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Published online before print February 14, 2008
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From the Divisions of Comparative Pathology,*Immunology,and Bacteriology and Parasitology,Tulane National Primate Research Center, Tulane University Health Sciences Center, Covington, Louisiana
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Abstract |
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CD163 is a member of the scavenger receptor family with cysteine-rich domains (SCRC) identified as a receptor of haptoglobin-hemoglobin (Hp-Hb) and exclusively expressed in cells of monocyte-macrophage lineage.32
This 130-kDa transmembrane glycoprotein binds with high affinity to the Hp-Hb complex that forms in plasma when Hb is released from ruptured erythrocytes and is exposed to plasma Hp.33,34
In vitro, CD163 can be suppressed by proinflammatory mediators such as lipopolysaccharide, interferon-gamma, and tumor necrosis factor-
, whereas interleukin-6 and the anti-inflammatory cytokine interleukin-10 strongly up-regulate CD163.35-38
In vivo, CD163-positive macrophages and soluble CD163 are found during late acute and chronic phases of inflammation.39-42
Recently, in SIVE and HIVE, it has been reported that CD163 labels perivascular macrophages and amoeboid cells as well as a ramified gray matter microglia population that was not positive for other macrophage markers.10
In contrast, another report indicated that CD163 expression in encephalitic brains of HIV-infected patients and SIV-infected macaques was confined to perivascular macrophages and that viral antigen-positive cells were all positive for CD163.13
Considering that the cellular expression of CD163 is not fully characterized in normal or SIV-infected macaques we examined the expression of CD163 in vitro and in vivo by multiple techniques and at varying times after SIV infection and in animals with or without SIVE. Our data show that CD163 is expressed by cells of monocyte/macrophage lineage including perivascular macrophages but not microglia in normal and acutely SIV-infected animals. CD163 expression was detected in ramified but activated microglia surrounding SIVE lesions in chronically infected macaques with severe encephalitis in the presence of Hp-Hb complex in the tissue. CD163 expression could also be induced on microglia in vitro by stimulation with Hp-Hb. The presence of Hp-Hb complex in tissues is suggestive of leakage of the blood-brain barrier that is known to occur in SIVE and HIVE.43,44 We conclude that CD163 is a selective marker of perivascular macrophages in normal macaques and during the early phases of SIV infection. However, later in infection CD163 also labels microglia that have been activated probably as a result of vascular compromise.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Tissues from 23 SIV-infected and 2 uninfected Indian-origin rhesus macaques (Macaca mulatta) obtained from the pathology archives of the Tulane National Primate Research Center were used for these studies. Twelve of the animals were from a time course study. Three animals were euthanized at 7, 14, 21, and 50 days after inoculation. An additional 11 animals with terminal AIDS were selected based on the presence (n = 6) or absence (n = 5) of SIVE. All of the animals except one with SIVE were infected with either SIVmac239 or SIVmac251. The one remaining animal was infected with both SHIV162 and SHIV33A. All animals were infected intravenously with 50 ng p27 of virus. Details on the animals, including time after inoculation and major pathological findings, are provided in Table 1
. Additional information on some of these animals has been published.45
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Localization of SIV-Infected Cells
In situ hybridization for SIV was performed using both riboprobes and random primed DNA probes as described previously.45 Briefly, for RNA in situ hybridization formalin-fixed, paraffin-embedded tissue sections were pretreated in a microwave with citrate buffer (antigen unmasking solution; Vector Laboratories, Burlingame, CA) for 20 minutes at high power according to the manufacturer’s instructions. Thereafter, sections were thoroughly washed, placed in a humidified chamber, and prehybridized at 45°C with hybridization buffer (containing 50% formamide with denatured herring sperm DNA and yeast tRNA at 10 mg/ml each). SIV-digoxigenin-labeled antisense riboprobes (Lofstrand Laboratories, Gaithersburg, MD) were used at a concentration of 10 ng/slide in hybridization buffer and hybridized overnight at 45°C. After hybridization slides were washed with 2x standard saline citrate, 1x standard saline citrate, 0.1x standard saline citrate, and blocking solution was applied. Fab fragments of an anti-digoxigenin antibody from sheep, conjugated with alkaline phosphatase (Roche, Penzberg, Germany) were used to detect digoxigenin-labeled probes. Controls included matched tissues from known positives and negatives and hybridization with digoxigenin-labeled sense RNA labeled with digoxigenin.
