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Originally published online as doi:10.1189/jlb.0405215 on January 13, 2006

Published online before print January 13, 2006
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(Journal of Leukocyte Biology. 2006;79:444-452.)
© 2006 by Society for Leukocyte Biology

Shedding of PECAM-1 during HIV infection: a potential role for soluble PECAM-1 in the pathogenesis of NeuroAIDS

E. A. Eugenin*, R. Gamss*, C. Buckner*, D. Buono{dagger}, R. S. Klein{dagger},{ddagger}, E. E. Schoenbaum{dagger},{ddagger}, T. M. Calderon* and J. W. Berman*,§,1

* Departments of Pathology and
§ Microbiology and Immunology, Albert Einstein College of Medicine, and Departments of
{dagger} Epidemiology and Population Health and
{ddagger} Medicine, Montefiore Medical Center/Albert Einstein College of Medicine, Bronx, New York

1 Correspondence: Dept. of Pathology, F727, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. E-mail: berman{at}aecom.yu.edu

ABSTRACT

Human immunodeficiency virus (HIV) infection is characterized by viral entry into the central nervous system (CNS), which is mediated, in part, by the transmigration of HIV-infected monocytes into the brain. The elaboration of chemokines and other factors by these infected cells contributes to CNS inflammation and cognitive impairment in a significant number of HIV-infected individuals. Recently, we demonstrated that HIV-infected monocyte transmigration into the CNS is enhanced greatly by the chemokine CC chemokine ligand 2 (CCL2)/monocyte chemoattractant protein-1. Platelet endothelial cell adhesion molecule-1 (PECAM-1) plays an important role in leukocyte transmigration across the endothelium of the systemic vasculature by mediating homophilic interactions between endothelial cells (EC)-EC and EC-leukocytes, thus preserving vessel integrity. The role of PECAM-1 in HIV-infected leukocyte transmigration across the blood brain barrier (BBB) and NeuroAIDS has not been characterized. We demonstrate that in brain tissue from individuals with HIV encephalitis, there is an accumulation of cleaved, soluble forms of the extracellular region of PECAM-1 (sPECAM-1). In addition, HIV-infected individuals have elevated levels of sPECAM-1 in their sera. Our in vitro data demonstrate that HIV-infected leukocytes, when treated with CCL2, shed sPECAM-1, suggesting a mechanism of extracellular PECAM-1 cleavage and release dependent on HIV infection and CCL2. We hypothesize that sPECAM-1 production by HIV-infected leukocytes, resulting in the accumulation of sPECAM-1 within the CNS vasculature and the generation of truncated, intracellular forms of PECAM-1 within leukocytes, alters PECAM-1 interactions between EC-EC and EC-leukocytes, thus contributing to enhanced transmigration of HIV-infected leukocytes into the CNS and changes in BBB permeability during the pathogenesis of NeuroAIDS.

Key Words: encephalitis • leukocytes • transmigration • CCL2

INTRODUCTION

Human immunodeficiency virus type 1 (HIV-1) entry into the brain is an early event after primary infection [1 ]. Even with the advent of highly active antiretroviral therapy (HAART), a low level of central nervous system (CNS) infection appears to persist [2 3 4 ]. A major complication of HIV infection in a significant percentage of affected individuals is HIV encephalitis (HIVE) and/or HIV-associated dementia (HAD). With the success of HAART, the chronic phase of CNS HIV infection has been prolonged, and the prevalence of cognitive and motor impairment as well as dementia is increasing [4 ]. The mechanisms that contribute to the development of these CNS complications are still not fully understood. Thus, it is critical to examine the early and chronic effects of viral infection within the CNS, which contribute cumulatively to the pathogenesis of NeuroAIDS. By doing so, it may be possible to develop interventional strategies, which would limit the development of cognitive impairment in a number of HIV-infected individuals.

Viral entry into the CNS is mediated, in part, by the transmigration of HIV-infected monocytes into the brain. Leukocyte transmigration, in general, has been characterized as a dynamic, multistep process involving the initial "rolling" of cells on vessel endothelium in response to locally produced inflammatory mediators and subsequent firm adhesion to, and diapedesis across, the systemic vasculature [5 ]. One of the adhesion molecules that participates in the process of diapedesis is platelet endothelial cell adhesion molecule-1 (PECAM-1), which is a 130-kDa glycoprotein expressed on the surface of platelets, endothelial cells (EC), monocytes, neutrophils, and subsets of lymphocytes [6 7 8 9 10 ]. PECAM-1 plays an important role in the transmigration of neutrophils, monocytes, and natural killer cells across the systemic vasculature [11 12 13 14 ] and has also been postulated to play an important regulatory role in the trafficking of monocytes and T lymphocytes into the CNS [15 , 16 ]. In confluent EC monolayers, PECAM-1 is concentrated at cell-cell borders [6 7 8 ]. Experiments with blocking antibodies to the extracellular portion of PECAM-1 selectively reduced diapedesis, but not the adhesion or activation, of leukocytes in peripheral blood vessels [11 , 17 18 19 20 ]. Thus, homophilic interactions between PECAM-1 proteins expressed on EC and on leukocytes, including monocytes, are critical for leukocyte transmigration through interendothelial junctions.

