Fig. 4

Exogenous HSP60 attracts both neutrophils and macrophages. (a) GroEL injected into the brain ventricle attracted mpx-positive neutrophils. Pink region outlined in first panel shows tracer dye in the zebrafish brain ventricle. Red arrows in the other three panels indicate injection site in the uninjected, control protein injected, or GroEL/human HSP60 injected embryos. (b) Quantification of neutrophil infiltration stimulated by GroEL. There is a significant increase in neutrophil numbers between the indicated two groups (n=10, P<0.05 for all comparisons). (c) Quantification of neutrophil infiltration stimulated by human HSP60 injected into the brain ventricle. Human HSP60 attracts significantly more neutrophils than human GAPDH (n=12, P<0.001). (d) GroEL injected into the brain ventricle attracted mpeg-postive macrophages. (e) Quantification of macrophage infiltration stimulated by GroEL. There is a significant increase in Tg(mpeg:EGFP) cells upon GroEL injection (n=8, P=0.003). (f) Inhibitory peptide L-37pA blocks GroEL’s neutrophil chemoattraction. Injection for each condition was into the brain ventricle. GroEL elicited a strong chemoattractive response, but the inhibitor of CD36, L37pA, prevented infiltration. Pictures were taken 3 h post injection into the brain ventricle and neutrophils were labelled by Tg(mpx:EGFP). Red boxes demarcate the areas in which neutrophils were quantified. (g) Quantification of the experiments in f. The increase in Tg(mpx:EGFP) cells is significant upon GroEL injection (n=10, P<0.001), but not significant upon GroEL/L37pA co-injection (n=10, P=0.109) or L37pA injection alone (n=10, P=0.962). Asterisks in the graphs indicate a significant difference. Bars = 200 μm. BSA, bovine serum albumin.