Findings of granulomatous colitis and partial reduction in neutrophil oxidase activity as measured by DHR123 assay. (A) Diffuse aphthous ulcerations () were visible throughout the colon at the time of presentation. Biopsies showed prominent epithelioid granulomata in lymphoid aggregates (B, ) and in lamina propria (C) with giant cells (inset). Four-μm sections were stained using hematoxylin and eosin prior to microscopy and photomicrographs were taken with a Nikon Eclipse 80i microscope equipped with a Nikon Digital Sight DS-U1 camera using 20×/0.50NA and 40×/0.75NA objectives. (D) DHR123 fluorescence in control (black line) and patient (red line) neutrophils before and after stimulation with 0.6 μg/mL PMA (representative of 5 assays).

Analysis of NCF4 and patient pedigree. (A) Chromatograms corresponding to the wild-type and patient sequences in NCF4 exons 3 and 4. The alleles present in the mother and father are as indicated; nucleotides that are duplicated in 3957_3966dup (exon 3) and the point mutation g.6263G>A (exon 4) are outlined by a black line. Nucleotide positions are based on the gDNA sequence, reported as the NCBI Reference Sequence number NC_000022.³⁷ (B) Location of the NCF4 mutations in a schematic of p40^phox. Amino acid positions are based on NCBI Reference Sequence number NP_000622 (from NM_000631; for isoform 1, containing 339 amino acids).³⁷ Predicted functional domains shown include a PX (phagocyte oxidase homology) domain (position 19 to 140), SH3 domain (position 175 to 225), and PB1 (phagocyte oxidase and Bem1p) domain (position 285 to 306).² (C) Pedigree of the patient's family. Squares represent male family members and circle, the female family member; genotypes for the NCF4 alleles at the protein level are shown.

Translocation of YFP-p40^phox or YFP-p40^phoxR105Q during phagocytosis of SOZ. Confocal videomicroscopy was used to monitor phagosome translocation of YFP-p40^phox and YFP-p40^phoxR105Q expressed in PLB-p40 KD granulocytes. (A) Time-lapse images. Times shown are referenced to time of phagosome sealing. Arrow indicates a monitored SOZ phagosome; N, nucleus. (B) Phagosomes that showed accumulation of wild-type or mutant YFP-tagged p40^phox during phagosome cup formation were monitored for continued presence of the YFP-tagged proteins for 220 seconds after sealing. Solid line indicates YFP-p40^phoxWT; dashed line, YFP-p40^phoxR105Q. Data for YFP-p40^phoxR105Q and YFP-p40^phoxWT were collected from 10 and 5 phagosomes, respectively, as filmed in 3 independent experiments.

NADPH oxidase function and expression in patient and family members. (A) Neutrophil NADPH oxidase activity in response to PMA (200 ng/mL), fMLF (10 μM), or SOZ (2 mg/mL) as indicated, as monitored by cytochrome reduction or isoluminol for extracellular activity (PMA and fMLF, respectively) or luminol for intracellular activity (SOZ). RLU indicates relative light units. Color-coded symbols and bars indicate patient (P, red); mother (M, purple); father (F, green); brother (B, blue); or control (C, black). (Top row) Representative kinetic data; (bottom row) NADPH oxidase activity as the mean ± SD relative to control samples; results of all controls are shown in one bar. Dashed red line indicates background signal for patient neutrophils stimulated with PMA or fMLF in the presence of SOD, or for SOZ, unstimulated cells. PMA and SOZ assays were performed in patient (n = 3), mother (n = 3), father (n = 2), brother (n = 2), and controls (n = 6). *P < .05 NADPH oxidase activity versus controls. For fMLF, superoxide production was measured in the patient (n = 3) and controls (n = 6). (B) Results of a 1-step bactericidal assay at a neutrophil-bacteria ratio = 1:5. Mean ± SD of quadruplicate measurements of bacteria colony-forming units (CFUs) in the absence of neutrophils (blue line), or the presence of control (black line) or patient (red line) neutrophils. *P < .05 for the control versus patient at 20 minutes and 60 minutes. Results for neutrophils treated with the NADPH oxidase inhibitor diphenyliodonium (10 μM) are shown by dashed lines. (C) Immunoblot of NADPH oxidase subunits and densitometry of p40^phox and p67^phox expression (representative of 2 independently obtained sample sets).

Modeling and function of the p40^phoxR105Q mutant. (A) A model showing a close-up view of the Ptdlns(3)P binding cavity within the PX domain of wild-type p40^phox, and the R105Q p40^phox mutant (). The protein is shown in ribbon representation and colored in cyan. Residues that interact with Ptdlns(3)P are shown in capped-sticks rendering and color-coded based on atom types (C, N, O, and P in yellow, blue, red, and orange, respectively). Ptdlns(3)P is also color-coded based on atom types except that carbon is colored in white. Dashed lines show hydrogen bond interactions of R58, R60, V93, and R105 with Ptdlns(3)P. The interactions between amino acid 105 and Ptdlns(3)P are lost in the R105Q mutant (), as the distance between the Q105 amide oxygen and nitrogen atoms is greater than 5 Å, which precludes any hydrogen bonding. In addition, Q105 is neutral and therefore cannot compensate for the loss of the positive charge on R105, which contributes to stabilize interactions with Ptdlns(3)P. Images were created with program PyMOL (DeLano Scientific LLC). (B) Representative image collected during confocal videomicroscopy of nonstimulated (NS) PLB-985 granulocytes and during phagocytosis of SOZ, where cells are expressing the wild-type YFP-p40^phox PX domain (YFP-PX_40WT) or with R105Q mutation (YFP-PX_{40 R105Q}), as indicated. Representative of cells filmed in 3 experiments. (C-D) Results using granulocyte-differentiated PLB-p40 KD cells (1 or blue), PLB-p40 KD cells expressing p40^phoxR105Q (2 or red), PLB-p40 KD cells expressing wild-type p40^phoxwt (3 or green), and wild-type PLB-985 cells (4 or black). (C) Immunoblots probed with antibodies directed at the indicated proteins. Representative of 3 experiments. (D) PLB-985 granulocyte NADPH oxidase activity in response to PMA (200 ng/mL), fMLF (10 μM), or SOZ (2 mg/mL) as indicated, as monitored by isoluminol for extracellular activity (PMA and fMLF) or luminol for intracellular activity (SOZ). RLU indicates relative light units. (Top row) Representative kinetic data; (bottom row) NADPH oxidase activity as the mean ± SD (n = 3) relative to control wild-type PLB-985 granulocytes. Dashed blue line shows signal from PMA- or fMLF-stimulated PLB-p40 KD cells in the presence of SOD, or in the absence of stimulus for the SOZ data. *P < .05, PLB-p40 KD cells expressing p40^phoxR105Q versus PLB KD cells expressing wild-type p40^phox.