the death of oligodendrocytes and/or destroythe myelin.13,14 In addition, our laboratory has demonstratedthe present of TNF-α in optic nerves from AIDS patients,with and without CMV retinitis, supporting the theory thatTNF-α is a major mediator of AIDS-associated opticneuropathy.9 More recently, we showed that TNF-α had thecapacity to cause similar optic nerve pathology when injectedintravitreally in a rabbit model.4 Hence, we proposed thatpro-inflammatory mediators such as IL-1, IL-6, in additionto TNF-α, were mediators of neurodegeneration in AIDSpatients. The following pathophysiologic model was sug-gested: HIV virus is carried into the brain by infiltratingmacrophages, which pass through the blood-brain barrier.These macrophages and resident microglial cells harborvirus, which induces these cells to produce and to releasecytokines such as TNF-α and IL-1β. These cytokines con-sequently enhance viral replication and the release ofIL-6.2 This, in turn, stimulates astrocytes to become acti-vated and to secrete even larger amounts of IL-1β, IL-6, andTNF-α. The present study supports this model in that wedemonstrated intense labeling for both IL-1β and IL-6 inreactive astrocytes with lesser staining in macrophages bothin capillaries and in the parenchyma. Strong endotheliallabeling suggests an impairment of the blood brain barrierfacilitating the passage of the macrophages through thisbreach.

Others have also demonstrated immunoreactivity for IL-1 and IL-6 in astrocytes, macrophages, microglia and en-dothelial cells in the CNS.1,2 In addition, IL-1β has beenshown to activate expression of HIV-1 in astrocytes usinga reporter gene linked to an HIV-1 LTR promoter sequence.7

Our present results demonstrated that IL-1β and IL-6 arepresent in significant amounts in HIV infected optic nerve.These cytokines also have an autocrine effect, which leadsto further stimulation of the astrocyte to produce increasingamounts of cytokines. This explains the sharp contrast be-

tween the amount of staining in different astrocytes; thestrongly stained cells were activated and the faintly stainedcells were still non-reactive. Astrocyte migration and pro-liferation then occurs in response to IL-1β and IL-6, whichfurther inhibits axonal function. Other cells, including oli-godendrocytes, which do not stain intensely, may haveundergone apoptosis as a result of exposure to TNF-α andIL-1β produced by these reactive astroctyes. Consequently,damage to oligodendrocytes, may lead to myelin loss, ax-onal degeneration and related damage to neurons.

IL-1β may be directly and/or indirectly injurious andinduces apoptosis via the lymphocyte Fas antigen. IL-1βconverting enzyme (ICE) may regulate apoptosis both inneuronal development and in response to external injury.15

In addition, CNS administration of IL-1 receptor antagonist(IL1-ra) significantly inhibits neurodegeneration stimulatedby various insults in a rat model.16

IL-1β has also been shown in human astroglioma cellsto induce production of TNF-α gene expression.17 The Fasreceptor contains a death domain that can mediate apoptosis.18

Stimulation of apoptosis can also occur via the ceramidesignaling pathway after activation of the TNF-α receptor.In microglia, TNF-α receptor activation of NF-kappa Btranscription factor via an acid sphingomyelinase in thecytoplasm also mediates apoptosis.10,11 In rats, TNF-αstimulates the production of nitric oxide (NO) in microglialcells precipitating cell death.12 Nitric oxide production viaNMDA receptor activation is also described in various humanneurodegenerative disorders.19 Further, IL-1β has been shownto be a potent inducer of IL-6 release in astrocytoma celllines.20

Glial cells have demonstrated proliferation at a distancefrom CNS injury.1 IL-1 has been shown to induce theproduction of Nerve Growth Factor (NGF) in vitro.21 IL-1effects of inducing NGF have also been shown to be enhancedby arachidonic acid21 which is released from the plasmamembrane of cells normally in an inflammatory process.This may cause migration and proliferation of reactiveastrocytes as seen in astrocyte monolayer cell culture.22

Since in this present study IL-1β is strongly positive inreactive astrocytes (+4), this cytokine may provide the avenuefor increased replication of the virus in astroglial cells aswell as infiltrating macrophages.

Several investigators have also observed the effect of IL-1 and TNF-α causing damage in white matter, stimulatingastrogliosis, and vascular changes.3 Other studies describingthe effects of IL-1β on rabbit retina have shown hemor-rhage and inflammation of epiretinal vessels with the injec-tion of IL-1β.23 Increases in polymorphonuclear and mono-cytic cells also occurred and are related to the endothelialdamage and resultant increased permeability of the bloodbrain barrier. Endothelial cells may also be acted on bythese cytokines as they showed the presence of IL-1β andIL-6, (+2 to +3) in our study, although less intensely thanthe astrocytes. Endothelial cells, which were second onlyto astrocytes in the amount of IL-1β and IL-6 present, may

Table 1. Summary of cytokine immunoreactivity.

Cell types Reactivity Reactivityfor IL-1β for IL-6

Oligodendrocytes 0 to +1 0 to +1Macrophages +1 0 to +1Endothelial Cells +1 to +2 +2 to +3Astrocytes +3 to +4 +3 to +4

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also produce these cytokines which can cause self-injury aswell as a mechanism for the infected macrophages to enterthe optic nerve.

Additional studies may elucidate neuroprotective effectsfrom inhibiting cytokine expression. Tendinap, a nonste-roidal anti-inflammatory drug, has been shown in one studyto decrease the production of IL-6 induced by IL-1β by85% in astrocytoma cells. Tendinap was shown also tosignificantly inhibit IL-6 synthesis at the transcriptionallevel.20 Meloxicam and naproxen were able to decrease IL-1β-induced IL-6 synthesis by 15–20%. Recovery on steroidtreatment from bilateral optic neuropathy in a patient foundto be infected with (HIV-1) has also been described.24

In summary, our present investigation supports the no-tion that IL-1β and IL-6 are significant immune factorsinvolved in AIDS related optic neuropathy. Marked immu-noreactivity for IL-1β and IL-6 was found in reactiveastrocytes, endothelial cells and to a lesser extent macroph-ages and microglia. TNF-α and IL-1β exposure may producedeleterious effect on oligodendrocytes causing apoptosis,demyelination and possible neuronal damage. IL-1β andIL-6 exposure may also contribute to increased HIV-1replication in macrophages, microglia, and astrocytes. Certainmechanisms to inhibit the pro-inflammatory response mayprove to be neuroprotective and prolong visual and otherCNS capacities in patients with AIDS.

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