There have been no studies to date addressing the role of full-length, soluble or truncated forms of PECAM-1, expressed by blood brain barrier (BBB) EC and by leukocytes, on the transmigration of HIV-infected leukocytes across the CNS vasculature and possible resultant damage to, and breakdown of, the BBB. Aberrant expression of adhesion molecules, which are associated with leukocyte transmigration, may be an early step in the development of HIV neurologic disease and may precede clinical manifestations. An immunohistochemical study of HIV and simian immunodeficiency virus (SIV) encephalitic tissue showed increased expression of the adhesion proteins, vascular cell adhesion molecule 1 (V-CAM-1) and intercellular adhesion molecule 1 (ICAM-1), on EC and astrocytes [21 , 22 ], supporting the idea that during HIVE, alterations in adhesion molecules may facilitate leukocyte transmigration. Immunohistochemical staining demonstrated abnormal BBB structure and altered tight junction protein expression (zonula occluden 1 and occludin) on sites where leukocyte infiltration and HIV infection were detected [3 , 23 24 25 26 27 ]. All of these studies suggest that the interaction of HIV-infected leukocytes with BBB cells during the process of transmigration alters the expression of important regulators of leukocyte transmigration across the CNS vasculature.

In our tissue-culture model of leukocyte transmigration across the BBB, the presence of the chemokine CC chemokine ligand 2 [CCL2; monocyte chemoattractant protein-1 (MCP-1)] as a chemoattractant resulted in enhanced transmigration of HIV-infected peripheral blood mononuclear cells (PBMC) and BBB disruption [28 ] (E. A. Eugenin and J. W. Berman, unpublished data). We suggest that this is, in part, a result of altered leukocyte-EC interactions. In our BBB model, the presence of chemokines other than CCL2, including CCL3 [macrophage-inflammatory protein-1{alpha} (MIP-1{alpha})], CCL4 (MIP-1ß), CXC chemokine ligand 10 (CXCL10) [interferon-inducible protein-10 (IP-10)], or CCL5 [regulated on activation, normal T expressed and secreted (RANTES)], did not result in increased transmigration of infected cells or BBB disruption [28 ] (E. A. Eugenin and J. W. Berman, unpublished data). Thus, it is the unique combination of HIV infection and CCL2 that participates in aberrant EC-leukocyte interactions and changes in BBB permeability. We propose that these alterations are mediated, in part, by increased elaboration of cleaved soluble forms of the extracellular region of PECAM-1 (sPECAM-1) and the generation of truncated forms of intracellular PECAM-1 in leukocytes, which destabilize EC-EC and EC-leukocyte interactions, contributing to enhanced transmigration and increased BBB permeability.

MATERIALS AND METHODS

Materials
RPMI media, fetal bovine serum, penicillin/streptomycin, and trypsin-EDTA were purchased from Gibco-BRL (Grand Island, NY). Essentially immunoglobulin (Ig)-free bovine serum albumin (BSA) and fluorescein isothiocyanate (FITC)-conjugated anti-goat antibody were from Sigma Chemical Co. (St. Louis, MO). R-phycoerythrin (R-PE)-conjugated anti-rabbit antibody was from BD PharMingen (San Diego, CA). Cyanine 5 (Cy5)-conjugated anti-mouse F(ab)'2 antibody was from Jackson ImmunoResearch Laboratories (West Grove, PA).

Isolation of mononuclear cells
Anticoagulated blood was obtained from healthy volunteers or from study participants (see below), and PBMC were isolated by under-layering with Ficoll-Paque (Amersham Bioscience, Uppsala, Sweden) according to the procedure described by the manufacturer. Purified PBMC used in these studies were ~90% lymphocytes and 10% monocytes, as determined by incubation with premixed human CD45 and CD14 monoclonal antibodies conjugated to FITC and PE, respectively (1:50, Caltag Laboratories, Burlingame, CA), followed by fluorescence-activated cell sorter analysis (FACS).

HIV-1 infection of PBMC
PBMC were isolated and activated with phytohemagglutinin (PHA; 5 µg/ml) plus interleukin (IL)-2 (5%) in RPMI 1640 for 48 h in polypropylene tubes at a density of 2 x 106 cells/ml. Cells do not adhere to these tubes, and therefore, monocyte differentiation into adherent macrophages is inhibited. Aliquots of this activated cell population were incubated with high titers of HIV-1ADA virus for 1–2 h, washed thoroughly, resuspended in fresh medium, and maintained in polypropylene tubes for an additional 7 days to facilitate viral replication. HIV-1ADA is an R5 isolate, which infects human monocytes/macrophages, and was obtained from PBMC (Accession Number M60472). Cell-free viral inocula were obtained from the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program (Germantown, MD). Other aliquots of the original activated PBMC population were washed and maintained in polypropylene tubes in fresh media for an additional 7 days without any virus, thus serving as uninfected, activated controls. An additional, uninfected control was freshly isolated, unactivated, uninfected PBMC. To determine the levels of HIV infection, HIV-1 p24 values were determined by enzyme-linked immunosorbent assay (ELISA; PerkinElmer Life Science, Boston, MA) 7 days post-infection. Only virally infected cultures with p24 values that correspond to at least 2 logs greater than the viral 50% tissue culture infectious dose, as the NIH recommends, were used for sPECAM-1 experiments.

