Figure 4
Figure 4. NET formation by MPO-deficient cells is not rescued by extracellular products of MPO. (A) Neutrophils from a healthy and a completely MPO-deficient donor (donor 1) were incubated either separately or co-incubated in a transwell system. Upon PMA activation, control neutrophils formed NETs. MPO-deficient neutrophils did not form NETs, even in the presence of control cells on a transwell insert. (B) Neutrophils from a healthy and a completely MPO-deficient donor (donor 1) were either incubated separately or mixed at a 1:1 ratio and stimulated with PMA. Before mixing, cells were differentially stained using MitoTracker Green and Deep Red dyes to distinguish the 2 populations. NET formation by MPO-deficient neutrophils was not significantly increased in the presence of control neutrophils. (C) A representative image of the mixed cell culture from panel B. Red, MitoTracker Deep Red–stained neutrophils from a healthy donor; green, MitoTracker Green–stained neutrophils from donor 1 (CD); blue, DNA (Hoechst). Left panel, merge of all 3 colors; middle panel, merge of red and blue channels; right panel, merge of green and blue channels. Scale bar, 25 μm. (D-E) Histamine monochloramine and histamine dichloramine were generated using an in vitro enzymatic system, then applied to neutrophils from a completely MPO-deficient donor (donor 1) at the indicated concentrations. Neither chloramine tested rescued NET formation by the MPO-deficient cells. Statistical analysis: ***P < .0001; *P = .01-05; n.s., differences not significant. (A) Control versus donor 1***; control versus donor 1 plus control transwell***; donor 1 versus donor 1 plus control transwell, n.s. (D) Control, PMA versus donor 1, PMA***; control, PMA versus donor 1, 100μM ***; control, PMA versus donor 1, 1mM*; donor 1, PMA versus donor 1, 100μM, n.s.; donor 1, PMA versus donor 1, 1mM, n.s.; donor 1, 100μM versus donor 1, 1mM, n.s. (E) Control, PMA versus donor 1, PMA***; control, PMA versus donor 1, 100μM***; control, PMA versus donor 1, 1mM***; donor 1, PMA versus donor 1, 100μM, n.s.; donor 1, PMA versus donor 1, 1mM, n.s.; donor 1, 100μM versus donor 1, 1mM, n.s. For each sample, 100-500 cells were evaluated.

NET formation by MPO-deficient cells is not rescued by extracellular products of MPO. (A) Neutrophils from a healthy and a completely MPO-deficient donor (donor 1) were incubated either separately or co-incubated in a transwell system. Upon PMA activation, control neutrophils formed NETs. MPO-deficient neutrophils did not form NETs, even in the presence of control cells on a transwell insert. (B) Neutrophils from a healthy and a completely MPO-deficient donor (donor 1) were either incubated separately or mixed at a 1:1 ratio and stimulated with PMA. Before mixing, cells were differentially stained using MitoTracker Green and Deep Red dyes to distinguish the 2 populations. NET formation by MPO-deficient neutrophils was not significantly increased in the presence of control neutrophils. (C) A representative image of the mixed cell culture from panel B. Red, MitoTracker Deep Red–stained neutrophils from a healthy donor; green, MitoTracker Green–stained neutrophils from donor 1 (CD); blue, DNA (Hoechst). Left panel, merge of all 3 colors; middle panel, merge of red and blue channels; right panel, merge of green and blue channels. Scale bar, 25 μm. (D-E) Histamine monochloramine and histamine dichloramine were generated using an in vitro enzymatic system, then applied to neutrophils from a completely MPO-deficient donor (donor 1) at the indicated concentrations. Neither chloramine tested rescued NET formation by the MPO-deficient cells. Statistical analysis: ***P < .0001; *P = .01-05; n.s., differences not significant. (A) Control versus donor 1***; control versus donor 1 plus control transwell***; donor 1 versus donor 1 plus control transwell, n.s. (D) Control, PMA versus donor 1, PMA***; control, PMA versus donor 1, 100μM ***; control, PMA versus donor 1, 1mM*; donor 1, PMA versus donor 1, 100μM, n.s.; donor 1, PMA versus donor 1, 1mM, n.s.; donor 1, 100μM versus donor 1, 1mM, n.s. (E) Control, PMA versus donor 1, PMA***; control, PMA versus donor 1, 100μM***; control, PMA versus donor 1, 1mM***; donor 1, PMA versus donor 1, 100μM, n.s.; donor 1, PMA versus donor 1, 1mM, n.s.; donor 1, 100μM versus donor 1, 1mM, n.s. For each sample, 100-500 cells were evaluated.

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