Published online before print March 16, 2007
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* Molecular and Integrative Neurosciences Department and
Core Microscopy Facility, The Scripps Research Institute, La Jolla, California, USA
1 Correspondence: Molecular and Integrative Neuroscience Department, The Scripps Research Institute, 10550 North Torrey Pines Rd., SP30-2030, La Jolla, CA 92037, USA. E-mail: hsfox{at}scripps.edu
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key Words: HIV • AIDS • macrophage
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Macrophages, which can produce numerous products potentially harmful to the CNS, are prime candidates in mediating indirect damage to the CNS initiated by HIV infection. Macrophages in brain and other tissues originate from circulating blood monocytes, which in turn are generated from precursor cells in the bone marrow. In addition to the predominant population of blood monocytes (CD14+CD16–), a subset of monocytes also expresses CD16 (Fc
RIII). In HIV-1-associated dementia (HAD), an increase in this subset (CD14+CD16+) of monocytes is found in the blood [3
]. In the brain during HIV-1 encephalitis (HIVE), cells of a similar phenotype accumulate in perivascular locations (known as perivascular macrophages) and are often infected with HIV-1. Whether the CD16+ monocytes in the blood preferentially become CD16+ macrophages in tissue is not known. However, recent data reveal that such cells have a high ability to migrate across an endothelial cell layer, as well as reverse-transmigrate from an ablumenal to lumenal location [4
]. This monocyte subset is also intriguing in regards to neuropathogenesis, as it can produce high levels of the proinflammatory cytokines, such as TNF-
and IL-1 [5
6
7
], which may play a major role in neuronal death. TNF- and IL-1 increase the permeability of the blood-brain barrier (BBB), subsequently allowing additional HIV-infected monocytes to enter the brain [8
]. Moreover, TNF- up-regulates the expression and release of various chemokines in the CNS, such as MCP-1/CCL2, a potent chemoattractant for monocytes [8
, 9
].
Recent studies have shown that CD16+ monocytes express high levels of CX3CR1, the receptor for fractalkine/CX3CL1, and low levels of CCR2, a receptor for CCL2 [10 11 12 ]. These cells show efficient transendothelial migration in response to CX3CL1 but not CCL2 [10 , 11 ]. These studies identified CX3CL1 as a major chemokine and adhesion molecule, mediating CD16+ monocyte arrest and migration. However, it has also been shown that increased CCL2 expression in the cerebrospinal fluid (CSF) occurs in conjunction with HAD and HIVE. In vitro studies have revealed that CCL2 can induce the translocation of monocytes across a model of the BBB [13 14 15 ]. These chemokines likely contribute to an increase in monocyte entry, but their role, as well as that of other molecules, in the macrophage accumulation that occurs in neuroAIDS is unknown. Further unknown mechanisms may also participate in driving blood monocytes into the brain. Still, additional means likely contribute to the increased number of macrophages in the brain, such as mechanisms that prevent macrophage egress and means that prevent monocyte/macrophage death. Although monocyte entry into the brain is the subject of much focus, mechanisms that retain macrophages in the brain are under-explored.
Osteopontin (OPN) is an extracellular protein involved in differentiation and immune cell activation as well as cell attachment and migration [16
]. OPN has two receptors on monocytes/macrophages and other cellular targets: CD44 variant 6 (CD44v6) and certain integrins, most notably, the (V) and ß(1) integrins, which also play crucial roles in monocyte transmigration [17
]. CD44v6 mediates macrophage chemotactic migration in response to OPN [18
], as well as plays an important role in monocytes differentiating to macrophages [19
]. In addition to its involvement in migration, OPN prevents the apoptosis of endothelial cells, melanocytes, renal epithelial cells, and IL-3-dependent hematopoietic cells [16
, 20
21
22
].