Human tissue
Brain tissues were obtained at autopsy from 16 individuals, aged 6 months to 52 years old. Tissue sections from six individuals without HIV infection (uninfected), four individuals with HIV infection but no HIVE, and six individuals with HIVE were analyzed by immunohistochemistry. The criteria for encephalitic areas in tissue sections were infiltration and/or accumulation of macrophages and microglia, as detected by CD68 immunostaining (mouse monoclonal anti-CD68, Dako Corp., Carpinteria, CA, 1:300 dilution). For all tissues, paraffin-embedded sections were used, and three separate slides per case were examined.

Immunohistochemistry
Postmortem human tissue sections corresponding to uninfected, HIV-infected without HIVE and HIVE cases were analyzed by double immunohistochemical staining for the intracellular and extracellular domains of PECAM-1 using domain-specific antibodies and our protocol previously described [28 ]. Briefly, the sections were incubated in blocking solution (5 mM EDTA, 1% fish gelatin, 1% essentially Ig-free BSA, and 2% horse serum) for 30 min at room temperature and then incubated in diluted, primary polyclonal anti-PECAM-1 cytoplasmic domain antibody (goat polyclonal antibody, sc-1505, 1:500 dilution, Santa Cruz Biotechnology, CA), monoclonal anti-CD68 (Dako Corp., 1:300), or polyclonal anti-PECAM-1 extracellular domain antibody (rabbit polyclonal antibody, sc-8306, 1:800 dilution, Santa Cruz Biotechnology) overnight at 4°C. The sections were then washed with phosphate-buffered saline (PBS), incubated with FITC-conjugated anti-goat IgG or R-PE-conjugated anti-rabbit IgG for 1 h at room temperature, followed by another wash in PBS for 1 h. Samples were then mounted using Prolong Gold antifade reagent (Molecular Probes, Junction City, OR) and examined by confocal microscopy. Specificity was confirmed by replacing the primary antibody with the appropriate isotype-matched control reagent or the IgG fraction of normal rabbit or goat serum (Santa Cruz Biotechnology).

Participants in HIV study groups
Sera were collected from 97 consecutive participants enrolled and followed in two related prospective (parent) studies of HIV infection and substance abuse, which use similar methodology, one with women (MS study) and one with men (CHAMPS study) [29 30 31 ]. In these studies, research visits were conducted semi-annually, at which time, standardized interviews were administered eliciting substance abuse behaviors and medical history. Blood was drawn for HIV antibody levels, viral load, CD4 count, and medication history, including HAART. A fresh tube of heparinized blood (10 ml) was drawn separately from each participant for use in the study presented in this manuscript. In addition, sera from 10 HIV-negative volunteers who were not participants in these studies and did not have a history of substance abuse were used as controls. Among the 97 study participants, there were 40% women and 60% men. The prevalence of HIV within this group was ~51%. Substance abuse in the last 6 months was reported by 37 study participants (30% of HIV-positive and 47% of HIV-negative). Substance abuse was defined as use of heroin or cocaine (including crack) or any admixture (speedball) by any route. By design, these prospective studies enrolled ~50% HIV-infected and 50% uninfected persons. The HIV-infected individuals (n=50) consisted of a group who had taken substances of abuse in the last 6 months (DR, n=8), a group that was on HAART (HA, n=23), a group that had taken substances of abuse in the last 6 months and was on HAART (HA/DR, n=7), and a group that was neither on HAART nor taking substances of abuse (HIV alone, n=12). In the HIV-alone group, there were individuals with a high viral load (26,962±15,672, n=3) and a low viral load (770±1129, n=9) and CD4 counts averaging 329.8 ± 221 (n=12). In the DR group, there were individuals with a high viral load (10,923±3020, n=3) and a low viral load (338±468, n=5) and CD4 counts averaging 485 ± 236 (n=8). In the HA group, there were individuals with a high viral load (39,962±24,647, n=6) and a low viral load (267±457, n=17) and CD4 counts averaging 606 ± 255 (n=23). In the HA/DR group, there were individuals with a high viral load (53,847±70,089, n=5) and a low viral load (607±230, n=2) and CD4 counts averaging 261 ± 169 (n=7). Written, informed consent was obtained from all participants. Institutional Review Board approval was obtained from the Montefiore Medical Center and The Albert Einstein College of Medicine (AECOM; Bronx, NY).

ELISA for sPECAM-1 and sICAM-1
Sera obtained from participants in the HIV infection and substance abuse studies as well as from uninfected volunteers were analyzed for sPECAM-1 and sICAM-1 levels using the ZyQuik sPECAM-1 and sICAM-1 ELISA kits, according to the protocol suggested by the manufacturer (Zymed, San Francisco, CA). Tissue culture supernatants from uninfected PBMC or PBMC infected with HIV-1ADA in vitro, treated with or without CCL2 (100 ng/ml) for 24 h, were also assayed for sPECAM-1. The limit of detection for the sPECAM-1 and sICAM-1 assays was 0.1 ng/ml.