For DNA in situ hybridization the DNA probe used was a combination of two plasmids: a subclone of p239SpE3' in phosphate-buffered saline (PBS), which contains tat, rev, env, nef, and a small part of the 3' LTR; and p239SpSp5', which contains gag, pol, vif, vpx, vpr, and the 5'LTR in PBS. This combination provides essentially the entire SIVmac239 genome. The probes were labeled with digoxigenin-11-dUTP by random priming (Boehringer Mannheim, Indianapolis, IN), as described previously.46,47 Hybridization was performed under denaturing conditions to detect both viral DNA and RNA. Formalin-fixed, paraffin-embedded tissue sections were pretreated in a microwave with antigen unmasking solution (Vector Laboratories) for 20 minutes at high power and according to the manufacturer’s instructions. Thereafter, sections were thoroughly washed, placed in a humidified chamber, and prehybridized at 37°C with hybridization buffer (containing 50% of formamide with denatured herring sperm DNA and yeast tRNA at 10 mg/ml each) and washed with 2x standard saline citrate. SIV-digoxigenin-labeled DNA probes were used at 0.5 ng/µl in hybridization buffer and hybridized overnight at 37°C. After hybridization, sections were treated as previously described above for RNA in situ hybridization. Selected positive tissues and serial sections of the test tissues hybridized with plasmid pUC19 labeled with digoxigenin were used as controls.
When immunohistochemistry followed in situ hybridization, the slides were developed using nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate stock solution (Roche, Mannheim, Germany) or Vector blue (Vector Laboratories, Inc.) as chromogen. If immunofluorescence followed the in situ hybridization, 2-hydroxy-3-naphtoic acid fluorescent detection system (Boehringer Mannheim) was used. Briefly, slides were rinsed in detection buffer (0.1 mol/L Tris base, 0.1 mol/L NaCl, 0.01 mol/L MgCl2) and then 200 µl of HNPP/Fast Red TR (10 µg of HNPP in 1 ml of detection buffer plus 10 µl of Fast Red TR solution) was applied. This solution was filtered through a 0.2-µm nylon filter immediately before use, and then the slides were coverslipped and incubated for 30 minutes at room temperature in the dark as described previously.45
Immunophenotype of Brain Macrophages
To define the immunophenotype of SIV-infected and noninfected macrophages, we performed combined in situ hybridization/immunohistochemistry as described previously.21,45,48-50
After in situ hybridization for viral nucleic acid as described above, single- or double-label immunohistochemistry or immunofluorescence were performed using a variety of monocyte/macrophage and microglia-specific markers (Table 2)
.
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After in situ hybridization for SIV, sections were incubated sequentially with the primary, cell-type-specific antibody for 60 minutes at room temperature followed by biotinylated horse anti-mouse or goat anti-rabbit (Vector Laboratories) secondary antibodies, respectively. Finally, sections were incubated with avidin-biotin-complex (ABC Elite or alkaline phosphatase, Vector Laboratories), and the reaction was visualized with AEC (DAKO, Carpinteria, CA) or Vector Red (Vector Laboratories) as the chromogen. As negative control, serial sections were processed identically using equivalent concentrations of irrelevant primary antibodies of the same isotype.
To more carefully examine the cell types infected we performed additional multilabel techniques combining in situ hybridization with immunofluorescence for two cell-type-specific markers as described previously21,45,50
using combinations of antibodies as shown in Table 3
. For these multilabel techniques, in situ hybridization was performed as described above except that the results of in situ hybridization were visualized with HNPP/Fast Red, which fluoresces intensely red. In addition to CD163 and Iba1 we used several additional cell-type-specific antibodies to define specific cell types including NeuN (neuronal nuclei) and MAP2 (microtubule-associated protein-2) for neurons; GFAP (glial fibrillary acidic protein) and peripherin for astrocytes51
; MAB328 (myelin oligodendrocyte-specific protein) and CNPase (cyclic nucleotide phosphodiesterase) for oligodendrocytes; and Glut5 (glucose transporter-5) and RCA-1 (Ricinus communis agglutinin-1) for microglia. To differentiate resting cell populations from activated cell populations we used HLA-DR as an activation marker. To determine the presence of Hp in the brain parenchyma we used anti-human polyclonal Hp (Table 2)
. The cell-type-specific antibodies of differing isotypes or species origin were applied sequentially followed by isotype-specific anti-mouse or species-specific secondary antibodies (if the primary was not directly conjugated) applied simultaneously as previously described.45
The secondary antibodies were coupled with either Alexa 488 (green), Alexa 568 (red), or Alexa 633 (far red) (Molecular Probes, Eugene, OR). After antibody treatment, sections were washed twice for 15 minutes in phosphate-buffered saline (PBS) with 0.2% fish skin gelatin. Finally, the sections were rinsed in doubly distilled water and mounted with aqueous mounting medium.
