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CCR5 monkey business: SIV can bypass CD4
IMMUNOLOGY
Endothelial cells 'present' chemokines during i n f l a m m a t i o n Middleton, J., Neil, S., Wintle, J. et al. (1997) Transcytosis and surface presentation of IL-8 by venular endothelial cells Cell 91,385-395 Tissue-derived chemokines, such as interleukin 8 (IL-8), must find their way to the luminal surface of venular endothelial cells (ECs) if they are to initiate the chemotactic gradient that ultimately draws leukocytes from the circulation into inflammed tissues. Previously, this has been proposed to occur by simple diffusion of the chemokine through intercellular spaces between venular ECs. However, Middleton and colleagues show here that the prototype chemokine IL-8 is actively internalized by ECs, followed by transcytosis and 'presentation' at the luminal surface. IL-8 trafficking was assessed by immunoelectron microscopy after intradermal injection of radiolabelled IL-8 in rabbits. As soon as 30 rain after injection, IL-8 was present on the luminal surface of ECs, particularly on microvilli and other luminal projections. Importantly, IL-8 was shown to bind initially to the EC abluminal surface,
then incorporate into smooth-membranebound plasmalemmal vesicles, which then locate to the basal and apical portions of the EC cytoplasm. These vesicles then fuse with the apical cell membrane, allowing IL-8 to be displayed at the luminal surface. No IL-8 was detected in the junctions between ECs. The display and immobilization of IL-8 at the luminal surface appears to involve heparan sulphate, although the contribution of other molecules to IL-8 transcytosis is not clear. This series of in vivo and in situ observations is particularly important given that cultured EC lines do not similarly bind and traffic IL-8 in vitro. Thus, for IL-8 at least, there is an active process of chemokine transport from the tissues, across the EC barrier, to the venous circulation. Essential chemoattractants can therefore be 'presented' at the luminal EC surface as a means of establishhag a chemotactic gradient into the tissues.
C C R 5 m o n k e y business: S I V can bypass C D 4 Martin, K.A., Wyatt, R., Farzan, M. et al. (1997) CD4-independent binding of SIV gpl20 to rhesus CCR5 Science 278, 1470-1473 The entry of human immunodeficiency virus (HIV) into a cell is initiated by gp120 binding to CD4 on the target cell. Fusion of the viral and target cell membranes is subsequently facilitated by the binding of CCR5, a target cell co-receptor of the chemokine family that is thought to induce a conformational change in the HIV gp41 protein. Rhesus macaque CCR5 (CCR5rh) shares 98% sequence identity with human CCR5hu, and supports entry of either HIV or simian immunodeficiency virus (SIV) envelope glycoproteins expressed on recombinant virions. This study examines the CD4 dependency of these interactions. HEK293 cells were transiently transfected with plasmids expressing CCR5rh or CCRShu and tested for binding HIV and SIV gp120. HIV gpl20 (strain YU2) bound to both transfectants but only if soluble (s)CD4 was also present. By contrast, gp120 from
SIVmac239 was capable of binding CCR5rh transfectants in the absence of sCD4, while binding of SIV gp120 to CCR5hu remained CD4 dependent. The basis for this dependency was narrowed down to the N-terminal Asp13 residue in CCR5rh (CCR5hu has Asn13 at this position). A mutant CCR5rhDlgN, in which the Aspl3 residue was changed to Asnl3, bound SIV gpl20 only in the presence of sCD4, suggesting that this amino acid difference is responsible for the difference in CD4 dependency between the two chemokine receptor proteins. This was confirmed by creating the corresponding CCR5huN13D mutant, which showed CD4independent binding to SIV gp120. These observations suggest that some lenteviruses can use seven-transmembranespanning proteins, such as CCR5, as the sole receptor for cell entry. This has clear implications for immunotherapy.
