Anti ccl5

Rabbit polyclonal and mouse monoclonal Abs directed against CCL2 and CCL3, respectively, as well as control rabbit and mouse Abs, were purchased from PeproTech. Goat anti-CCL4, anti-CCL5 and control Abs were purchased from R&D Systems. At the concentration employed in the study, the anti-CCL3 and anti-CCL5 Abs effectively neutralized the endogenously produced chemokines, whereas the anti-CCL4 Ab inhibited CCL4-mediated ERK1/2 phosphorylation, as assessed by Elisa and western blot, respectively (data not shown). Recombinant human IFN-β and IFN-α were kindly provided by Serono and Schering-Plough, respectively. Sheep anti-human IFN-α (neutralizing titer is 1:3000000 against 8 units IFN-α) and calf anti-human IFN-β (neutralizing titer is 1:25000 against 10 units IFN-β) sera were kindly provided by Dr Vilcek. In a cytopathic effect reduction assay these sera have been shown to neutralize exogenous IFNs at the used concentrations (data not shown). Soluble LDLR was purchased from R&D Systems. The nucleoside analogue reverse-transcriptase inhibitor AZT and the fusion inhibitor T20 were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program.

Purified human eosinophils or mouse eosinophils at 2 × 10⁶ cells/mL or 3 × 10⁶ cells/mL in Ca²⁺/Mg²⁺ HBSS (HBSS^+/+; pH 7.4) were pre-treated with the PI3K inhibitors wortmannin (1 μM; Biomol) and LY294002 (10 μM; Cayman Chemicals), PKC inhibitor calphostin C (1 μM; Biomol), pertussis toxin (PTX; 100 ng/mL), neutralizing monoclonal antibodies anti-CCL5 (10 μg/mL) and anti-CCR3 (10 μg/mL) (both from R&D), the PAF receptor antagonist BN52021 (10 μM), PGD₂ receptor antagonists BWA868c (200 nM; DP1 receptor) and Cay10471 (200 nM; DP2 receptor, or PGD₂ synthesis inhibitors HQL-79 (10 μM; H-PGDS) and AT-56 (10 μM; L-PGDS) (all from Cayman Chemicals) at 37°C for 30 min before stimulation with human recombinant (hr) or mouse recombinant (mr) leptin (0.5, 5, or 50 nM, as indicated; Peprotech) for 15 or 60 min in a water bath (37°C). Alternatively, eosinophils were also stimulated with PAF (1-O-hexadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine; 1 μM; Cayman Chemicals), hr CCL5 (also known as RANTES−100 ng/mL; R&D) or PGD₂ (25 nM; Cayman Chemicals). Each experimental condition was repeated at least three times with eosinophils purified from different donors or differentiated in vitro from different mouse bone marrows.

For the transwell migration and invasion assay, FLS from WT (Epas1^+/+) or Epas1^+/− mice (C57BL/6) were seeded on membranes of inserts with 8.0 μm pores (Corning Costar, Corning, NY, USA ) and the lower chambers filled with CM, which served as the chemoattractant. Serum-free CM was prepared from primary cultures of chondrocytes or FLS isolated from WT or Epas1^+/− mice (C57BL/6). For neutralization of chemokines, 0.5 μg anti-IgG (control), anti-CXCL2, or anti-CCL5 (R&D Systems) were added to the lower chamber. For the invasion assay, inserts of the transwell were precoated with matrigel (BD Bioscience, San Jose, CA, USA). After 24 hours of incubation, transwell inserts were fixed with 4 % paraformaldehyde for 10 minutes and stained with 0.05 % crystal violet. Cells on the bottom layer were captured in five randomly selected fields at 200× magnification using a light microscope and quantified with Image J software (NIH, shareware) .