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Confocal microscopy was performed using a Leica TCS SP2 confocal microscope equipped with three lasers (Leica Microsystems, Exton, PA). Thirty-two to sixty-two optical slices were collected at 512 x 512 pixel resolution. Each individual slice represented 0.2 µm. NIH Image (version1.62; National Institutes of Health, Bethesda, MD) and Adobe Photoshop (version 7.0; Adobe, San Jose, CA) were used to assign colors to the four channels collected: HNPP/Fast Red, which fluoresces when exposed to a 568-nm wavelength laser, appears red; Alexa 488 (Molecular Probes) appears green; Alexa 633 (Molecular Probes) appears blue; and the differential interference contrast (DIC) image is gray scale. The four channels were collected simultaneously. In some tissues and to differentiate between individual cells, To-pro3 (nuclear marker, Molecular Probes) was used at 1 µg/ml, incubated for 5 minutes, and tissues were then washed in PBS. Co-localization of antigens is demonstrated by the addition of colors as indicated in the figure legend.
Microglial Culture to Assess CD163 Expression
Microglia were isolated from normal adult brain as previously described.52 After 1 week in culture the microglia were removed from the flask by gentle rotation on an environmental incubator at 100 rpm for 1 hour. The cells were collected and centrifuged at 400 x g and the cell pellet was resuspended and plated in eight-well chambers (Nagle Nunc International, Rochester, NY) for microscopy studies and in 75-cm2 cell culture flasks for RNA and protein studies (Corning Inc., Corning, NY) as described below. The cells were plated at the same density (same number of cells) in each 75-cm2 flask. The resulting cultures were 99% microglia as evidenced by Iba1 labeling. The remaining cells (less than 1%) were GFAP+ astrocytes (data not shown).
Hp-Hb complex was prepared as follows: blood from normal rhesus macaques was collected in two vacutainer tubes, one containing sodium heparin and the other containing silica clot activator and polymer gel to obtain serum. The red blood cells were separated from the plasma and rinsed twice with isotonic PBS. The packed red blood cells were lysed mechanically by adding 2-mm glass beads and vigorously mixing the tube for 5 to 10 minutes on a vortex (Fisher Scientific, Inc., Pittsburgh, PA). Disruption of the red blood cells was confirmed by microscopic examination. The blood lysate was centrifuged at 15,000 rpm in a microcentrifuge (International Equipment Company, Needham Heights, MA) and the supernatant (soluble Hb) was separated from the particulate pellet. The soluble Hb was mixed at a 1:10 ratio with serum from the same animal. The mixture was left at room temperature for 10 minutes to allow binding of Hp to the free Hb (Hp-Hb complex formation). The complex was further diluted 1:100 in RPMI media before addition to tissue culture media.
After 24 hours of culture, the microglia were treated with Hp-Hb complex, prepared as described above, for either 2 hours followed by culture for 0, 6, 12, or 18 hours or constantly with Hp-Hb complex for 18 hours. In addition, one eight-well chamber culture dish was cultured for 48 hours after 2 hours of incubation with Hp-Hb complex. In those cultures that were only exposed to Hp-Hb complex for 2 hours, at the end of this time the cultures were rinsed three times with tissue culture media (RPMI-10% fetal calf serum) to remove the Hp-Hb complex. The control microglia tissue culture received the same serum used to make the Hp-Hb complex at the same concentration (final concentration 1:1000 in the tissue culture media). After the culture period the cell cultures in chambers for microscopy were washed with warm PBS two times and fixed in 2% paraformaldehyde for 10 minutes, and immunostained as described above with anti-CD163 and anti-Iba1 antibodies to identify macrophages13 and microglia,53 respectively. This experiment was performed using cells from three different animals and each microglia culture was examined in quadruplicate. For RNA and protein extraction one 75-cm2 flask was used for each assay at each time point. The extraction of RNA and protein were performed as described below.
