The chemokine receptor CCR8 mediates human endothelial cell chemotaxis induced by I-309 and Kaposi sarcoma herpesvirus-encoded vMIP-I and by lipoprotein(a)-stimulated endothelial cell conditioned medium by Nasreen S. Haque, John T. Fallon, Mark B. Taubman, and Peter C. Harpel Blood Volume 97(1):39-45 January 1, 2001 ©2001 by American Society of Hematology

I-309, vMIP-I, and the CM from 6K apo(a) derivative incubated with HUVECs are chemotactic for endothelial cells.I-309 or vMIP-I (100 ng/mL) was placed in both top and bottom wells, only in the top well (containing the HUVECs), or only in the bottom wells of... I-309, vMIP-I, and the CM from 6K apo(a) derivative incubated with HUVECs are chemotactic for endothelial cells.I-309 or vMIP-I (100 ng/mL) was placed in both top and bottom wells, only in the top well (containing the HUVECs), or only in the bottom wells of the chemotaxis chamber. A recombinant apo(a) derivative containing 6 type 4 kringles, kringle V, and the protease domain of apo(a) (40 μg/mL) was incubated 6 hours with HUVECs and the CM (K) was tested for chemotactic activity as described for the purified chemokines. The − indicates medium 199; I, I-309; v, vMIP-I; K, medium from HUVECs treated with 6K apo(a) derivative. Significant activity was observed when the agonist was in the bottom chamber only indicating that the endothelial transmigration was due to chemotaxis and not due to chemokinesis. hpf, high power field. Nasreen S. Haque et al. Blood 2001;97:39-45 ©2001 by American Society of Hematology

Polyclonal goat antibody against CCR8 inhibits the chemotaxis of HUVECs in response to I-309 and vMIP-I.I-309 (▨; 100 ng/mL) and vMIP-I (▪; 100 ng/mL) were tested for endothelial cell chemotactic activity as detailed in “Materials and methods.” Prior to ass... Polyclonal goat antibody against CCR8 inhibits the chemotaxis of HUVECs in response to I-309 and vMIP-I.I-309 (▨; 100 ng/mL) and vMIP-I (▪; 100 ng/mL) were tested for endothelial cell chemotactic activity as detailed in “Materials and methods.” Prior to assay, the indicator HUVECs were incubated either with normal goat IgG, goat polyclonal antibody against CCR8 (anti-CCR8), or goat polyclonal antibody against CCR2 (anti-CCR2) (1 μg/mL) for 30 minutes at 37°C. The anti-CCR8 significantly inhibited endothelial chemotaxis as induced by I-309 and vMIP-I (*P < .001). hpf, high power field. Nasreen S. Haque et al. Blood 2001;97:39-45 ©2001 by American Society of Hematology

The CM resulting from incubation of Lp(a) (LCM) or recombinant 6K apo(a) (KCM) with HUVECs induces chemotaxis of HUVECs that is inhibited by polyclonal antibodies against I-309 or CCR8.The CM obtained following incubation of medium 199 (CM), Lp(a) (150 μg/m... The CM resulting from incubation of Lp(a) (LCM) or recombinant 6K apo(a) (KCM) with HUVECs induces chemotaxis of HUVECs that is inhibited by polyclonal antibodies against I-309 or CCR8.The CM obtained following incubation of medium 199 (CM), Lp(a) (150 μg/mL; LCM), or a recombinant apo(a) derivative containing 6 type 4 kringles, kringle V, and the protease domain (40 μg/mL; KCM) with HUVECs for 6 hours at 37°C, were tested for HUVECs transmigration as described in “Materials and methods.” Medium 199 (M), Lp(a) (150 μg/mL), or the apo(a) derivative (6K; 40 μg/mL) was used as a control. Polyclonal goat anti-I-309 (1 μg/mL) was added to the CM, or polyclonal goat anti-CCR8 (1 μg/mL) was added to the indicator endothelial cells prior to testing as indicated in the figure. Neither Lp(a) nor the 6K apo(a) derivative directly induced HUVECs transmigration as compared to the medium 199 control. LCM and KCM induced endothelial cell transmigration as compared to the CM (*P < .001). Anti-I-309 and anti-CCR8 inhibited endothelial cell transmigration induced by LCM and KCM to the constitutive activity induced by the HUVEC CM. hpf, high power field. Nasreen S. Haque et al. Blood 2001;97:39-45 ©2001 by American Society of Hematology

Murine monoclonal antibody against CCR8 inhibits chemotaxis of HUVECs in response to I-309, vMIP-I, and LCM.I-309, vMIP-I, or SDF-1 (100 ng/mL each) was used to induce endothelial chemotaxis. Murine monoclonal antibody against CCR8 inhibits chemotaxis of HUVECs in response to I-309, vMIP-I, and LCM.I-309, vMIP-I, or SDF-1 (100 ng/mL each) was used to induce endothelial chemotaxis. The HUVECs were used without treatment (No treatment) or were incubated 30 minutes with 1 μg murine monoclonal antibody against CCR8 (Mab > CCR8), an isotype monoclonal IgG1 control (IgG1), or with a murine monoclonal antibody against CCR2 (isotype IgG1), the MCP-1 receptor (Mab > CCR2). The CM obtained following incubation of Lp(a) (150 μg/mL) with HUVECs for 6 hours (LCM) was also tested using HUVECs from a different donor. These cells had lower background activity when tested using medium 199 (Control). The monoclonal anti-CCR8 inhibited endothelial transmigration stimulated by I-309, vMIP-I, and LCM, but did not inhibit migration in response to SDF-1 (*P < .001). Neither the control murine immunoglobulin nor the monoclonal anti-CCR2 inhibited endothelial chemotaxis in response to the 3 chemokines tested. hpf, high power field. Nasreen S. Haque et al. Blood 2001;97:39-45 ©2001 by American Society of Hematology

