Fig. 4.

The effect of early and late CnCda4 products and A6/D6 controls on the secretion of TNFα by macrophages and the cleavage products generated by recombinant human chitotriosidase (ChT). (A) Activation of human peripheral blood-derived macrophages (HPBMs) by different chitin and chitosan hexamers (1 µg/mL). Secretion of TNFα into the supernatant was measured 6 h after stimulation. Data from five independent experiments with different batches of macrophages were normalized to the negative control (starvation medium, 0%) and to the positive control (10 ng/mL LPS, 100%). Data are presented as means ± SD (B) Activity of the human ChT measured in the supernatant after stimulation with the different chitin and chitosan hexamers (1 µg/mL). The concentration of ChT in 5 µL supernatant was determined by adding 20 mM of the ChT-specific substrate 4-methylumbelliferyl-β-d-N,N′-diacetylchitobioside hydrate in 100 mL McIlvain buffer (100 mM citric acid, 200 mM sodium phosphate, pH 5.2). The enzyme reaction was stopped after 20 min at 37 °C by adding 200 mL of 300 mM glycine-NaOH buffer, pH 10.5. Converted substrate was measured by fluorimetry at 450 nm and related to a standard of recombinant ChT. Values are means of three independent experiments ±SD. (C) Relative ChT activity on the oligomeric substrates GlcNAc₆ (A6), GlcN₆ (D6), and the early and late hexameric products of CnCda4. Isolated ChT (50 ng) was incubated with the oligomeric substrates (2.5 µg) for 24 h at 37 °C in 50 mM ammonium acetate (pH 4.5). Products were detected and quantified by HPLC-MS analysis, and ChT activity was quantified by the amount of hydrolyzed substrate, described as the degree of scission (α), where 100% equals complete degradation of the substrate. All data are based on five independent experiments (n = 5) and are given as means ± SD. Statistical analysis was performed using the t test (*P < 0.5).