Role of CCRL2 in neutrophil functions. (A) Elastase release of WT and CCRL2-deficient neutrophils in response to CXCL8 and CXCL1 evaluated as elastase activity in cell supernatants. Representative results of 1 of 3 independent experiments performed in triplicate are shown as the mean ± SEM. (B) Migration of BM neutrophils in response to LTB4 (100 nM), platelet-activating factor (PAF) (100 nM), CXCL1 (100 ng/mL), CXCL8 (100 ng/mL), CCL3 (100 ng/mL), or chemerin (100 pM) evaluated in Boyden chambers as previously described.³⁴  Results are expressed as the mean number of migrated cells in 5 high-power fields (100×). Data are shown as the mean ± SEM of 3 independent experiments performed in triplicate. (C) Migration of BM-purified neutrophils from WT or CCRL2-deficient (KO) mice assessed by time-lapse microscopy. Representative tracking analyses of resting WT and CCRL2-deficient neutrophils in response to CXCL8 are shown in the left panel. Single-cell speed toward CXCL8 of cells stimulated with LPS (100 ng/mL) or left untreated is shown in the right panel. Single-cell directionality and speed were analyzed with ImageJ software, and data were re-elaborated by using TimeLapseAnalyser open source software (http://www.informatik.uni-ulm.de/ni/staff/HKestler/tla/). In the right panel, the mean ± SEM of the speed recorded in 3 independent experiments is shown. *P < .05 by one-way ANOVA; **P < .01 by Student t test.