Statistical analysis
ANOVA and two-tailed Student’s t-test were used to compare sPECAM-1 and sICAM-1 levels among the different study participant groups or to compare media from infected or uninfected PBMC with or without CCL2. A value of P< 0.05 was considered significant.

RESULTS

HIV-encephalitic human tissue shows altered expression of PECAM-1 in EC, CNS parenchymal cells, and leukocytes as compared with CNS tissue from uninfected or nonencephalitic, HIV-positive individuals, suggesting shedding of PECAM-1 occurs in vivo
Sections of brain tissue from six uninfected individuals (normal), four individuals with HIV infection but no HIVE, and six individuals with HIVE were analyzed by double immunohistochemical staining to determine the distribution of full-length and cleaved forms of PECAM-1. Figure 1 is a schematic drawing of the PECAM-1 protein, illustrating the extracellular region of PECAM-1 (ePECAM-1) and the intracellular portion of PECAM-1 (iPECAM-1). We used two different polyclonal anti-PECAM-1 antibodies in our immunohistochemical analyses: one specific for iPECAM-1 (C-terminal domain) and the other specific for ePECAM-1 (N-terminal domain). Secondary antibody conjugated to FITC (green staining) was used to detect anti-iPECAM-1 reactivity, and secondary antibody conjugated to R-PE (red staining) was used to detect anti-ePECAM-1 reactivity. Sections were then examined by confocal microscopy. iPECAM-1 (green staining) and ePECAM-1 (red staining) in normal, uninfected tissue were mainly expressed by brain EC (Fig. 2B and2C , low-power, and Fig. 2 , F and G, high-power magnification of blood vessel in region indicated in Fig. 2A 2J and 2K , high-power magnification of blood vessel), with some reactivity in a few parenchymal cells, as determined by macrophage/microglia morphology (Fig. 2B and 2C) and by CD68 immunoreactivity, as detected with a Cy5-conjugated secondary antibody (cyan staining; Fig. 2L ). In brain EC and parenchymal cells (Fig. 2D and 2H) , there was almost-perfect colocalization (Merge, yellow staining) between staining obtained with the antibodies recognizing iPECAM-1 (green staining) and ePECAM (red staining) in all six cases examined, suggesting that in normal brain, full-length PECAM-1 protein is expressed. Colocalization of CD68, iPECAM-1, and ePECAM-1 staining is shown in Figure 2M . All of the sections of brain tissue from HIV-infected individuals, without HIVE, exhibited colocalization of ePECAM-1 and iPECAM-1 staining as well. These sections were indistinguishable from the normal, uninfected tissue sections described above (data not shown).


Figure 1
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  		Figure 1. Schematic diagram showing the membrane topology of full-length PECAM-1 and its extracellular (ePECAM-1) and intracellular (iPECAM-1) portions. Cleaved forms of the ePECAM-1 can be shed to generate sPECAM-1 and a truncated, intracellular membrane-bound protien (tPECAM-1). The role of these different forms of PECAM-1 in the inflammatory response is still under investigation. P represents potential sites of phosphorolation.

 

Figure 2
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  		Figure 2. In normal brain tissue, iPECAM-1 and ePECAM-1 colocalize. Immunohistochemical analysis of normal human brain tissue sections with antibodies to iPECAM-1 (green staining) and ePECAM-1 (red staining) was performed and analyzed by phase contrast and confocal microscopy. Our results indicated that in normal brain tissue, reactivity for both antibodies colocalizes (yellow staining, Merge) and is present mainly in brain blood vessels (BV) and some CNS parenchymal cells, as detected by confocal microscopy. (A, Phase contrast and B–D; confocal) Low magnification of CNS tissue stained for iPECAM-1, ePECAM-1, and colocalization (Merge). (E, phase contrast, and F–H, confocal) An enlargement of the area indicated in A (dotted square) to show the colocalization of both PECAM-1 epitopes in BV. In all cases, almost perfect colocalization of both epitopes was found. (I, phase contrast, and J–M, confocal) The presence and distribution of iPECAM-1 and ePECAM-1 in CNS macrophage/microglia, as indicated by CD68 immunoreactivity (cyan staining; L). (I, Inset) 4',6-Diamidino-2-phenylindole (DAPI) staining. (M) Colocalization of iPECAM-1, ePECAM-1, and CD68. Original bars, 170 µm (A–D), 25 µm (E–H), and 65 µm (I–M).

 
In contrast, all of the cases of HIVE tissue showed bright staining in encephalitic areas with the antibody recognizing ePECAM-1 (red staining; Fig. 3C 3G, and3O ) and little colocalization (Merge, yellow staining; Fig. 3D and 3H ) with staining obtained with the antibody recognizing iPECAM-1 (green staining; Fig. 3B 3F and 3N ). The staining for iPECAM-1 was similar in sections from uninfected tissue, tissue from HIV-infected individuals without HIVE, and HIV-encephalitic tissue, suggesting that the differences in the staining for ePECAM-1 were not a result of differences in full-length PECAM-1 protein expression. The abundant presence of ePECAM-1 (red staining), concentrated on EC and parenchymal cells with macrophage and/or microglia morphology (Fig. 3C and 3G) , which does not colocalize (Fig. 3D and 3H) with iPECAM-1 (green staining; Fig. 3B and 3F ), suggests significant accumulation of cleaved forms of ePECAM-1 (sPECAM-1) (see Fig. 1 ) and fewer full-length PECAM-1 molecules. Parenchymal cells positive for ePECAM-1 staining (Fig. 3O) were identified as microglia and/or macrophages as a result of their CD68 immunoreactivity, as detected with a Cy5-conjugated secondary antibody (cyan staining; Fig. 3P ). Colocalization of CD68, iPECAM-1, and ePECAM-1 staining is shown in Figure 3Q .