TODAY
Skin kin: P S G L - I becomes C L A for dermal homing Fuhlbrigge, R.C., Keitfer,J.D., Armerdin~ D. and Kupper, T.S. (I 997) Cutaneous lymphocyte antigen is a specialised form of PSGL-I expressed on skin-homing T cells Nature 389, 978-981 T cells from inflammatory or malignant skin diseases express a unique skin-homing receptor, cutaneous lymphocyte antigen (CLA). CLA is a carbohydrate moiety that binds E-selectin and facilitates targeting of T cells to inflammed skin. However, the structure of CLA has remained elusive, until now. Here, Fuhlbrigge and colleagues report that CLA is an inducible modification of a constitutive marker of human T cells: P-selectin glycoprotein 1 (PSGL-1). T cells expresing the CLA modification can bind both E- and P-selectin, suggesting that differential regulation of selectin binding can regulate tissue specifid~. Using serum-free culture conditions, it is possible to maintain elevated levels of CLA on a high proportion of peripheral blood T cells. Using a parallel-plate flow chamber to reproduce physiological shear conditions, CLA expression was shown to facilitate E-selectin tethering and rolling, whereas PSGL-1 expression allowed P-selectin binding only. Immunoblot characterization of CLA from serum-free cell cultures reveals that, under reducing conditions, two CLAspecific bands migrate similarly to PSGL-1, suggesting the possibility of a common protein core structure. This was confirmed using monomeric recombinant (mr)PSGL-1 produced in cells cotransfected with fucosyltransferase. The mrPSGL-1 has both Eand P-selectin-binding ability, has a molecular weight common with the CLA heavy chain and reacts with the CLA-specific monoclonal antibody HECA-452. This strongly suggests that the CLA epitopes are a result of fucosyltransferase-mediated post-translational modification of the PSGL-1 backbone. That a single surface structure can be modified to bind E- and P-selectin differentially adds a dimension to the control of T-cell homing to specific tissues, in this case the skin. The physiological variables that control such differential expression will be scrutinized closely in the search for novel immunotherapies for T-cell-mediated inflammation.
J A N U A RY Vol.
I 9
No.
I 9 9 8 I
I
Endothelial cells 'present' chemokines during i n f l a m m a t i o n Middleton, J., Neil, S., Wintle, J. et al. (1997) Transcytosis and surface presentation of IL-8 by venular endothelial cells Cell 91,385-395 Tissue-derived chemokines, such as interleukin 8 (IL-8), must find their way to the luminal surface of venular endothelial cells (ECs) if they are to initiate the chemotactic gradient that ultimately draws leukocytes from the circulation into inflammed tissues. Previously, this has been proposed to occur by simple diffusion of the chemokine through intercellular spaces between venular ECs. However, Middleton and colleagues show here that the prototype chemokine IL-8 is actively internalized by ECs, followed by transcytosis and 'presentation' at the luminal surface. IL-8 trafficking was assessed by immunoelectron microscopy after intradermal injection of radiolabelled IL-8 in rabbits. As soon as 30 rain after injection, IL-8 was present on the luminal surface of ECs, particularly on microvilli and other luminal projections. Importantly, IL-8 was shown to bind initially to the EC abluminal surface,
then incorporate into smooth-membranebound plasmalemmal vesicles, which then locate to the basal and apical portions of the EC cytoplasm. These vesicles then fuse with the apical cell membrane, allowing IL-8 to be displayed at the luminal surface. No IL-8 was detected in the junctions between ECs. The display and immobilization of IL-8 at the luminal surface appears to involve heparan sulphate, although the contribution of other molecules to IL-8 transcytosis is not clear. This series of in vivo and in situ observations is particularly important given that cultured EC lines do not similarly bind and traffic IL-8 in vitro. Thus, for IL-8 at least, there is an active process of chemokine transport from the tissues, across the EC barrier, to the venous circulation. Essential chemoattractants can therefore be 'presented' at the luminal EC surface as a means of establishhag a chemotactic gradient into the tissues.