We have found recently that expression of OPN was increased in the brains of monkeys with SIV encephalitis (SIVE), a nonhuman, primate model of HIV-induced brain disease [23 ]. Here, we report that OPN is also increased in the plasma and CSF of animals with SIVE. We hypothesized that OPN was involved in the accumulation of macrophages in the brain in HIV-induced CNS disease and here, have analyzed the role of OPN in three distinct aspects of monocyte/macrophage biology: chemotaxis, prevention from recirculation, and protection from apoptosis. In these ways, OPN could aid in monocyte/macrophage infiltration and accumulation in the brain, the pathological substrate of HAD.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Rhesus monkey plasma and CSF collection
Rhesus macaques had plasma and CSF samples collected under ketamine anesthesia. For plasma, blood was placed in EDTA-treated tubes and centrifuged for separation from cells and erythrocytes. CSF samples were also centrifuged for elimination of cells.
OPN measurement
The levels of OPN in plasma and CSF at the time of necropsy from monkeys were measured using a human OPN ELISA (Immuno-Biological Laboratories, IBL-America, Minneapolis, MN, USA). Samples were analyzed in duplicate according to the manufacturer’s instructions. Plasma and CSF (n=30) samples were also analyzed from different animals prior to infection to establish baseline OPN levels.
Isolation of PBMCs and purification of human monocytes
Normal human blood was obtained from The Scripps Research Institute, Normal Blood Donor Service (NBD; La Jolla, CA, USA) into an EDTA-containing syringe (1 ml 7.4% EDTA per 60 ml blood drawn). To qualify for the NBD pool, volunteers were between 18 and 65 years of age, weighed at least 110 pounds (50 kg), had blood pressure between 90 and 180 mm Hg systolic and 50 and 100 mm Hg diastolic, have not had major surgery within the past 6 months, had an adequate hematocrit on recent testing, and had negative screening for HIV and Hepatitis B and C at initial screening and annually thereafter. Blood was processed using OptiPrep (OptiPrepTM cell separation media, maximum density=1.320 g/ml; maximum osmolality=170 mOsm, Accurate Chemical, Westbury, NY, USA) using the manufacturer’s recommendations for enhancement of monocytes but scaled up for isolation of more blood. The monocyte-enriched PBMCs were then washed with RPMI with 2% FCS and pelleted, platelets were removed, and cells were quantified using the Z2 series Coulter Counter (Beckman Coulter, Fullerton, CA, USA). The resulting cell suspension contained between 40% and 60% monocytes (determined by CD14 APC FACS staining), and the remaining cells were mostly T and B cells. For some experiments, we used these monocyte-enriched PBMCs, but for others, we proceeded to isolate pure monocytes from the enriched PBMCs using the MACS Human Monocyte Isolation Kit II (Miltenyi Biotec, Auburn, CA, USA) using the manufacturer’s instructions. Monocytes were counted and assayed for purity by FACS using the mouse antihuman CD14 APC antibody (Beckman Coulter/Immunotech; purity >95%).
Chemotaxis assay
Monocyte-enriched PBMCs (using the OptiPrep method above) were used for the chemotaxis assays. Cells were resuspended at 106 cells per ml RPMI, and 3 ml was added to the top of each six-well transwell (3 µm pore size, Corning, Corning, NY, USA). Below the insert, 3 ml RPMI, containing the indicated concentration of chemokine (CCL2, CX3CL1, or OPN, all purchased from R&D Systems, Minneapolis, MN, USA), was added. The cells were allowed to migrate for 3 h (37°C, 5% CO2). Cells were collected from the bottom chamber, counted, stained with CD14 APC and mouse antihuman CD16 FITC antibody (BD PharMingen, San Diego, CA, USA), and examined by FACS. The migration indices were measured as fold-over-control (no chemokine) migration.