Figure 3
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  		Figure 3. In HIVE tissue, ePECAM-1 is abundant and minimally colocalizes with the intracellular epitope, suggesting an active shedding process. Immunohistochemical analysis of human HIVE brain tissue sections with antibodies to iPECAM-1 (green staining) and ePECAM-1 (red staining) was performed and analyzed by phase contrast and confocal microscopy. Our results indicated that in encephalitic areas, high amounts of ePECAM-1 (red staining), which colocalized minimally (D and H, Merge, yellow staining) with iPECAM-1 (green staining), were concentrated in CNS blood vessels (BV), parenchymal macrophages, and/or microglia and infiltrated leukocytes as detected by confocal microscopy. (A, phase contrast, and B–D, confocal) Low magnification of HIVE tissue stained for iPECAM-1, ePECAM-1, and colocalization (Merge). (E, phase contrast, and F–H, confocal) An enlargement of the area indicated in A (dotted square) to show the accumulation of ePECAM-1 as compared with iPECAM-1 immunoreactivity in BV. (I, phase contrast, and J–L, confocal) Circulating leukocytes in BV, which have high levels of iPECAM-1 and reduced levels of ePECAM-1 staining, suggesting that an active process of PECAM-1 shedding from leukocytes occurs, resulting in leukocytes with only truncated iPECAM-1 and the accumulation of sPECAM-1 in vessels in encephalitic areas. (M, phase contrast, and N–Q, confocal) The presence and distribution of iPECAM-1 and ePECAM-1 in CNS macrophage/microglia. Note the accumulation of ePECAM-1 staining in the encephalitic areas (O). (M, Inset) DAPI staining. We characterized the CNS parenchymal cells, which are positive for ePECAM-1, as macrophages and/or microglia by their immunoreactivity to CD68 (P, cyan staining). (Q) Colocalization of iPECAM-1, ePECAM-1, and CD68. Original bars, 25 µm (A–D), 170 µm (E–L), and 65 µm (M–Q).

 
These results suggest that the enhanced ePECAM-1 staining, which colocalizes minimally with iPECAM-1 staining, may be a result of shedding of cleaved forms of ePECAM-1 (sPECAM-1) and their accumulation within encephalitic areas of brain tissue. These experiments do not identify the source of the sPECAM-1. However, in blood vessels in encephalitic areas of HIVE brain tissue sections, circulating leukocytes exhibited mostly iPECAM-1 staining (Fig. 3J) with reduced levels of ePECAM-1 staining (Fig. 3K) and minimal colocalization (Fig. 3L) . These data suggest that leukocytes in these vessels may produce sPECAM-1, and the shedding of cleaved forms of ePECAM-1 would generate the formation of truncated intracellular molecules (tPECAM-1) in these cells composed of the transmembrane and cytoplasmic domains of PECAM-1 (see Fig. 1 ). To control for the specificity of staining, the IgG fraction of normal goat or rabbit serum at the same concentration as the primary antibodies was used. There was no specific staining under these control conditions (data not shown). At least three sections per case were analyzed to confirm the PECAM-1-staining patterns.

Treatment of HIV-infected PBMC with CCL2 induces release of sPECAM-1
Our immunohistochemical data indicated that increased amounts of sPECAM-1, possibly produced by leukocytes, were concentrated in encephalitic areas of brain tissue sections from individuals with HIVE. Based on our previous data that CCL2, but not other chemokines, enhanced the transmigration of HIV-infected PBMC across the BBB and increased BBB permeability [28 ] (E. A. Eugenin and J. W. Berman, unpublished data), we hypothesized that CCL2 may play a role in the shedding of cleaved, soluble forms of ePECAM-1 from HIV-infected leukocytes at sites of increased transmigration across the CNS vasculature.