C C R 5 m o n k e y business: S I V can bypass C D 4 Martin, K.A., Wyatt, R., Farzan, M. et al. (1997) CD4-independent binding of SIV gpl20 to rhesus CCR5 Science 278, 1470-1473 The entry of human immunodeficiency virus (HIV) into a cell is initiated by gp120 binding to CD4 on the target cell. Fusion of the viral and target cell membranes is subsequently facilitated by the binding of CCR5, a target cell co-receptor of the chemokine family that is thought to induce a conformational change in the HIV gp41 protein. Rhesus macaque CCR5 (CCR5rh) shares 98% sequence identity with human CCR5hu, and supports entry of either HIV or simian immunodeficiency virus (SIV) envelope glycoproteins expressed on recombinant virions. This study examines the CD4 dependency of these interactions. HEK293 cells were transiently transfected with plasmids expressing CCR5rh or CCRShu and tested for binding HIV and SIV gp120. HIV gpl20 (strain YU2) bound to both transfectants but only if soluble (s)CD4 was also present. By contrast, gp120 from
SIVmac239 was capable of binding CCR5rh transfectants in the absence of sCD4, while binding of SIV gp120 to CCR5hu remained CD4 dependent. The basis for this dependency was narrowed down to the N-terminal Asp13 residue in CCR5rh (CCR5hu has Asn13 at this position). A mutant CCR5rhDlgN, in which the Aspl3 residue was changed to Asnl3, bound SIV gpl20 only in the presence of sCD4, suggesting that this amino acid difference is responsible for the difference in CD4 dependency between the two chemokine receptor proteins. This was confirmed by creating the corresponding CCR5huN13D mutant, which showed CD4independent binding to SIV gp120. These observations suggest that some lenteviruses can use seven-transmembranespanning proteins, such as CCR5, as the sole receptor for cell entry. This has clear implications for immunotherapy.
TODAY
Skin kin: P S G L - I becomes C L A for dermal homing Fuhlbrigge, R.C., Keitfer,J.D., Armerdin~ D. and Kupper, T.S. (I 997) Cutaneous lymphocyte antigen is a specialised form of PSGL-I expressed on skin-homing T cells Nature 389, 978-981 T cells from inflammatory or malignant skin diseases express a unique skin-homing receptor, cutaneous lymphocyte antigen (CLA). CLA is a carbohydrate moiety that binds E-selectin and facilitates targeting of T cells to inflammed skin. However, the structure of CLA has remained elusive, until now. Here, Fuhlbrigge and colleagues report that CLA is an inducible modification of a constitutive marker of human T cells: P-selectin glycoprotein 1 (PSGL-1). T cells expresing the CLA modification can bind both E- and P-selectin, suggesting that differential regulation of selectin binding can regulate tissue specifid~. Using serum-free culture conditions, it is possible to maintain elevated levels of CLA on a high proportion of peripheral blood T cells. Using a parallel-plate flow chamber to reproduce physiological shear conditions, CLA expression was shown to facilitate E-selectin tethering and rolling, whereas PSGL-1 expression allowed P-selectin binding only. Immunoblot characterization of CLA from serum-free cell cultures reveals that, under reducing conditions, two CLAspecific bands migrate similarly to PSGL-1, suggesting the possibility of a common protein core structure. This was confirmed using monomeric recombinant (mr)PSGL-1 produced in cells cotransfected with fucosyltransferase. The mrPSGL-1 has both Eand P-selectin-binding ability, has a molecular weight common with the CLA heavy chain and reacts with the CLA-specific monoclonal antibody HECA-452. This strongly suggests that the CLA epitopes are a result of fucosyltransferase-mediated post-translational modification of the PSGL-1 backbone. That a single surface structure can be modified to bind E- and P-selectin differentially adds a dimension to the control of T-cell homing to specific tissues, in this case the skin. The physiological variables that control such differential expression will be scrutinized closely in the search for novel immunotherapies for T-cell-mediated inflammation.
J A N U A RY Vol.
I 9
No.
I 9 9 8 I
I