1. No category

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1995 85: 231-241
A new X-linked variant of chronic granulomatous disease
characterized by the existence of a normal clone of respiratory
burst-competent phagocytic cells
RC Woodman, PE Newburger, P Anklesaria, RW Erickson, J Rae, MS Cohen and JT Curnutte
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A New X-Linked Variant of Chronic Granulomatous Disease Characterized
by the Existence of a Normal Clone of Respiratory Burst-Competent
Phagocytic Cells
By Richard C. Woodman, Peter E. Newburger, Pervin Anklesaria, Richard W. Erickson, Julie Rae, Myron
and John T. Curnutte
Chronic granulomatous disease (CGD) is characterized by
recurrent infections, and is usually associated with a complete i n a b i l i i of phagocytic cells to generate superoxide
anion (0;).Rarely, variant forms of CGD have beenreported
in which there is reduced, but detectable, 0; production by
phagocytic cells. We describe three adult males in two kindreds with a unique form of X-linked cytochrome b,,-deficient (X91-) CGD not previously reported. All three patients
had two distinct populations of phagocytic cells, with one
subset capable of normal respiratory burst activity and the
other larger subset inactive, as in classic CGD (X91"). The
respiratory burst activity in neutrophils purified from each
patient was -10% of normal as determined by 0: producspectroscopy, and
tion, 0, consumption, cytochrome bra
membrane oxidase activity using a cell-free activation system. In contrast with other patients with X91"variant CGD,
the unique feature of these patients is the presence of a
small but significant population (5% t o 15%) of circulating
C
HRONIC GRANULOMATOUS disease (CGD) is a
rare inherited group of disorders characterized by recurrent, sometimes life-threatening, bacterial and fungal infections.' The impaired microbicidal activity occurs because
of an inability of phagocytic cells (neutrophils, monocytes,
macrophages, and eosinophils) to generate superoxide (0;)
from molecular oxygen by the enzyme referred to as the
respiratory burst oxidase.' Although 0; itself is not microbicidal, it is the precursor for all other microbicidal oxidants
produced by phagocytic c e h 2
From the Division of Experimental Hematology, Department of
Molecular and Experimental Medicine, Research Institute of Scripps
Clinic, LaJolla, CA; the Departments of Pediatrics and Radiation
Oncology, University of Massachusetts Medical School, Worcester,
MA; and theDepartment of Medicine, University of North Carolina,
Chapel Hill.
Submitted February 7, 1994: accepted September 12, 1994.
Supported in partby
Public Service Grants No. A124838,
RR00833, A133346, A120866, and AI28412 from the National Institutes of Health: the Canadian government, and the Division of Hematology, University of Calgary. R.C. W. was a recipient of a Research Grant from the Department of Medicine, University of
Calgary, Calgary,Alberta, Canada and is currently a Clinical fnvestigator of the Alberta Heritage Foundation for Medical Research.
J.T.C. is an Established Investigator of (he American Heart Association.
Published in part in abstract form in Blood 74:401a, 1989 (suppl)
and presented at the 1982 Interscience Conference on Antimicrobial
Agents and Chemotherapy, New Orleans.
Address reprint requests to Richard C. Woodman, MD, Health
Sciences Centre, University of Calgary, 3330Hospital Dr NW, C&gary, Alberta T2N 4N1, Canada.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
0006~4971/95/8501-0014$3.00/0
Blood, Vol 85, No 1 (January), 1995: pp 231-241
S.Cohen,
neutrophils and monocyteswith completely normal respiratory burst activity asassessed by nitroblue tetrazolium
(NBT) reduction and flow-cytometric measurement of dihydrorhodamine oxidation. NBT reduction of peripheral blood
granulocyte-macrophage progenitor cells also showed the
presence of a subset of coloniesderived from myeloid progenitor cells that had normal respiratory burst capabilities. A
mosaic XXchromosome karyotype and anunstable oxidase
complex that might occur during myeloid maturation were
both excluded as possible explanations. In these families,
the molecular defect in the gp9l-phox gene, which is currently under investigation, appears to prevent expression of
the gene in the majority of neutrophils, but not in a small
subset. Our studies suggest that commitment t o either a
respiratory burst-competent or -incompetent phagocyticcell
occurs at the level of the myeloid progenitor cell.
0 1995 by The American Societyof Hematology.
Recently, it has become apparent that the active oxidase
is actually a complex multicomponent enzyme system that
is comprised of several membrane and cytosolic proteins.