To determine whether CCL2 induced sPECAM-1 production by HIV-infected leukocytes, the media from human PBMC cultures infected with HIV-1ADA in vitro were assayed for sPECAM-1 by ELISA. Assays were performed on media from the following PBMC cultures after 24 h of treatment with CCL2 (W/CCL2; 100 ng/ml) or without CCL2 (WO/CCL2): PHA + IL-2-activated cells infected with HIV-1ADA and cultured for 7 days to facilitate viral replication (HIV-1ADA-infected cells), uninfected PHA + IL-2-activated cells cultured for 7 days (activated/uninfected cells), and freshly isolated, unactivated, uninfected cells (unactivated/uninfected cells). Our results demonstrated that without CCL2 treatment, unactivated/uninfected, activated/uninfected, and HIV-1ADA-infected cells secrete similar, low amounts of sPECAM-1 into the tissue culture media (Fig. 4 ). However, when HIV-1ADA-infected PBMC, but not uninfected PBMC, were exposed to CCL2 for 24 h, a significant amount of sPECAM-1 was released into the media. The increased presence of sPECAM-1 in the media cannot be explained by release of PECAM-1 isoforms as a result of increased cell death in CCL2-treated, HIV-infected PBMC, as all of the cell cultures examined had the same low level of trypan blue-positive cells. These data suggest that the balance between full-length and cleaved forms of PECAM-1 in leukocytes may be dysregulated by HIV infection and CCL2 exposure, contributing to increased transmigration of these cells into the brain in response to CCL2 and to resultant changes in BBB permeability.


Figure 4
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  		Figure 4. CCL2 treatment induces the shedding of sPECAM-1 from HIV-infected PBMC as detected by ELISA. Uninfected/unactivated and uninfected/activated (PHA+IL-2) PBMC release low levels of sPECAM-1 into the culture media, with CCL2 (W/CCL2; 100 ng/ml) or without CCL2 (WO/CCL2) treatment for 24 h. HIV-1ADA-infected PBMC without CCL2 treatment release similar amounts of sPECAM-1 as uninfected cells. CCL2 treatment of HIV-1ADA-infected PBMC, but not uninfected PBMC, induces a significant increase in the release of sPECAM-1. *, P < 0.05, compared with uninfected cells or HIV-infected cells without CCL2 treatment (n=3).

 
sPECAM-1 levels are elevated in the sera of HIV-infected individuals
Based on our data that HIV-infected PBMC exposed to CCL2 release sPECAM-1 and that HIVE tissues have increased sPECAM-1, we examined the levels of sPECAM-1 in the sera of HIV-infected and uninfected individuals by ELISA. In addition, we determined the serum levels of sICAM-1 to assess whether the possible cleavage and shedding of ePECAM-1 are regulated specifically by HIV infection or whether general proteolytic events or increased levels of cell death in circulating leukocytes of HIV-infected individuals nonspecifically increase the shedding of soluble forms of several cell surface proteins, including PECAM-1 and ICAM-1.

Sera were collected from participants enrolled and followed in two related, prospective studies of HIV infection and substance abuse, which are described in Materials and Methods. Some individuals in these studies were HIV-negative, with and without a recent history of substance abuse, and other participants were HIV-positive, with and without a recent history of substance abuse and/or HAART therapy (HIV). In addition, sera from 10 healthy, uninfected volunteers without a history of substance abuse, who were not enrolled in these studies, were used as normal controls. A highly significant increase in sPECAM-1 (Fig. 5A , *, P<2.8x10–11) was observed in sera of HIV-infected individuals (HIV) when compared with the normal control group of uninfected volunteers (N) and uninfected individuals from the studies, with or without a recent history of substance abuse (C). When we further grouped the HIV-infected study participants into those who had taken substances of abuse in the last 6 months (DR) and/or were undergoing HAART (HA), we did not detect differences in the levels of sPECAM-1 among these different subgroups of HIV-infected individuals (Fig. 5B) . In addition, the levels of sPECAM-1 in each of these subgroups were still increased significantly when compared with the normal control group of uninfected volunteers (N) and the uninfected study participants (Control), grouped according to their recent history of substance abuse (DR). Thus, HIV-infected individuals compared with uninfected individuals, have elevated sera levels of sPECAM-1, which did not appear to be altered during the chronic phase of HIV infection by substance abuse and/or HAART.


Figure 5
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  		Figure 5. Sera from HIV-infected individuals have significant levels of sPECAM-1 as determined by ELISA. Levels of sPECAM-1 in the sera of HIV-infected individuals were assayed by ELISA. Sera from uninfected volunteers without a history of substance abuse (Normal, N) and uninfected participants enrolled in two HIV/substance abuse studies (Control, C) were used as uninfected controls. Increases in serum levels of sPECAM-1 were highly significant in HIV-infected participants in the HIV/substance abuse studies (HIV) compared with uninfected individuals. (A) *, P < 2.8 x 10–11, compared with uninfected volunteers (N) and to uninfected study participants, some of which have a recent history of substance abuse (C). When the population was subdivided further into those who used substances of abuse recently (DR) or were on HAART (HA) or both (DR/HA), we did not detect any additional differences in sPECAM-1 levels (B).

 
In contrast, there was no significant difference in serum levels of sICAM-1 in HIV-infected individuals when compared with uninfected individuals (Fig. 6A ). In addition, when participants from the studies were grouped for recent substance abuse and/or HAART, significant differences in sICAM-1 levels were still not detected (Fig. 6B) .


Figure 6
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  		Figure 6. Sera from HIV-infected individuals did not show any significant differences in sICAM-1 levels as determined by ELISA. Levels of sICAM-1 in the sera of HIV-infected individuals (HIV) with a recent history of substance abuse (DR), on HAART (HA) or both (DR/HA) were assayed. Uninfected volunteers without a history of substance abuse (N) and uninfected participants enrolled in the HIV/substance abuse studies (Control, C) were included as uninfected controls. We did not detect any significant differences in sICAM-1 levels between HIV-infected and uninfected individuals (A) or among the subdivided groups (B).