Quantitative Real-Time SYBR Green One-Step Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Gene expression for CD163 in cultured microglia from three animals was evaluated by quantitative real-time SYBR green one-step RT-PCR assay (QRT-PCR). Total RNA was extracted from untreated and Hp-Hb-treated microglial cultures for 0, 6, 12, or 18 hours or constantly with Hp-Hb complex for 18 hours and was assayed in triplicate wells. Each QRT-PCR reaction (25 µl) contained the following: 2x Master mix without uracil-N-glycosylase (12.5 µl), reverse transcriptase (0.25 µl), target forward and reverse primer, and total RNA (25 ng) quantified spectrophotometrically based on A260:A280 ratios. Forward and reverse primer sequence, concentration, and product size including β-actin are shown in Table 4
. The PCR amplification was performed in the ABI Prism 7700 sequence detection system (PE Applied Biosystems, Foster City, CA). Thermal cycling conditions were 50°C for 30 minutes, 95°C for 15 minutes, followed by 40 repetitive cycles of 95°C for 15 seconds, 55°C for 30 seconds, 72°C for 30 seconds. As a normalization control for RNA loading, parallel reactions in the same multiwell plate were performed using β-actin mRNA.
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CT value was determined by subtracting the β-actin CT value for each sample from the target CT value of that sample. Calculation of CT involved using the highest sample CT value (ie, sample with the lowest target expression) as an arbitrary constant to subtract from all other CT sample values. Fold changes in the relative gene expression of target was determined by evaluating the expression, 2–CT. Immunoprecipitation and Western Blotting
Cultured microglial cells were dislodged, pelleted, and protein extraction was performed in 
250 µl of lysis buffer (Cell Signaling Technology, Inc., Beverly, MA) containing 20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L Na2EDTA, 1 mmol/L EGTA, 1% Triton, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β-glycerophosphate, 1 mmol/L Na3VO4, 1 µg/ml leupeptin, protease inhibitor cocktail, and phosphatase inhibitor cocktail (Sigma Chemical Co., St. Louis, MO). The protein lysates were first precleared with 5 µl of normal rabbit immunoglobulin for 1 hour and then immunoprecipitated with 10 µl of a rabbit polyclonal antibody against CD163 (Santa Cruz Biotechnology, Santa Cruz, CA), overnight at 4°C followed by incubation with 30 µl (50% w/v) of protein G agarose beads (Millipore Corp., Billerica, MA) at 4°C for 4 to 5 hours. The supernatant was removed and transferred to a separate 1.5-ml microcentrifuge tube and immunoprecipitated using a goat polyclonal antibody against β-actin (5 µl) (Santa Cruz Biotechnology). The immunoprecipitation for β-actin was performed at 4°C overnight on a shaker. Immunoprecipitated CD163 and β-actin proteins were heat denatured for 5 minutes at 100°C in sample loading buffer containing 62.5 mmol/L Tris-HCl, 5% 2-mercaptoethanol, 10% glycerol, 2% sodium dodecyl sulfate, and bromophenol blue, resolved on 8% and 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, respectively, and transferred to 0.2-µm nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). The membranes were probed with a rabbit polyclonal primary antibody against CD163 (Santa Cruz Biotechnology), and β-actin (Santa Cruz Biotechnology) followed by a anti-rabbit polyclonal horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology). Membranes were treated with West Pico chemiluminescent substrate (Pierce Biotechnology Inc., Rockford, IL) for 5 minutes and the signal was developed by exposing the membrane to Kodak X-OMAT film (Eastman-Kodak, Rochester, NY) for 5 and 10 minutes for CD163 and β-actin, respectively.
