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. 2006 Mar;79(3):444-52.
doi: 10.1189/jlb.0405215. Epub 2006 Jan 13.

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

E A Eugenin  1 , R Gamss, C Buckner, D Buono, R S Klein, E E Schoenbaum, T M Calderon, J W Berman
Affiliations

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

E A Eugenin et al. J Leukoc Biol. 2006 Mar.

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.

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Figures

Fig. 1
Fig. 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.
Fig. 2
Fig. 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).
Fig. 3
Fig. 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).
Fig. 4
Fig. 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).
Fig. 5
Fig. 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 × 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).
Fig. 6
Fig. 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).
Fig. 7
Fig. 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.
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