 
DISCUSSION

Leukocyte transmigration necessitates the opening of interendothelial cell junctions to allow for cell passage between tightly apposed EC, yet the barrier properties of the endothelium must be preserved to inhibit the uncontrolled transit of cells and fluids through the vessel wall into parenchymal tissue. In contrast to most other adhesion molecules, PECAM-1 can establish homophilic adhesive interactions between EC-EC and EC-leukocytes [32 , 33 ]. In this way, PECAM-1 acts like a cellular "zipper" that allows for tight intercellular interactions between transmigrating leukocytes and surrounding blood vessel endothelium, thus preserving barrier properties [34 , 35 ]. PECAM-1, like other adhesion molecules, has circulating, soluble isoforms, which can be generated by shedding of cleaved forms of the extracellular portion of cell membrane-bound protein, a process that is mediated by metalloproteinases and caspases, or by alternative splicing [36 , 37 ]. One of the proposed functions for these soluble adhesion proteins, including sPECAM-1, is to act as competitive inhibitors of the full-length, membrane-bound protein, thereby reducing leukocyte adhesion and transmigration [36 , 38 39 40 ].

In contrast, under specific pathological conditions, the lack of PECAM-1 appears to enhance transendothelial migration of leukocytes to sites of inflammation. When PECAM-1-deficient mice were sensitized to develop experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, an accelerated disease course occurred as a result of an earlier onset of mononuclear cell migration into the brain and spinal cord. EC isolated from these mice supported increased transmigration of T lymphocytes and exhibited increased and prolonged permeability in response to histamine in vitro [16 ]. In another study, lipopolysaccharide challenge of PECAM-1-deficient mice resulted in an exaggerated, systemic, inflammatory response and in increased peripheral vascular permeability [41 ]. However, these types of vascular effects seem to occur only after the animal is subjected to a particular physical or pathological challenge, as no structural or functional abnormalities have been reported in the vasculature of PECAM-1-deficient mice under basal conditions, and these mice exhibit normal leukocyte migration into the peritoneal cavity after challenge with IL-1ß or thioglycolate [42 ].

In this study, our data in vitro indicated that HIV infection plus exposure to CCL2 induced the shedding of sPECAM-1 by PBMC. In addition, our data in vivo, obtained by immunohistochemical analyses of HIVE brain tissue, showed increased levels of sPECAM-1, which correlated with inflammation and encephalitis, as evidenced by enhanced CD68 reactivity in the tissue. In HIVE tissue, sPECAM-1 was mainly localized to CNS blood vessel endothelium and to a lesser degree, to parenchymal macrophages and/or microglia. Leukocytes within the lumen of vessels in HIVE tissue areas with significant leukocyte infiltration showed high expression of iPECAM-1 staining with little ePECAM-1 staining, suggesting that an active process of shedding by leukocytes in vessels within these tissue areas occurs in vivo. This process would compromise full-length PECAM-1 expression in leukocytes as well as result in the production of sPECAM-1 within the lumen of CNS vessels. Platelets within CNS vessels are another possible cellular source of sPECAM-1. We had demonstrated previously that in the same HIVE brain tissue used in this study, CCL2 was also expressed significantly [43 ]. In addition, we had demonstrated previously that CCL2 specifically mediates high levels of transmigration of HIV-infected leukocytes across a tissue culture model of the BBB [28 ] (E. A. Eugenin and J. W. Berman, unpublished data). We propose that local release of CCL2 in the brain may be an important signal to trigger HIV-infected leukocyte transmigration and the shedding of sPECAM-1 from these cells. Thus, the balance between full-length and cleaved forms of membrane-bound PECAM-1 in leukocytes, as well as the release of sPECAM-1, may be dysregulated by HIV infection and CCL2 exposure, contributing to increased transmigration of these cells into the brain in response to CCL2 and to resultant changes in BBB permeability. These changes, in conjunction with possible alterations in other junctional proteins that may facilitate leukocyte diapedesis, contribute to the CNS inflammation, which characterizes HIVE and many cases of HAD.

Although the majority of sPECAM-1 appeared to be localized to CNS endothelium in HIVE tissue, it is not known whether cleaved forms of ePECAM-1 are actively being shed by EC. Perhaps the enhanced staining is a result of binding of sPECAM-1, produced by leukocytes, to ligands of PECAM-1 expressed by EC, such as PECAM-1 itself, the integrin {alpha}vß3 [44 , 45 ], or cell surface glycosaminoglycans [46 ]. In addition, sPECAM-1 was detected on some parenchymal macrophages and/or microglia. As many of the leukocytes in the lumen of CNS vessels in encephalitic tissue areas maintained expression of cleaved membrane-bound iPECAM-1 only, it seems unlikely that after monocytes transmigrated across the BBB and differentiated to macrophages within the parenchyma, these cells would be positive for sPECAM-1. A possible explanation for the parenchymal expression of sPECAM-1 is that leukocyte transmigration across the BBB in response to CCL2 altered BBB permeability, resulting in the efflux of sPECAM-1 from the lumen of CNS vessels to the parenchyma, where it may bind to PECAM-1 ligands expressed by macrophages and/or microglia. In addition, HIV-infected microglia within the CNS parenchyma may shed sPECAM-1 in the presence of CCL2, as was seen with leukocytes.