Flow Cytometry
Flow cytometry on peripheral blood was used to confirm CD163 expression on circulating monocytes. One hundred µl of EDTA-treated fresh whole blood from eight uninfected and six SIVmac239- or SIVmac251-infected rhesus macaques were stained with phycoerythrin-conjugated anti-human CD163 monoclonal antibody (mAb) (Mac2–158; Trillium Diagnostics, LLC., Scarborough, ME) and PerCP-Cy5.5-conjugated anti-human CD14 mAb (M5E2; BD Pharmingen, San Jose, CA) for 15 minutes at room temperature. Red blood cells were then lysed using a Coulter Immunoprep reagent system and a TQ-prep Workstation (Beckman Coulter, Hialeah, FL). The cells were then washed with 2% fetal calf serum-PBS, fixed with 1% formaldehyde-PBS, and were analyzed on a FACSCalibur (Becton Dickinson, Franklin Lakes, NJ).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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As indicated in Table 1
, of the 11 animals with AIDS 6 had SIVE characterized by nodular perivascular aggregates of mononuclear cells and multinucleated giant cells along with variable areas of gliosis as described previously (Supplemental Figure S1A at http:
ajp.amjpathol.org).21,54-56
The SIVE lesions were observed in both gray and white matter of brain but predominantly in white matter. The remaining five animals with AIDS had no histopathological abnormalities in the CNS, however, all 11 animals with AIDS had AIDS-defining lesions such as multiple opportunistic infections and generalized lymphoid depletion. The 12 animals from which tissues were collected within 50 days of infection had mild perivascular cuffs in the CNS (Supplemental Figure S1B at http:ajp.amjpathol.org) as described previously.21,54,55,57
CD163 and SIV Infection in Vivo
Localization of SIV-infected cells in brain was examined by in situ hybridization using both riboprobes and random primed DNA probes to detect viral RNA and/or DNA. To define the immunophenotype of infected brain macrophages, we performed single- and double-labeled immunohistochemistry or multilabel confocal microscopy for CD163 and a variety of cell markers combined with in situ hybridization for SIV (Tables 2 and 3)
. We also confirmed that CD163 was expressed exclusively on circulating CD14+ monocytes of both normal and SIV-infected macaques (Supplemental Figure S2 at http:ajp.amjpathol.org).
In Normal Brain
In normal brain the expression of CD163 was observed in meningeal, choroid plexus, and perivascular macrophages (Figure 1, A–C)
. No labeling of cells in the brain parenchyma where microglia reside was detected in normal controls (Figure 1C)
. In contrast, using a variety of microglial markers (Iba1, Glut-5, and RCA-1, which label resting and activated microglia) we were able to clearly label microglia with elongated nuclei, scanty perikaryon, and multiple fine cytoplasmic processes characteristic of resting microglia (Figure 1D)
. Double-label immunohistochemistry using CD163 plus Iba1 clearly demonstrate that the Iba1+ cells in the parenchyma were not CD163+ (Figure 1E)
.
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Similar to normal brain, in acutely SIV-infected macaques, the expression of CD163 was observed in meningeal, choroid plexus, and perivascular macrophages and no positive cells resembling microglia were observed in the brain parenchyma. In addition, consistent with previous observations using other macrophage markers58
increased numbers of CD163+ cells consistent with perivascular macrophages were present corresponding to the mild perivascular cuffs observed (Supplemental Figure S1B at http:ajp.amjpathol.org).
In Terminal AIDS without SIVE
In animals with terminal AIDS but no histological evidence of SIVE the expression of CD163 was the same as for acutely SIV-infected animals.
In Terminal AIDS with SIVE
In contrast to the other groups of animals, in animals with SIVE the expression of CD163 in the CNS was not limited to the three populations of brain macrophages, but was also observed in cells morphologically compatible with activated microglia (Figure 1F)
. Generally, the staining pattern of putative activated microglia was focal or focally extensive but always near a SIVE lesion. This was true in both the gray and white matter. Most of the CD163+ cells had short crenellated cytoplasmic projections and there appeared to be progressive diminution of cytoplasmic projections the closer the proximity to an SIVE lesion. No such labeling was seen in brains of normal animals nor acutely or chronically SIV-infected animals without encephalitis. In brain of acutely infected macaques and in macaques with terminal AIDS without SIVE, the expression of CD163 was restricted to meningeal, choroid plexus, and perivascular macrophages (similar to normal brain). To confirm the impression that CD163 was labeling microglia in the vicinity of SIVE lesions we used double-label immunofluorescence plus confocal microscopy combining CD163 and Iba1; this demonstrated co-localization of both markers indicating that CD163 was labeling microglia in encephalitic brain (Figure 2A)
.