These include several recently recognized guanosine triphosphate (GTP) binding protein^^.^ important in oxidase regulation and cytochrome
a unique membrane-associatedheterodimeric cytochrome b that functions as the terminal
electron transport carrier for oxidase. The cytochrome b558
consists of a 91-kD glycoprotein subunit (gp91-phox; phox
for phagocyte oxidase) and a 22-kD polypeptide (p22-phox)
subunit, both of which are required for the stable expression
of the entire heterodimer."" Two cytosolic components of
the oxidase, referred to as p67- and p47-phox, have been
identified, although the involvement of other cytosolic proteins has not been ex~1uded.l'"~
Recent evidence suggests
that the phosphorylated form of p47-phox is responsible for
assembling the cytosolic components into a large complex
(a260-kD) that is translocated to the membraneI5 and binds
near the C-terminus of the g p 9 1 - p h o ~In
' ~addition, p47- and
p67-phox both contain regions that could bind the activated
membrane oxidase with the cytoskeleton of the ell.'^,'^
The vast majority of CGD patients have no detectable
0;production (<1% of normal) by their phagocytic cells.'
Occasionally (<10% of cases), patients with an X-linked
variant form of CGD have been reported in which there is
diminished, but measurable, 0; production (2% to 35% of
Nitroblue tetrazolium (NBT) reduction by neutrophils from these patients usually shows a homogeneous
population of cells with weak staining compared with normal; ie, each celI has only a few grains of blue formazan
precipitate rather than the heavy deposits normally seen.
NBT reduction by neutrophils from carriers with these variant X-linked forms of CGD are also unique inthat two
populations of NBT-positive cells are seen, one with weak
staining identical to the patients, and one with intense reduction similar to normal.'9s22324
In several of the cases reported,
231
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232
WOODMAN ET AL
Famiry P
I
' I
II
6
2
b
2
1
3
l
Famiry B
I
IT
1
2
3
4
Fig 1. Pedigrees of family P and family B. Individuals P+,P-M,,B-
IZ,and B-lI1were proven to be carriers forX-linked COD based onthe
NBT slide test (resultsnot shown). For each carrier,the NBT-positive
neutrophils were strongly positive while the negative neutrophils
completely lacked NBT reduction. In addition, these individuals all
had intermediate superoxide rates and cytochrome b measurements
(results not shown). In contrast, individuals P-&, P-1Iz,B+,B+,
and
B-ll, were asymptomaticand had normal NBT reduction, cytochrome
b spectroscopy, superoxide production or chemiluminescence meanormal
.
surements. A slash indicatesdeceased; (U), normal male; (0)
female; IO), proven female carrier; (m),affected male.
lococcus aureus hepatic abscess. The postoperative course was complicated by a Serratia marcescens wound abscess. At the time of
diagnosis, =3% of his neutrophils were reported to heNBT-positive.
Infections since diagnosis have included S marcescens pharyngitis,
submandibular lymphadenitis, recurrent pleural effusions, and bilateral pneumonias. The patient has been on daily prophylactic trimethoprim-sulfamethoxazole since age 4, and since age 18, he has also
received prophylactic interferon y (IFNy; 0.05 mg/m2) subcutaneously three times per week. Since starting IFNy, the patient has not
had any serious infections requiring hospitalization and parenteral
antibiotics. Red blood cell (RBC) phenotyping showed normal expression of the Kell antigens. X-linked inheritance was established
based on studies in the mother (P-I,) and sister (P-I12) in which a
mosaic pattern of NBT-positive and NBT-negative neutrophils was
found and intermediate levels of 0; production and cytochrome b,,,
(20% to 30% of normal) were measured. The proband's mother (PI,) has a history of discoid lupus with weakly positive antinuclear
antibodies but negative anti-DNA antibodies.
Family B. Brothers B-11, and B-I13 (Fig l), were diagnosed with
CGD at ages 14 and 12, respectively. B-U?, a 34-year-old man, has
a life-long history of infections that includes: (1) recurrent S aureus
hepatic abscesses; (2) several episodes of culture-negative pneumonia involving both lungs; (3) a myocardial abscess; (4) septic arthritis
of the left knee complicated by cellulitis and septic thrombophlebitis:
% his
and ( 5 ) recurrent aphthous stomatitis. At diagnosis, ~ 3 of
neutrophils were reported to be NBT+. B-II,, a 32-year-old man, has
also experienced multiple infections that include: ( 1 ) right humeral
osteomyelitis after a fracture that eventually required surgical debridement and open reduction; (2) two separate episodes of S marcescens bursitis, one involving the right elbow and the other the right
calcaneus; (3) bacterial conjunctivitis and blepharitis; (4) multiple
episodes of gram-negative and Staphylococcal soft-tissue abscesses;
and ( 5 ) recurrent pneumonia. In addition, B-I12 has experienced
erythema multiforme, recurrent aphthous stomatitis, severe periodontal disease, and chronic interstitial pneumonitis. Because both
patients are allergic to sulfonamides, they have generally used
dicloxacillin or cephalexin for daily prophylaxis. Both B-I12 and B113 have received prophylactic recombinant IFNy (0.05 mg/mz/dose)
subcutaneously three times per week for the past 33 months. Neither
patient has had any serious infections since starting IFNy. Both
patients have normal RBC expression of the Kell antigens. Intermediate levels of neutrophil chemiluminescence and a dimorphic population ofNBT' and NBT" neutrophils in the mother (B-12)established the X-linked inheritance.