To characterize further the expression of sPECAM-1 in vivo during HIV pathogenesis, sera from HIV-infected and uninfected individuals enrolled in two studies of HIV infection and substance abuse were assayed for sPECAM-1. Our data indicated that the levels of sPECAM-1, but not sICAM-1, were increased significantly in the sera of HIV-infected individuals when compared with sera from uninfected individuals. When the HIV-infected group was subdivided into individuals with a recent history of substance abuse and/or current HAART therapy, no differences in sPECAM-1 levels in the sera of these different subgroups were evident. This may be a result of the fact that these individuals have been infected for a long period of time (2–5 years), and the effects of substances of abuse may occur earlier in the disease course, resulting in alterations of disease progression. In addition, in all of the groups analyzed, including those on HAART, there were individuals with high or low viral loads, making it difficult to determine the effects of viral replication on sPECAM-1 production. We will now analyze sera from additional study participants, who will return every 6 months, and assess whether there are changes in sPECAM-1 expression with time. Also, using minimental scores for each patient to evaluate CNS impairment, we will be better able to determine the contribution of substance abuse and/or HAART over time to the mental status of these individuals and to determine if there is a correlation between the generation of sPECAM-1 and HIV-related neurological deficits.

The results of this study led us to hypothesize that shed sPECAM-1 enhances the transmigration of HIV-infected leukocytes into the brain by destabilizing the normal transmigration process. The mechanism(s) by which sPECAM-1 would contribute to enhanced CCL2-mediated, HIV-infected leukocyte transmigration and to increased BBB permeability has not been characterized. The destabilization of normal transmigration may be, in part, a result of disruption of homophilic PECAM-1-PECAM-1 interactions between EC-EC and EC-leukocytes (Fig. 7 , schematic model). The loss of full-length PECAM-1 on leukocytes may have similar consequences as those observed in PECAM-1-deficient mice sensitized to develop EAE [16 ], suggesting that alterations in PECAM-1 adhesive interactions and/or signaling within leukocytes and/or EC are important contributors to enhanced leukocyte transmigration and increased vascular permeability during the CNS inflammation, which occurs in the pathogenesis of NeuroAIDS. Previous studies showed that anti-PECAM-1 antibody or sPECAM-1 inhibited monocyte transmigration through the systemic vasculature [11 , 17 18 19 20 ]. However, in these studies, PECAM-1 was not cleaved and shed from leukocytes, so that the contribution of altered PECAM-1 signaling mediated by truncated iPECAM-1 was not assessed. The ability of PECAM-1 to function as a signaling molecule is dependent on the interaction of immunoregulatory tyrosine-based activation motif domains in the cytoplasmic domain of PECAM-1 with multiple adaptor proteins [47 ]. In a previous study, truncated iPECAM-1 was shown to exhibit different binding interactions with intracellular molecules when compared with full-length PECAM-1 [37 ], perhaps contributing to altered signaling.


Figure 7
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  		Figure 7. Proposed mechanism of transmigration of HIV-infected cells across the BBB in response to CCL2 and the role of sPECAM-1 during this process. We propose that local CCL2 production in the CNS and subsequent inflammatory cell infiltration induce the release of sPECAM-1 from HIV-infected leukocytes. Focal accumulation of sPECAM-1 at the BBB contributes to the dysregulation of PECAM-1-PECAM-1 interactions between CNS blood vessel EC and between transmigrating leukocytes and EC, resulting in opening of the BBB. Increased BBB permeability contributes to enhanced transmigration of HIV-infected leukocytes into the CNS and to sPECAM-1 influx into the brain parenchyma. CCR2, CC chemokine receptor 2.

 
The unique combination of HIV-infected leukocytes and CCL2 within the CNS vasculature may contribute to a mechanism, whereby low levels of leukocyte transmigration occur in the presence of sPECAM-1, thus opening up EC-EC junctions to allow sPECAM-1 to bind to PECAM-1 on EC. This may inhibit the formation of the tight seal that develops around leukocytes as they traverse interendothelial cell junctions, resulting in BBB compromise and enhanced leukocyte transmigration and permeability. Additional studies in our laboratory are addressing the mechanisms by which PECAM-1, sPECAM-1, and truncated forms of iPECAM-1 participate in the different stages of NeuroAIDS.

ACKNOWLEDGEMENTS

This work was supported by the National Institute of Mental Health Grants MH052974 and MH070297, NIH Grant NS11920, National Institute on Drug Abuse Grants DA13564 and DA014998, NIH Centers for AIDS Research Grant AI-051519, and the Neuropathology Training Grant NIH NS07098 (C. B.). We greatly appreciate the services of the virology, flow cytometry, and immunology and pathology cores of the AECOM Center for AIDS Research. We especially thank K. Osieki for her virology work.

Received April 24, 2005; revised October 28, 2005; accepted November 24, 2005.

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