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ajp.amjpathol.org illustrates this for astrocytes) providing additional support to the notion that the CD163+ cells were microglia.
In acutely infected macaques and in macaques with terminal AIDS without SIVE, SIV-infected cells were infrequent whereas in animals with SIVE large numbers of infected mononuclear cells and multinucleated giant cells were present within lesions as described previously.21,54,55,57,58
In the lesions most of the individual macrophages and multinucleated giant cells expressed CD163 and these cells were often infected with SIV (Figure 2B)
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CD163 Is Expressed on Activated Microglia
We hypothesized that the CD163+ microglia were activated based on their morphology. To further evaluate this we examined the expression of HLA-DR (human leukocyte antigen) on multiple cell types. In normal brain, HLA-DR expression was restricted to perivascular cells whereas in SIVE a large number of HLA-DR+ cells were observed, which were associated with SIVE lesions (Figure 2C)
. HLA-DR+ cells had a round to oval morphology within the lesions and a more ramified morphology compatible with microglia in the area around the lesions. We then performed triple-label studies with Iba1, HLA-DR, and CD163 (Figure 3)
. This revealed that the CD163+ microglia (defined by morphology and Iba1+) were also positive for HLA-DR indicating that CD163 was expressed by activated microglia.
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We hypothesized that expression of CD163 on microglia was a result of damage to the blood-brain barrier and formation of Hp-Hb complexes that up-regulated CD163 expression. To assess this possibility we examined the presence of Hp in relation to the presence of activated microglia by single-, double- and triple-label immunofluorescence. In brain of normal animals, acutely SIV-infected animals and SIV-infected animals without SIVE, labeling for Hp was limited to the lumen of vessels (Figure 4A)
. In contrast, in encephalitic brain, Hp labeling also occurred in the brain parenchyma in areas where CD163 was expressed by microglia (Iba1+ CD163+) (Figure 4, B–D)
. Clear co-localization of Hp and Iba1 (Figure 4B)
and Hp and CD163 (Figure 4C)
were observed. Moreover, clear co-localization of CD163, Iba1, and Hp (Figure 4D)
was also observed. These data strongly suggested that breakdown of the blood-brain barrier was associated with the activation of microglia and their expression of CD163.
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To further evaluate the connection between Hp-Hb and CD163 expression on microglia we performed in vitro experiments on the effect of Hp-Hb on microglia. Microglia were prepared and cultured in eight-well chambers with or without Hp-Hb complex as described in the Materials and Methods. At all times the cultures were 99 to 100% positive for Iba1 (Figure 5A)
. In contrast, expression of CD163 was observed only in cells treated with the Hp-Hb complex for at least 2 hours (Figure 5C)
. No CD163 expression was observed in untreated wells or in wells treated with serum only. This result suggests that the interaction of the Hp-Hb complex is required to trigger the up-regulation of CD163, the receptor for Hp-Hb complex in cultured microglia in vitro.
