P2Y₆ receptor expression after intravenous LPS treatment and attenuated inflammatory responses in P2Y6^−/− mice after intravenous LPS exposure. (A-B) C57BL/6 mice received an intravenous injection of LPS (300 μg; E coli O26:B6) or vehicle control (phosphate-buffered saline [PBS]). Induction of P2Y₆ mRNA was seen in kidney (A) and heart (B). (C) C57BL/6 mice received an intravenous injection of LPS (300 μg; E coli O26:B6) or vehicle control (PBS). Abdominal aorta was stained with antibodies specific for murine P2Y₆ receptor and Alexa Fluor 488–coupled secondary antibody (green) or isotype controls and Alexa Fluor 488–coupled secondary antibody or Alexa Fluor 488–coupled secondary antibody only. DAPI (4′,6-diamidino-2-phenylindole) was used as nuclear counterstain (blue). Slides were kept on ice before image acquisition. Probes were analyzed by confocal microscopy with the use of Zeiss Laser Scanning Microscope LSM 710 and the Plan-Apochromat 20×/0.8 M27 objective lens with oil immersion. Zen software was used for acquisition and image processing (γ adjustment) applied equally to all images. White arrows indicate luminal side. One representative image of 3 is displayed. (D) Determination of plasma KC levels by enzyme-linked immunoabsorbent assay (ELISA) in C57Bl/6 wild-type mice treated with MRS 2578 and DMSO (solvent of MRS 2578) 2 hours after intravenous injection of LPS (300 μg; E coli O26:B6) or vehicle control (PBS) (wild-type control, n = 8; wild-type LPS, n = 8; P2Y₆^−/− control, n = 6; P2Y₆^−/− LPS, n = 6). (E) Determination of plasma KC levels by ELISA in C57Bl/6 wild-type versus P2Y₆^−/− mice 2 hours after intravenous injection of LPS (300 μg; E coli O26:B6) or vehicle control (PBS) (wild-type control, n = 10; wild-type LPS, n = 10; P2Y₆^−/− control, n = 8; P2Y₆^−/− LPS, n = 14). (F-G) Two hours after the LPS injection, mice were killed, and vascular organs (heart, kidney) were harvested. Transcript levels of VCAM were determined by real-time RT-PCR relative to the housekeeping gene β-actin in vascular organs (F) kidney (control, n = 3; LPS, n = 6) or (G) heart (control, n = 4; LPS, n = 6). All results are displayed as mean ± SD. (H-I) Determination of Evans blue concentration in kidney and heart tissue of C57Bl/6 wild-type versus P2Y₆^−/− mice 2 hours after intravenous challenge with 300 μg of LPS. (H) Kidney (wild-type control, n = 4; wild-type LPS, n = 10; P2Y₆^−/− control, n = 6; P2Y₆^−/− LPS, n = 12). (I) Heart (wild-type control, n = 4; wild-type LPS, n = 10; P2Y₆^−/− control, n = 6; P2Y₆^−/− LPS, n = 11).