Preparation of collagen and HUVECs
Millicell six-well CM (hydrophilic polytetrafluoroethylene) cell culture inserts (Millipore, Billerica, MA, USA) with a 0.4-µm pore size were used. Monomeric collagen (Cohesion Vitrogen 100, Invitrogen/Gibco, Palo Alto, CA, USA) was prepared as recommended (8x Vitrogen, 1x 10x PBS, and 1x 0.1 N NaOH, to a final pH 7.0), and 1.5 ml collagen was added to the top of the CM filters and was allowed to polymerize at 37°C for 1.5 h. Gels were coated with 1 ml fibronectin at 37°C for 15 min (Invitrogen/Gibco; 50 µg/ml in normal saline). HUVEC (Cascade Biologics, Portland, OR, USA) monolayers were plated at 250,000 cells per well (50% confluency) and grown in Medium 200 with low serum growth supplement (Cascade Biologics) on the collagen for 2–3 days until confluent (see 
Fig. 2A
for schematic).
 View larger version (18K): [in a new window] |
Figure 1. OPN is not a classic chemokine for monocytes. Control is shown in hatched bars, OPN in shaded bars, CX3CL1 (fractalkine) in solid bars, and CCL2 in open bars. Values are relative to control, and shown is one representative experiment out of three performed. (A) Shown are the CD14+CD16+ monocyte migration indices. CX3CL1 is known to be a potent chemoattractant for CD14+CD16+ monocytes. All OPN indices shown are similar to control. (B) Shown are the CD14+CD16– monocyte migration indices. CCL2 is known to be a potent chemoattractant for CD14+CD16– monocytes. All OPN indices shown are similar to control. OPN (1, 5, and 20 nM) correspond to concentrations of 65, 325, and 1300 ng/ml; 1, 5, and 20 nM CX3CL1 corresponds to concentrations of 90, 450, and 1800 ng/ml; 1, 4, and 20 nM CCL2 corresponds to concentrations of 8.7, 35, and 87 ng/ml.
|
 View larger version (98K): [in a new window] |
Figure 2. In vitro model for the examination of the reverse transmigration of human monocytes. (A) Diagram of the in vitro filter model to study reverse transmigration. (B and C) Tight junctions between adjacent HUVECs; original calibration bars: 100 nm and 200 nm, respectively. (Individual insets show enlargements of the junctions.) (D–F) Three figures illustrating monocytes in transit through the layer of endothelial cells (*) into the collagen matrix; original calibration bars: 2 µm, 2 µm, and 1 µm respectively.
|
Electron microscopy (EM)
The same procedure was used to set up the collagen and migration/reverse migration procedure but was scaled down to use the CM filters in 24-well plates. Samples were fixed for EM based on a standard protocol [30
]. Following the designated time interval for experimental treatment(s), the culture medium was removed, and the entire filter complex was fixed in 2.5% glutaraldehyde in 0.1 M Na cacodylate buffer (pH 7.3), washed in buffer, and then fixed in 1% osmium tetroxide in 0.1 M Na cacodylate buffer. The intact filters were subsequently treated with 0.5% tannic acid followed by 1% sodium sulfate, washed in buffer, and then dehydrated in a graded ethanol series. During the initial 50% ethanol step, the filter holders were inverted, and the filter with its associated sandwich of cells and collagen was removed carefully from the plastic frame by cutting around the perimeter of the frame-base using a #11 scalpel blade. The intact filter "sandwich" was fully dehydrated, cleared in propylene oxide, and infiltrated overnight with Epon/Araldite resin (Electron Microscopy Sciences, Hatfield, PA, USA). The circular filters with intact sample layers still on top were then sliced into long strips with a #11 scalpel blade and polymerized overnight in embedding molds. Semithick (1–2 µm) sections were stained with toluidine blue for general assessment in the light microscope. Thin sections (70 nm) were cut on a Reichert Ultracut E (Leica, Deerfield, IL, USA) using a diamond knife (Diatome, Electron Microscopy Sciences), mounted on parlodion-coated, copper slot grids, and stained in uranyl acetate and lead citrate. Sections were examined on a Philips CM100 transmission electron microscope (FEI, Hillsbrough, OR, USA) and data documented on Kodak SO-163 film for later analysis. Negatives were scanned at 600 lpi using a Fuji FineScan 2750xl (Enovation Graphics, Chicago, IL, USA) and converted to tiff format for subsequent handling in Adobe Photoshop.