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To confirm that the activation of microglia was associated with the presence and up-regulation of CD163 mRNA and protein we treated in vitro-cultured microglia isolated from rhesus macaque brain (frontal cortex and mid brain) with Hp-Hb complexes (100 µl) for either 2 hours followed by culture for 0, 6, 12, 18 hours or constantly for 18 hours and performed quantitative real-time SYBR Green one-step RT-PCR and immunoprecipitation/Western blotting, respectively, for CD163 mRNA and protein analysis. Treatment for either 2 hours followed by culture for 0, 6, 12, or 18 hours or 18 hours constant treatment with Hp-Hb complexes did not have any effect on CD163 mRNA transcription (data not shown). The data indicates that CD163 mRNA is constitutively expressed and is not influenced by the presence of Hp-Hb complexes. However, these findings along with the fact that the CD163 protein was detectable by immunofluorescence/confocal microscopy only after treatment with Hp-Hb complexes (Figure 5C)
led us to hypothesize the presence of posttranscriptional regulation. To confirm the latter hypothesis, total protein was extracted at each time point and changes in CD163 protein expression was evaluated using immunoprecipitation/Western blotting. Figure 5B
shows the band density for CD163 and β-actin at each time point and the bars represent the ratio of CD163 to β-actin levels. Interestingly, we observed no significant increase in CD163 protein expression in microglia cultured in vitro for 0, 6, 12, or 18 hours after a 2-hour treatment with Hp-Hb complexes. In contrast, constant treatment of in vitro-cultured microglia with Hp-Hb complexes for 18 hours resulted in a significant increase (3.4-fold) in CD163 protein expression compared to the 2-hour treatment/incubation time points. The data clearly indicates that continued stimulation of in vitro-cultured microglia with Hp-Hb complexes (100 µl) results in increased translation of the CD163 protein from a message (mRNA) that is basally expressed, implying posttranscriptional regulation.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Previous studies in macaques with SIV encephalitis demonstrated that CD163 was expressed by perivascular macrophages and/or a population of hyperramified microglia in the gray matter.10,13 In the present study, we examined the expression of CD163 in brain of normal and SIV-infected macaques with or without encephalitis. We determined that the expression of CD163 in normal animals and SIV-infected animals without encephalitis is limited to perivascular macrophages. In contrast, in animals with SIVE, CD163 was expressed by perivascular macrophages as well as activated microglia. Using multilabel confocal microscopy we demonstrated co-localization of CD163 with Iba1 and HLA-DR, indicating that CD163 was up-regulated on activated microglia surrounding SIVE lesions.
Based on this we hypothesized that the localized expression of CD163 on microglia was a result of immune activation possibly associated with damage to the blood-brain barrier, which would facilitate formation of Hp-Hb complexes and could increase CD163 expression via the high affinity between Hp-Hb complexes and CD163.34 To assess this possibility we examined the presence of Hp in the CNS of normal macaques and SIV-infected animals with and without SIVE. In brain of normal macaques, acutely SIV-infected animals and SIV-infected animals without SIVE, the presence of Hp was limited to the lumen of vessels. In contrast, in animals with SIVE, Hp was also present in the brain parenchyma in areas where CD163 was up-regulated by microglia (Iba1+ CD163+).
To examine the relationship between extravascular Hp-Hb complexes and up-regulation of CD163 we performed in vitro studies. This work demonstrated that CD163 protein was undetectable on microglia by immunofluorescence under normal culture conditions but was detectable by immunoprecipitation/Western blotting. After treatment of microglia with Hp-Hb complexes up-regulation of CD163 was shown by immunoprecipitation/Western blotting and CD163 became detectable by immunofluorescence. Together these data strongly suggest that Hp-Hb complexes bind to existing, but rare, CD163 molecules on microglia that results in up-regulation of CD163 to a level detectable by immunofluorescence and immunohistochemistry. This finding prompted us to further investigate the mechanisms by which Hp-Hb complexes regulate CD163 protein expression in microglia. Interestingly, despite evidence from two different techniques that up-regulation of CD163 occurs in microglia as a result of exposure to Hp-Hb complexes, no changes in CD163 mRNA expression were observed suggesting the presence of posttranscriptional or translational regulation or alterations in intracellular trafficking or degradation of CD163. Similar increases in protein synthesis in the absence of a concomitant increase in mRNA synthesis have been previously described for ferritin and insulin-like growth factor-1.61,62 Hb released from ruptured erythrocytes is a molecule with toxic and proinflammatory properties and consequently requires immediate clearance from the circulation and areas of tissue damage.63 In the present study, the strong expression of CD163 on macrophages and microglia in encephalitic brain may enable these phagocytic cells to clear Hb/Hp complexes and in doing so perform an anti-inflammatory role. Finally, the up-regulation of CD163 protein expression in response to Hp-Hb complexes without any increase in its cognate mRNA represents a highly efficient mechanism in the brain and can be attributed to a need to hasten the clearance of Hb and protect the brain from its untoward toxic effects.
We conclude that CD163 is a selective immunohistochemical marker of perivascular macrophages in normal macaques and during the early phases of SIV infection. However, later in infection in animals with encephalitis, CD163 also labels microglia, probably activated as a result of vascular compromise and the generation of Hp-Hb complexes.
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Acknowledgements |
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Footnotes |
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Supported in part by the Public Health Service (grants RR00164, RR016930, RR019607, and NS30769).
Supplemental material for this article can be found on http://ajp.amjpathol.org.
Accepted for publication December 6, 2007.
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