Defective respiratory burst in the seting of CD40L deficiency and its relation to
neutrophil microbicidal activity. A, Neutrophils from
CD40L-deficient patients (n = 6) exhibit normal phagocytic capacity
compared with those from healthy control subjects. B and
C, The neutrophil respiratory burst from CD40L-deficient
patients was assessed by using dihydrorhodamine (DHR) analysis
in response to P brasiliensis (Fig 1, B), as
well as PMA, fMLP, or S aureus (Fig 1, C),
from 6 independent experiments comparing each CD40L-deficient patient with
different healthy control subjects. Results were expressed as MFI (n =
6; *P ≤ .05, Mann-Whitney test).
D, Respiratory bursts from neutrophils from healthy control
subjects treated with various concentrations of catalase were analyzed by using
luminol-enhanced chemiluminescence; values were expressed as relative light
units (RLU). E, After challenging neutrophils with
P brasiliensis (ratio of 2 neutrophils/1 fungus),
microbicidal activity was assessed based on CFU values from recovering
internalized fungi. CFU values (percentage of controls) were determined in
relation to CFU numbers of untreated neutrophils from healthy control subjects.
Both the respiratory burst and microbicidal activity of neutrophils from healthy
control subjects were assessed in the presence of different doses of catalase
(100, 300, and 1000 U/mL). F, Correlation between respiratory burst
(data set from Fig 1, D) and microbicidal activity (data set
from Fig 1, E) reduction was assessed by using Pearson
correlation analysis (n = 3). G, Quantification of NETs
release by isolated neutrophils from CD40L-deficient patients in comparison with
healthy control subjects. After incubation for 4 hours in the presence of PMA,
NET release (n = 5) was analyzed by using Sytox Orange, and results were
expressed in relative fluorescence units (RFU), as previously
described.62 Patient 4
died before NET analysis was available. Significant differences are denoted by
asterisks at a P value of .05 or less (Mann-Whitney test).