Defective CXCL8-dependent β2-integrin activation and signaling in CCRL2-deficient mice. (A) Intravital microscopy of the interaction between leukocytes and endothelial cells in the synovial microvasculature in WT and CCRL2-deficient (KO) mice previously immunized with metBSA. A leukocyte was considered adherent when it was stationary for at least 30 seconds, and total leukocyte adhesion was quantified as the number of adherent cells within a 100-μm length of venule in 5 minutes. Left panel: representative images captured after antigen (left) and saline (right) injection into the knees. Scale bar represents 20 μm. Right panel: quantitative analysis of cells adherent to the synovial endothelium (n = 7 mice per group). ***P < .001 by Student t test. (B) Under flow adhesion of freshly purified BM neutrophils from WT and CCRL2-deficient (KO) mice to immobilized E-selectin, ICAM-1, and CXCL8. Where indicated, WT neutrophils were pretreated with an anti-CCRL2 or isotype control moAb for 30 minutes. The behavior of interacting neutrophils was recorded on a digital drive with fast charge-coupled device video camera (25 frames per second, capable of one-half subframes per 20 millisecond recording) and analyzed subframe by subframe. Single areas of 0.2 mm² were recorded for at least 60 seconds. Interactions of 40 milliseconds or longer were considered significant and scored.²⁴  Cells that remained firmly adherent for at least 1 second were considered fully arrested. Cells arrested for at least 1 second and then detached or arrested for 10 seconds and then remained adherent were scored separately and plotted as independent groups. Data are shown as mean ± SEM of 3 experiments performed in triplicate. ***P < .001 WT vs CCRL2 KO or WT+anti-CCRL2 by Student t test. (C) ERK1/2 phosphorylation evaluated in CD11b⁺/Ly6G⁺-gated freshly isolated BM cells stimulated with 100 ng/mL CXCL8 at the indicated time points. Results are expressed as percent of increase of MFI of stimulated over unstimulated (unst) cells. Shown is the mean ± SEM for 8 mice per group in duplicates. *P < .05 by Student t test. The activation of (D) RhoA and (E) Rac1 was evaluated in CD11b⁺/Ly6G⁺-gated freshly isolated BM cells. Data are expressed as fold of increase of MFI of CXCL8 100 ng/mL stimulated over unstimulated cells (time 0) at the indicated time points. The mean ± SEM of 10 RhoA and 8 Rac1 mice per group are shown. *P < .05; **P < .01 by Student t test. (F) Calcium fluxes of CXCL8-stimulated WT and CCLR2-deficient neutrophils. Fluo-8 NW-loaded freshly isolated BM neutrophils were exposed to increasing concentrations of CXCL8; calcium traces are reported as ΔF/F₀ above time (left 2 panels), where ΔF/F₀ is the difference between the relative fluorescence units and the basal fluorescence at time 0 (F₀) normalized for F₀. Each curve represents the mean of 4 replicate wells. Right panel: concentration-response curves obtained by calculating the calcium response as ΔF/F₀, where ΔF represents the difference between the maximum fluorescence signal in a selected time window (9-65 seconds) and the minimum fluorescence signal occurring at 11 seconds normalized for the basal fluorescence at F₀. Half maximal effective concentration values were 125 nM and 251 nM for WT and CCLR2-deficient neutrophils, respectively. Data are shown as mean ± SEM (n = 4). P < .0001 by Student t test.