Fig. 2

Macrophage chemoattraction by Cxcr3.2-independent factors. (A–H) Chemoattraction of macrophages by LTB₄ and fMLP. cxcr3.2^+/+ (A–C) and cxcr3.2^−/− (D–F) embryos at 30 hpf were locally injected into the hindbrain cavity with 10.1 ng/ml (30 nM) LTB₄ (representative images B and E) or with 0.2 mg/ml (0.5 mM) of fMLP (representative images C and F). Mock control injections with the solvents were 0.02% EtOH in PBS for the LTB₄ treatment and 5% DMSO in PBS for the fMLP treatment (representative images A and D). Lp-stained cells accumulated in 3 hours within the hindbrain limits (dotted line) were counted as macrophages, as neutrophils do not significantly contribute to the total number of leukocytes recruited to the hindbrain at this developmental stage (supplementary material Fig. S3). LTB₄ and fMLP stimulation resulted in significantly increased macrophage recruitment, compared with the mock injection, and this was independent of cxcr3.2 mutation (G,H). Scale bar in A–F: 100 μm. Data were accumulated from three independent experiments. Sample size (n) in G: 82, 70, 86, 100; Sample size (n) in H: 47, 30, 39, 46. Error bars: median and interquartile range. (I) Local macrophage recruitment to CuSO₄ chemically-induced inflammation. Copper sulphate treatment was performed on 3 dpf embryos and macrophages accumulated in 3 hours at the damaged neuromasts of the lateral line were counted as Lp-stained cells minus Lp/Mpx double-positive cells, representing neutrophils. Similar numbers of macrophages were recruited to inflamed areas in both cxcr3.2^+/+ and cxcr3.2^−/− embryos. Sample size (n): 24, 25. Error bars: median and interquartile range. ns, non-significant; **P<0.01; ***P<0.001.