MATERIALS AND METHODS
Chemicals. Cytochrome c (horse heart, type VI), bovine erythrothe reduced respiratory burst activity was associated with
cyte superoxide dismutase (SOD), diisopropylphosphofluoridate,
abnormal oxidase kinetics, specifically a reduced affinity for
phorbol myristate acetate (PMA), catalase, NBT (type III), dimethyl
nicotinamide adenine dinucleotide phosphate
(NADPH).'8,19.22 sulfoxide (DMSO), and P-NADPH were obtained from Sigma
We describe three patients from two kindreds with a gp91Chemical Co. (St Louis, MO). PMA stock solutions were made in
phox-deficient variant of CGD not previously reported. In
DMSO at a concentration of 2 mg/mL, stored in small aliquots, and
contrast with the other reported cases of variant X-linked
diluted to 80 pg/mL in DMSO just before use. Sodium dodecyl
sulfate (SDS) and gelatin were purchased from Bio-Rad Laboratories
CGD, these patients have two distinct populations of phago(Richmond, CA). Ficoll-Paque andPercoll were purchased from
cytic cells with varying respiratory burst capabilities. The
Pharmacia Fine Chemicals (Piscataway, NJ). Dihydrorhodamine
majority of cells were completely unable to generate OF, but
(DHR) was purchased from Molecular Probes (Eugene, OR), disthere was a small distinct subset (5% to 15%) of cells with
solved in DMSO at a concentration of 1 mmol/L and stored at -20°C
normal respiratory burst activity. This population of phagointhe dark under Nz (gas) andthen diluted inphosphate buffer
cytic cells appeared to be entirely responsible for the residbefore use. Recombinant human granulocyte colony-stimulating facual 0; production that was 10% to 15% of normal.
tor (rhG-CSF) and recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) were provided by Amgen, Inc
CASEREPORTS
(Thousand Oaks, CA). rhIFNy was obtained from Genentech, Inc
(South San Francisco, CA). Other reagents were of the best grade
Family P. The proband (P-&) (Fig l), a 22-year-old man, was
commercially available and were used without further purification.
diagnosed with CGD at age 4 years, after presentation with a Sraphy-
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NEW X-LINKED VARIANT OF CMO-B-DEFICIENT CGD
233
Table 1. Intact Neutrophil Respiratory Burst Activity in Patienta With Variant X-Linked CGD
Control
0; production* (nmol/min/lO’ cells)
PMA
FMLP
FMLP + DHCB
119.3 2 30.2 (n = 9)
38.5 2 15.3 (n = 5)
82.1 t 44.5 (n = 3)
o2consumptiont (nmol/min/10’ cells)5.4
(n = 2) 6.3
50.1
Cytochrome 4=
Intact cells (pm0ls/10~/cells)
64.8 2 21.2 (n = 6)
Membranes (pmols/lO’ cell equivalent)
(n = 2)
6.5
73.9
Flow cytometryS (% positive)
DHR (n = 2)
a3
NBT Slide Test5 (% positive)
100 (n = 15)
Neutrophils
100
Monocytes
P4I3
Ell2
B-llo
14.4 t 5.3 (n = 9)
0.0 (n = 5)
0.0 (n = 3)
9.9 t 6.8 (n = 5)
7.5 2 2.2 (n = 4)
1.9 (n = 2)
0.0 (n = 2)
0.0 (n = 2)
0.0 (n = 2)
0.0
4.2
2
1.3 (n = 6)
6.0 f 1.4 (n = 3)
ND
4.4 t 1.1 (n = 3)
ND
6
8 t 4 (n = 12)
752
6
723(n=3)
721
5
9t2(n=3)
11 2 2
Unless indicated results are represented as mean t 1 SD. Each experiment is an average of two measurements.
Abbreviations: DHCB, dihydrocytochalasin B; ND, not determined; n, number of experiments.
As indicated, cells were activated with either PMA (200 ng/mL), FMLP ( W 5 mol/L), or preincubated with DHCB 110 pmol/L) at 37°C for 10
minutes followed by activation with
mol/L FMLP.
t Activated with 100 pL heat-killed S aureus.
