Human neutrophil immunodeficiency syndrome is associated with an inhibitory Rac2 mutation

Abstract

A 5-week-old male infant presented with severe bacterial infections and poor wound healing, suggesting a neutrophil defect. Neutrophils from this patient exhibited decreased chemotaxis, polarization, azurophilic granule secretion, and superoxide anion (O(2)(-)) production but had normal expression and up-regulation of CD11b. Rac2, which constitutes >96% of the Rac in neutrophils, is a member of the Rho family of GTPases that regulates the actin cytoskeleton and O(2)(-) production. Western blot analysis of lysates from patient neutrophils demonstrated decreased levels of Rac2 protein. Addition of recombinant Rac to extracts of the patient neutrophils reconstituted O(2)(-) production in an in vitro assay system. Molecular analysis identified a point mutation in one allele of the Rac2 gene resulting in the substitution of Asp57 by an Asn (Rac2(D57N)). Asp57 is invariant in all defined GTP-binding proteins. Rac2(D57N) binds GDP but not GTP and inhibits oxidase activation and O(2)(-) production in vitro. These data represent the description of an inhibitory mutation in a member of the Rho family of GTPases associated with a human immunodeficiency syndrome.

Figure 1

(A) Impaired chemotaxis in the presence of normal CD11b up-regulation. Migration of the leading edge of neutrophils was measured in response to buffer only (random migration) or 10% zymosan activated serum (ZAS) (directed migration) in a modified Boyden chamber assay. Data are presented as the mean distance migrated in microns (±SEM) from three independent isolations. The P values were calculated by using Student's t test (*, P < 0.05; ***, P < 0.001). (B) Up-regulation of CD11b expression in response to 200 ng/ml PMA, 1 μM fMLP, or 1 μM PAF was determined by flow cytometry. The data represent the mean fold increase in CD11b surface expression (±SEM) from three to five experiments. The mean values of resting CD11b expression in mean channel fluorescence (MCF) units ± SEM were: control, 9.64 ± 1.52 (n = 5), and patient, 7.80 ± 3.09 (n = 3).

Figure 2

Functional activities of patient and control neutrophils. The release of azurophilic granules and specific granules in response to buffer only, 1 μM fMLP, or 200 ng/ml PMA in the presence of 5 μg/ml cytochalasin b was measured by the presence of either myloperoxidase (A) or lactoferrin (B) in the supernatant. The data represent the mean supernatant myloperoxidase or lactoferrin as a percentage of total cellular marker protein (±SEM) from three independent experiments. The P values were calculated by using Student's t test (*, P < 0.05). (C) The production of O₂⁻ by intact neutrophils in response to 200 ng/ml PMA, 1 μM fMLP, 1 μM PAF plus 1 μM fMLP, or 1 mg/ml opsonized zymosan (OZ) was measured as SOD-inhibitable cytochrome c reduction. The data represent the mean O₂⁻ production in nmol/min (±SEM) from 5–11 independent experiments. The P values were calculated by using Student's t test (***, P < 0.005). (D) Neutrophil polarization in response to buffer only or 25 nM fMLP. The images shown are representative of three independent experiments. Numerical values represent the mean of f-actin staining with NBD-phallacidin in MCF units ± SEM in neutrophils stimulated with 100 nM fMLP from three to six independent experiments.

Figure 3

Patient neutrophil cytosol is defective in reconstituting a cell-free NADPH oxidase system. Control (C) or patient (P) neutrophils were fractionated into plasma membranes (Mem) and cytosol (Cyt) by nitrogen cavitation and ultracentrifugation. The production of O₂⁻ was measured as SOD-inhibitable cytochrome c reduction in the SDS-dependent cell-free system. (A) The data shown are the mean values of O₂⁻ production as nmol/min⋅μg protein (±SEM) from four independent fractionations of patient and control neutrophils. The P value was calculated by using Student's t test (*, P < 0.05). (B) A representative experiment of four independent membrane and cytosol mixing experiments with patient and control fractions is shown. (C) Western blot analysis of the oxidase components p47-phox and p67-phox was performed on cytosol fractions from patient and control neutrophils. The blots shown are representative of three separate fractionations. Western blot analysis of Rac2 and p38 MAP kinase was performed on whole-cell lysates from patient and control neutrophils. The blot shown contains two separate lysates representative of five separate, whole-cell lysates. The amount of immunoreactive Rac2 protein from patient whole neutrophil lysate was 41 ± 18% of control.

Figure 4

Rac1⋅GTP[γS] restores O₂⁻ generation. Subcellular fractions were prepared as described in the legend to Fig. 3. The data represent the mean value of O₂⁻ production as nmol/min⋅μg protein (±SEM) from four independent experiments. The P value was calculated by using Student's t test (*, P < 0.05).

Figure 5

(A) Molecular analysis of the Rac2 gene. Genomic DNA was prepared from both patient and control mononuclear cells and EBV-transformed B cells. Using specific primers, all six exons and exon–intron boundaries were sequenced. The genomic structure of the Rac2 locus is shown, with black boxes representing exons at their approximate position within the locus. One allele has a single-nucleotide transition, G → A, in exon 3 in the first nucleotide position of codon 57. (B) Binding of [³⁵S]GTP[γS] and [³H]GDP to wild-type GST-Rac2 and GST-Rac2^D57N. Equal amounts of wild-type and mutant Rac2 (1 μg/tube) were used in [³⁵S]GTP[γS]- and [³H]GDP-binding experiments. These data are representative of three such experiments performed in duplicate, where the difference between replicates varied less than 5%. □, Wild-type GST-Rac2; ▵, GST-Rac2^D57N. (C) Inhibition of O₂⁻ production by Rac2^D57N. The production of O₂⁻ was measured as SOD-inhibitable cytochrome c reduction with the addition of 1 μg of membrane and 5 μg of cytosol from control neutrophils, 600 ng of recombinant p47-phox, 600 ng of p67-phox, and 200 ng of wild-type (WT) or D57N GST-Rac2^D57N that had been preincubated with GTP[γS]. The data are presented as the percentage of O₂⁻ production in the presence of WT Rac2. A representative result from three such experiments is shown. Inhibition of Rac1⋅GTP[γS] stimulated O₂⁻ production by Rac2^D57N. The production of O₂⁻ was measured as noted above except for the addition of Rac1 (173 ng) preincubated with GTP[γS] instead of cytosol in the presence of 173 ng of either WT or D57N GST-Rac2 preincubated with GTP[γS]. The data are presented as the percentage of O₂⁻ production in the presence of added recombinant WT Rac2. A representative experiment of three such experiments is shown.