Pertussis toxin inhibits HUVECs chemotaxis induced by I-309, vMIP-I, and LCM.I-309, vMIP-I, and LCM, prepared as detailed in the legend for Figure3, were tested for chemotactic activity using HUVECs pretreated with either pertussis or cholera toxin as descr... Pertussis toxin inhibits HUVECs chemotaxis induced by I-309, vMIP-I, and LCM.I-309, vMIP-I, and LCM, prepared as detailed in the legend for Figure3, were tested for chemotactic activity using HUVECs pretreated with either pertussis or cholera toxin as described in “Materials and methods.” Pertussis toxin inhibited HUVEC chemotaxis in response to all 3 agonists (*P < .001), whereas cholera toxin did not inhibit. hpf, high power field. Nasreen S. Haque et al. Blood 2001;97:39-45 ©2001 by American Society of Hematology

RNA blot analysis of CCR8 mRNA in HUVECs RNA blot analysis of CCR8 mRNA in HUVECs.Aliquots (10 μg) of total RNA from Jurkat cells (Ju) or from confluent HUVECs (Hu) were size-fractionated on agarose gels and hybridized to 32P-labeled human CCR8 cDNA as described in “Materials and methods.” GAPDH (... RNA blot analysis of CCR8 mRNA in HUVECs.Aliquots (10 μg) of total RNA from Jurkat cells (Ju) or from confluent HUVECs (Hu) were size-fractionated on agarose gels and hybridized to 32P-labeled human CCR8 cDNA as described in “Materials and methods.” GAPDH (GAP) is shown as a control for loading of samples. The cDNA was cloned as detailed in “Materials and methods” and the identity of the fragment as CCR8 mRNA was confirmed by sequence analysis. Nasreen S. Haque et al. Blood 2001;97:39-45 ©2001 by American Society of Hematology

CCR8 is found on the endothelium in human coronary endarterectomy specimens.Sections of human coronary endarterectomy specimens were stained with polyclonal antibody for CCR8 (A), I-309 (B), murine monoclonal antibody against CCR8 (C), vWF antigen (D), mono... CCR8 is found on the endothelium in human coronary endarterectomy specimens.Sections of human coronary endarterectomy specimens were stained with polyclonal antibody for CCR8 (A), I-309 (B), murine monoclonal antibody against CCR8 (C), vWF antigen (D), monocyte/macrophage antigen CD-68 (E), and the IgG1 isotype control for the monoclonal antibody against CCR8 (F). Endothelial cells in the lesion, identified by their staining for vWF antigen (D, arrow) stain positively for CCR8 with both polyclonal and monoclonal antibodies (A,C, arrows), and for I-309 (B, arrow). Anti-CD-68 did not stain the endothelium (E, arrow); however, it stained cells in the subendothelial neointima. The murine IgG1 isotype control did not stain the endothelial surface (F, arrow) or the underlying tissue. (DAB-peroxidase, hematoxylin counterstain, original magnification × 400). Nasreen S. Haque et al. Blood 2001;97:39-45 ©2001 by American Society of Hematology

Kaposi sarcoma shows diffuse staining for CCR8 and I-309 Kaposi sarcoma shows diffuse staining for CCR8 and I-309.Biopsies of Kaposi sarcoma were stained for multiple antigens as detailed in “Materials and methods.” Shown is a Kaposi sarcoma patch stained with goat polyclonal anti-CCR8 (A), anti-I-309 (B), anti-v... Kaposi sarcoma shows diffuse staining for CCR8 and I-309.Biopsies of Kaposi sarcoma were stained for multiple antigens as detailed in “Materials and methods.” Shown is a Kaposi sarcoma patch stained with goat polyclonal anti-CCR8 (A), anti-I-309 (B), anti-vWF (C), murine monoclonal anti-CCR8 (Mab > CCR8; D), anti-CD-68 (E), anti-α-actin (F), secondary antibody alone (Control, G), and isotype control IgG1 (H). The Kaposi sarcoma spindle cells stain intensely for CCR8 with both the polyclonal and monoclonal antibodies (A,D). The spindle cells also stain diffusely for vWF and CD-68 (C,E). Pericytes in small vessels stain for α-actin (F). There is no staining when the primary antibody is omitted (G), and only a trace staining with the mouse IgG1 isotype control (H). (DAB-peroxidase, hematoxylin counterstain, original magnification × 200). Nasreen S. Haque et al. Blood 2001;97:39-45 ©2001 by American Society of Hematology