Apoptosis assay and FACS
Pure monocytes were grown in serum-free RPMI media at 106 cells/ml under nonadherent conditions in VueLife (fluorinated ethylene propylene) bags (American Fluoroseal Corp., Gaithersburg, MD, USA) for 24 h (37°C, 5% CO2), with or without the addition of hrOPN (750 ng/ml). Control protein (HSA, 750 ng/ml) was also used in some experiments for the cultures without OPN and yielded results indistinguishable from those without albumin. After 24 h, cells were harvested and stained by FACS using the Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences, Franklin Lakes, NJ, USA). The percents of alive [Annexin V-FITC-negative and propidium iodide (PI)-negative] and apoptotic (Annexin V-FITC-positive and PI-positive) cells were measured. In addition, monocytes were stained with mouse antihuman CD44v6 biotin-conjugated mAb (Invitrogen/Biosource) and mouse antihuman integrin Vß3 biotin-conjugated mAb (Chemicon, El Segundo, CA, USA), followed by streptavidin-allophycocyanin conjugate (BD PharMingen), and the geometric mean fluorescent intensities were measured by FACS. The fluorescent intensity measurements were calibrated by the use of isotype control (mouse IgG1 biotin control). Cells were acquired on a FACSCalibur using a zero-threshold for cell size.
Statistical analysis
Statistical analyses used the Prism 4 software (GraphPad Software, Inc., San Diego, CA, USA) and Delta Graph (Red Rock Software, Salt Lake City, UT, USA).
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
View this table: [in a new window] |
Table 1. OPN Levels in the Plasma and CSF of Rhesus Monkeys
|
Under the hypothesis that OPN does not directly influence the accumulation of macrophages in the brain as a chemokine, we sought to examine other potential mechanisms, for instance, the ability of OPN in preventing macrophages from leaving the brain to recirculate in the body. Macrophage accumulation and retention in the brain were investigated using a model system of endothelial cells grown on a collagen gel (Fig. 2A
), in which monocytes can migrate beneath the endothelial cell layer as well as reverse-transmigrate back, modified from a characterized model [4
, 26
27
28
29
] (see Materials and Methods). Monocytes were added to the top (endothelial surface) layer and allowed to transmigrate for 90 min, at which time 
70% of the monocytes migrated across the monolayer into the collagen. Examination by EM demonstrated a confluent layer of endothelial cells, which possessed tight and gap junctions between continuous cells (Fig. 2B
and 2C)
as well as indications of basal lamina formation. Monocytes were readily visible above and below the HUVEC layer. It is more important that monocytes were found in the process of migrating between endothelial cells of this confluent layer (Fig. 2D
2E
2F)
.
Following the 90-min monocyte migration, cultures were then washed extensively. Media were replaced and OPN (at 750 ng/ml) added to the top or bottom chamber. Cultures were maintained for an additional 2 days to allow for reverse transmigration (monocytes moving from the ablumenal (tissue side) to the luminal (blood side) of the in vitro endothelial monolayer. Cells on both sides of the endothelial layer were recovered, quantified, and phenotyped for CD14 and CD16 expression.
In control wells, an average of 39% of total monocytes reverse-migrated, whereas in the presence of OPN in the bottom chamber, 20% of cells reverse-transmigrated (Fig. 3A ). Thus, OPN significantly decreased the percentage of cells that recrossed the endothelial layer compared with control (Fig. 3A ; Student’s t-test, P=0.02). Although reverse transmigration of the CD14+CD16– and CD14+CD16+ subsets of monocytes appeared to decline, only the inflammatory CD14+CD16+ subset reached statistical significance with a 51% reduction (Fig. 3C ; Student’s t-test, P=0.01). Thus, more monocytes were trapped in the collagen when OPN was added underneath the collagen. When OPN was added to the top chamber (above the endothelial layer), no difference between control and OPN was found (data not shown).
