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Figure 4
Cytokine release, chemotaxis, and toxicity of Gran1: (A,B) 0.5 × 106 macrophages or PBMC were incubated overnight with Gran1 or positive controls (LPS, 100 ng/mL or PHA, 20 µg/mL, for CCL-5) and the release of the cytokines (left panels) or chemokines (right panels) was determined after overnight incubation with enzyme-linked immunosorbent assay (ELISA). Background of CCL-2 and CCL-3 signals in unstimulated samples were deducted. The small numbers left from the black bars indicate the sensitivity of each ELISA in ng/mL. Bars represent the mean cytokine release + SEM (n = 7). (C) PBMC were incubated for 3 h in a chemotaxis chamber in the presence of Gran1 or SLC (5 µg/mL). The chemotaxis index was calculated by comparison with the number of migrated cells in stimulate samples to the number of migrated cells in un-stimulated samples. Bars represent the mean chemotaxis index + SD of five independent donors. Samples were compared to medium control using a paired t-test (SLC: p = 0.0240). (D) 0.1 × 106 macrophages were incubated with Gran1 for 24 h. Cell viability was measured by PrestoBlue assay. Bars show the mean viability (%) + SEM of five independent experiments. (E) Zebrafish embryos were investigated for viability and phenotypes indicative of sublethal toxicity at 48 hpf after exposure for 24 h to Gran1 (1 µM), NRC-03 (positive control causing cytotoxicity), and Aqua ad injectabilia (negative control). The graph shows the affected zebrafish embryos (%) of two independent experiments with n = 30 embryos in each group. “Severe damage” indicates lysis or widespread necrosis, which precluded analysis of more specific sublethal phenotypes. “Altered phenotype” includes less severe lysis or necrosis (cytotoxicity), reduced or absent circulation or heart edema (cardiovascular toxicity), developmental delay or malformation (developmental toxicity), and reduced or absent touch response (neurotoxicity).