Figure 4. Activation of neutrophil-like cells by HOCl-treated serum albumin is mediated by reversible N-chlorination.

Treatment with a 50-fold molar excess of HOCl (HSA _50xHOCl) converted HSA into an efficient inducer of the neutrophil respiratory burst, reflected by the increased production and release of oxidants that induce lucigenin chemiluminescence. This activating function of HSA _50xHOCl could be reversed by reduction with the antioxidant ascorbate (HSA _50xHOCl/Asc) and was abrogated by methylation of its basic amino acid side chains prior to HOCl exposure (HSA _Met/50xHOCl). (a) Extracellular oxidant production by neutrophil NADPH oxidase was measured in one- to two-minutes intervals over 90 min at 37°C using lucigenin-enhanced chemiluminescence. Phorbol 12-myristate 13-acetate (PMA; final concentration, 0.2 µM), untreated and variously treated HSA samples (final concentration, 3 mg · mL⁻¹) from the previous citrate synthase aggregation assays (see above) or PBS buffer (basal oxidant production) were added to (a) differentiated PLB-985 cells in PBS buffer or (b) cell-free PBS puffer containing 400 µM lucigenin immediately prior to chemiluminescence measurement. (c) Results shown in a are expressed as integrated total counts (means and standard deviations of three independent measurements) higher than buffer control. Student’s t-test: **p<0.01, ***p<0.001. PMA-induced activation of NADPH-oxidase was set to 100%.

Figure 4—figure supplement 1. Activation of neutrophil-like cells by HOCl-treated serum albumin.

Treatment with a 50-fold molar excess of HOCl converted HSA into an efficient inducer of the neutrophil respiratory burst, irrespective of the preparation (1 mM HSA treated with 50 mM HOCl or 0.3 mM HSA treated with 15 mM HOCl). (a) Extracellular oxidant production by neutrophil NADPH oxidase was measured in one- to two-minutes intervals over 90 min at 37°C using lucigenin-enhanced chemiluminescence. Phorbol 12-myristate 13-acetate (PMA; final concentration, 0.2 µM), untreated and differently treated HSA samples (final concentration, 3 mg · mL⁻¹) were added to differentiated PLB-985 cells in PBS buffer. (b) Results shown in a are expressed as integrated total counts (means and standard deviations of three independent measurements) higher than buffer control. Student’s t-test: ***p<0.001. PMA-induced activation of NADPH-oxidase was set to 100%.

Figure 4—figure supplement 2. N-chlorinated serum albumin converts 7‘-dichlorodihydrofluorescein diacetate (H₂DCF-DA) to the fluorescent 2’, 7’-dichlorofluorescein (DCF).

H₂DCF-DA is a commonly used fluorescent probe for monitoring intracellular production of reactive oxygen species. H₂DCF-DA was preincubated with 1xPBS for 15 min prior to the addition of native HSA (HSA _UT), HOCl-modified HSA (HSA _HOCl) or buffer (PBS). The fluorescence intensity of DCF was recorded for 45 min.

Figure 4—figure supplement 3. Taurine N-chloride and hydrogen peroxide-treated HSA do not induce the respiratory burst in neutrophil-like cells.

(a) The model N-chloramine prepared in different ways (5 mM of taurine treated with either 50 µM or 10 mM of HOCl) at different molar excesses (1- or 99-fold the molarity of HSA used) did not lead to significant activation of the respiratory burst in differentiated PLB-985 cells. (b) Likewise, it did not have an effect on lucigenin in the absence of cells. (c) Results shown in a are expressed as integrated total counts (means and standard deviations of three independent measurements) higher (or in case of 99 x Tau 5 mM +10 mM HOCl lower) than buffer control. PMA-induced activation of NADPH-oxidase was set to 100%.