 View larger version (12K): [in a new window] |
Figure 3. OPN decreased the percent of monocytes that reverse-transmigrated. (A) OPN (750 ng/ml) significantly decreased the percent of total monocytes, which reverse-transmigrated compared with control (Student’s t-test, P=0.02). (B) There is a decrease (however nonsignificant, P>0.05) in the percent of CD14+CD16– monocytes that reverse-transmigrated compared with control. (C) A significant decrease in the amount of CD14+CD16+ monocytes that reverse-transmigrated was demonstrated between OPN and control (Student’s t-test, P=0.01). The mean of three experiments with SEM is shown.
|
 View larger version (24K): [in a new window] |
Figure 4. OPN protects monocytes from apoptosis. (A) In eight separate experiments (EXP 1–8), human monocytes were grown in nonadherent culture conditions for 24 h. Shown is the percent of living cells (Annexin V- and PI-negative cell population) in RPMI or cultured with 750 ng/ml OPN. The fold is the increase in OPN compared with RPMI only. (B) The averages and SEM are shown. OPN increased the percentage of viable monocytes significantly (4.7-fold, P=0.0005).
|
 View larger version (79K): [in a new window] |
Figure 5. FACS staining showed OPN increased the percent of live cells. Annexin V-FITC and PI staining of two representative patients are shown (A and B). The left panel shows the cells cultured in RPMI for 24 h, and the right panel shows cells, which were cultured in RPMI with 750 ng/ml OPN for 24 h. Note that OPN increases live cell percentages on the lower-left quadrant compared with control.
|
Vß3, was also examined on the monocytes after 24 h in culture in RPMI or with OPN (Fig. 6A
and 6B
). CD44v6 and integrin Vß3 expression was increased consistently when monocytes were cultured with OPN, although levels were variable between individuals.
 View larger version (38K): [in a new window] |
Figure 6. OPN increases CD44v6 and integrin expression. Arrows indicate the histograms for cells cultured in RPMI media (gray line) and OPN (black line). (A) CD44v6 expression in monocytes, which were gated for Annexin V-negative and PI-negative (alive) cells after 24 h of culture, is shown in three patients. (B) Integrin Vß3 in monocytes, which were gated for Annexin V-negative and PI-negative (alive) cells after 24 h of culture, is shown in three patients.
|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
A normal, physiological trafficking of monocytes into the brain occurs to replenish perivascular macrophages, which turn over on a regular basis [35 ]. Monocytes can also enter the brain through chemokine-induced entry during inflammation, such as following CCL2 or CX3CL1 expression [14 ]. Although monocyte entry into the brain is the subject of much study [1 , 35 ], mechanisms that maintain macrophages in the brain themselves can also lead to macrophage accumulation, as increased entry is only one side of the equation in increased cell number. Here, we have shown two additional mechanisms of macrophage accumulation: prevention of monocyte/macrophage egress from the brain and prevention of monocyte death.
In normal conditions, monocytes continually enter the brain to become perivascular macrophages; however, the fate of the older perivascular macrophages is unknown. They may senesce, although apoptosis has not been observed [36 , 37 ], or they may become parenchymal microglia, which is likely a rare event [38 ]. The perivascular macrophages may re-enter the bloodstream or traverse the pial surface to enter lymphatics; however, examining these possibilities has been challenging experimentally. Regardless, preventing re-entry or death, as we have shown here with OPN, will lead to cell accumulation.
Many studies have examined chemokine-induced migration of monocytes across vessels. Although many studies in neuroAIDS have focused on CCL2, CCL2 is not effective in inducing transmigration of the CD16+ monocyte subset, which has been linked to neuroAIDS, whereas CX3CL1 is capable of inducing their transendothelial migration but has not been examined extensively in this disease [11 , 18 ]. Although several studies suggest that OPN facilitates monocyte infiltration during infection or injury [39 40 41 ], we have not been able to confirm this in our model. Our findings that OPN induces a decrease in monocyte reverse transmigration and protects monocytes from apoptosis provide mechanisms by which OPN expression can lead to macrophage accumulation. This may be the case in conditions in which OPN is increased, such as in neuroAIDS.
