The next day, the cells were washed twice with PBS and resuspended in PBS. serum IgE responses for GPR17-deficient mice as compared to wild-type mice and reduced responses in the genotypes lacking CysLT1R. These findings reveal a constitutive unfavorable regulation of CysLT1R functions by GPR17 in both the antigen presentation and downstream phases of allergic pulmonary inflammation. (Greer Laboratories, Lenoir, NC) intranasally twice per wk for 3 wks as described (27). 2 d after the last injection, mice were killed by i.p. injection of pentobarbital. BAL Fluid FIPI Cell Analysis 2 d after the last intranasal injection, the trachea was cannulated and BAL fluid was obtained by three repeated lavages with 0.75 ml of Ca2+- and Mg2+-free PBS with 1 mM EDTA. The BAL fluid was centrifuged at 500 x for 5 min. Cells were resuspended in 0.2 ml of PBS with 1% BSA, and the total cells were counted manually with a hemocytometer. For the differential cell counts of macrophages, neutrophils, eosinophils, and lymphocytes, the cells were cytospun onto a glass slide and stained with Diff-Quik, and cell types in a total of 200 cells were identified by morphologic criteria. Histology The lung tissues were excised, and the left lung was fixed and stained as described previously (16). For general morphology, tissue sections were stained with hematoxylin and eosin (H&E). The extent of cellular infiltration in the bronchovascular bundles was assessed in a blinded manner. Congo red staining was used to identify eosinophils, and periodic acid-Schiff staining was used to assess mucus and goblet cells. To assess vascular easy muscle hyperplasia, some tissue sections were stained with mouse anti-smooth muscle actin mAb (clone 1A4, Covance, Princeton, NJ) and with LSAB+ System-alkaline phosphatase (Dako, Carpinteria, CA) according to the manufacturers instructions. The total easy muscle cell numbers and the thickness of arteriolar medial muscular layer (tunica media) FIPI were quantified by Image J (National Institutes of Health image analysis software) on easy muscle actin-stained sections as previously described (27). Briefly, digital pictures of 5 medium-sized arterioles with a diameter of 30- to 50-m were taken from each slide, and the readouts are presented as the mean number of easy muscle cells per 100-m basement membrane and the mean thickness of the medial arteriolar walls in 5 arterioles from 3-5 mice per group. For immunohistochemistry to detect CysLT1R, mouse lung tissues were embedded in Tissue-Tek? OCT compound and cut into 5-m-thick sections. Frozen sections were fixed with 4% paraformaldehyde, washed, and incubated with polyclonal rabbit anti-CysLT1R IgG (RB34, against the conserved sequence DEKNNTKCFEPPQNN of extracellular loop 3 of both mouse and human CysLT1R, 30 g/ml) (23). Immunoreactivities were visualized with the rabbit ABC-peroxidase staining system (Santa Cruz Biotechnology, Santa Cruz, CA) according to the manufacturers instructions. The slides were analyzed with a Leica DM LB2 microscope (Leica Microsystems, Germany). The pictures were taken by a Nikon digital camera DXM 1200 with Nikon ACT-1 (version 2.70) image acquisition software. Measurement of Total IgE and and incubated with diluted serum followed by alkaline phosphatase-conjugated anti-mouse IgG1 (SouthernBiotech, Birmingham, AL) and p-nitrophenyl phosphate substrate (Sigma-Aldrich, St. Louis, MO). Measurement of Cytokine mRNA Expression in the Lung Total RNA was isolated from the right lungs with TRIzol reagent (Invitrogen, Carlsbad, CA), according to the manufacturers protocol. Quantities of mRNA for Rabbit polyclonal to ENO1 IL-4, IL-5, IL-13, IL-17A, and IFN- were measured relative to GAPDH using the Mx3000P Real-Time PCR System (Agilent Technologies, Santa Clara, CA) with gene-specific primers. Flow Cytometry Macrophages were harvested by peritoneal lavage with ice-cold PBS, washed, and incubated in RPMI medium made up of 10% FBS FIPI for 3 h at 37C in a humidified atmosphere with 5% CO2. Adherent FIPI cells were detached with 4 mg/ml lidocaine and 5 mM EDTA in PBS and were incubated with polyclonal rabbit anti-CysLT1R IgG (RB34, 5 g/ml) and allophycocyanin-conjugated donkey anti-rabbit IgG. Nonspecific rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) was used as a control. Analyses were performed on a FACSCanto flow cytometer (BD Biosciences), and data were analyzed with the FlowJo software (Tree FIPI Star, Ashland, OR). Cytokine Production by Parabronchial LN cells after ex vivo Restimulation with for 5 min at room temperature, and resuspended in RPMI1640 medium made up of heat-inactivated 10% FBS. After the total number of cells was counted for each mouse, cells were cultured at 2 106 cells/ml (100 l) in the presence of 20 g/ml in a 96-well plate for 72 h. The concentrations of IL-4, IL-5, IL-17A, and IFN- in the supernatants were measured with ELISA kits (eBiosciences, San Diego, CA). Transfer of at a concentration of 1 1 106 cells/ml in a 35-mm culture dish (Sumilon Celltight X, Sumitomo Bakelite, Japan) for 24.