It is interesting that an intracellular form of OPN has been described, which if absent, impairs macrophage chemotaxis when chemokines working through G-protein-coupled receptors are used [42 ]. Here, extracellular OPN was added, limiting the exploration of this possibility. Thus, it remains possible that during neuroAIDS, monocytes in the periphery may have higher levels of intracellular OPN, which primes them to enter more readily and take residence in the brain.
Recent studies have shown that basal-to-apical transendothelial migration is dependent on p-glycoprotein multidrug-resistance 1 (MDR-1) [28 ]. Antibodies to MDR-1 were identified as potent inhibitors of reverse transmigration of mononuclear phagocytes in an in vitro model of a vessel wall. However, the exact mechanism by which MDR-1 acts to facilitate migration remains unclear. Here, we have demonstrated another means of altering reverse transmigration of macrophages through OPN, which may use adhesion properties through interaction with its receptor CD44v6, which is found on macrophages, to trap cells in tissues.
Although this manuscript was in review, a study was published online, which showed OPN promoted the survival of activated mouse T cells and linked increased OPN to disease progression in models of multiple sclerosis (MS) [43 ]. That study used 2000 ng/ml or 10,000 ng/ml OPN to demonstrate survival of T cells, whereas here, we used 750 ng/ml, similar to the levels of OPN, which we found in the plasma during SIVE (600.1±122.5 ng/ml), to demonstrate that OPN promotes the survival of human monocytes. It is interesting that CSF levels of OPN during SIVE are higher (1244.4±337.1 ng/ml). It will be important in future studies to determine the levels of OPN, which immune and other cells are exposed to in the brain itself.
We have also found that OPN increased the survival of monocytes. It is known that soluble and extracellular matrix-immobilized OPN can protect against apoptosis but acting through different receptors and mechanisms [16 , 44 ]. As the antiapoptotic effect of soluble OPN in IL-3-dependent hematopoietic cells was shown to be mediated through its interaction with the CD44 receptor and activation of the PI-3K/Akt signaling pathway [16 ], this is a candidate mechanism.
The role of OPN in decreasing the amount of macrophages returning to circulation and increasing the survival of those cells can lead to the accumulation of macrophages in the brain. Thus, OPN provides a strong amplifying force in brain macrophage infiltration and accumulation, the pathological substrate of neuroAIDS. The properties of OPN described here provide a novel explanation for accumulation of macrophages in the brain during neuroAIDS and other diseases such as MS. To prevent this accumulation and further damage caused by activated macrophages, therapeutic strategies may focus on interfering with the production or actions of OPN.
Received November 30, 2006; revised February 3, 2007; accepted February 16, 2007.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
RIII(+)) subset of human monocytes preferentially becomes migratory dendritic cells in a model tissue setting J. Exp. Med. 196,517-527 and IL-1ß in HIV-associated dementia Eur. J. Clin. Invest. 36,447-458[CrossRef][Medline] and transforming growth factor ß upregulate astrocyte expression of monocyte chemoattractant protein-1 J. Neuroimmunol. 57,193-198[CrossRef][Medline]
B mediates vß3 integrin-induced endothelial cell survival J. Cell Biol. 141,1083-1093This article has been cited by other articles:
 |
|
 |
|
  L. Schack, R. Stapulionis, B. Christensen, E. Kofod-Olsen, U. B. Skov Sorensen, T. Vorup-Jensen, E. S. Sorensen, and P. Hollsberg Osteopontin Enhances Phagocytosis through a Novel Osteopontin Receptor, the {alpha}X{beta}2 Integrin J. Immunol., June 1, 2009; 182(11): 6943 - 6950. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