Refers to percentage of cells with an increased mean fluorescence value after 20 minutes of activation with PMA (final concentration 100
nglmL).
5 Activated with PMA (final concentration 200 ng/mL). NBT slide test results for unstimulated cells not shown.
*
Cell preparation and fractionation. Neutrophils were isolated
from whole blood by dextran sedimentation, hypotonic water lysis
and Histopaque density-gradient ~entrifugation.’~
Cytosol and solubilized membrane for 0; measurements in the cell-free activation
system were prepared from unstimulated neutrophils collected by
leukapheresis and fractionated using a previously published
method.26
Cytochrome
The quantity of cytochrome bSssin neutrophil
cell pellets ( lo7 cells total) was determined by dithionite difference
spectros~opy.~~
Cytochrome
spectroscopy was normally performed on the same day as neutrophil isolation. All measurements
were done in duplicate unless otherwise indicated. In patient P113, cytochrome b spectroscopy was also performed on solubilized
neutrophil membranes using the same method. These measurements
were expressed as picomoles per lo7 cell equivalents of membrane.
0;production and oxygen consumption. The rates of 0; production by intact neutrophils and in the cell-free activation system”
were measured using the SOD-inhibitable reduction of fenicytochrome c. For intact neutrophils, cells were stimulated with PMA
(final concentration, 200 ng/mL).29 All measurements were done in
duplicate unless otherwise indicated. The cell-free activation system
consisted of membrane and cytosol fractions of neutrophils, NADPH
(160 pmoVL) and was activated by the addition of SDS (40 pmoll
L) without any preincubation. Cytosol activity was determined by
mixing neutrophil membranes from normal donors with partially
purified cytosol from the patients. Membrane activity was determined by mixing neutrophil cytosol from normal donors with membranes from the patients. All measurements were done in triplicate.
The lag time was calculated from the intercept of the back-extrapolated linear portion of the curve with the preactivation baseline of
zero absorption change.
Neutrophil oxygen consumption was measured polarographically
with an electronic oxygen monitoring system (Yellow Springs Instruments CO, Yellow Springs, OH) using a previously published
method.” Neutrophils were activated with 100 pL of heat-killed S
aureus that had been opsonized with sera from normal donors; oxygen consumption was monitored over 5 minutes.
NBT reduction. The ability of individual circulating neutrophils
and monocytes to reduce NBT after stimulation with PMA (200 ngl
mL) was determined using a previously published method?’ The
percentage of NBT-positive cells was determined by light microscopy after counting 2200 neutrophils (or 2 1 0 0 monocytes). The
staining intensity of the patient’s NBT-positive cells compared with
control cells was also determined.
Flow cytometry. Flow cytometric analysis of neutrophil respiratory burst activity was measured using a modification ofa previously
published method.32Neutrophils were suspended in phoshate-buffered saline containing 5 mmoVL glucose and0.1 % gelatin at a
concentration of 1 X IO6cells/mL and preloaded with DHR (1 pmoll
L) by incubating the cells with DHR in a waterbath at 37°C for 15
minutes, with gentle mixing every 5 minutes. After incubation with
DHR, catalase (275 U/mL final concentration) was added to the
neutrophil suspension. Aliquots of cells were then removed and
incubated with either PMA (100 ng/mL final concentration) or
DMSO at room temperature for 10 and 20 minutes. Samples were
then analyzed on a FACScan flow cytometer (Becton Dickinson,
San Jose, CA). A total of 10,ooO cells from each sample were
collected, and green fluorescence emission between 530 and 550 nm
was monitored. Fluorescence intensity was measured on a logarithmic scale and the mean fluorescence of the analyzed cells was calculated using FACScan Research software (Becton Dickinson). Respiratory burst-dependent fluorescence was determined by subtracting
the mean channel fluorescence of cells exposed to DMSO alone
from that measured in cells stimulated with PMA.
Granulocyte-macrophage colony assays. Human peripheral
blood mononuclear cells were prepared by Histopaque (Sigma
Chemical CO, St Louis, MO) centrif~gation,~~
frozen in 10% DMSO
in a programmable cell freezer, then later thawed, counted, and used
for colony assays as follows. Cells were resuspended in culture
media supplemented with 30% fetal calf serum (FCS), then plated at
106/mLin 1.1% methylcellulose containing 30% FCS, 1.2% bovine
serum albumin, 5 X lo4 m o m &mercaptoethanol, 10 ng/mL human
recombinant interleukin-3 (IL-3; Immunex Corp, Seattle, WA), and
1,OOO U/mL of G-CSF (Amgen). Cultures were incubated in humidified chambers at 37°Cand 5% CO2 for 14 days. The functional
capacity of mature granulocytes in the colonies was then evaluated
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WOODMAN ET AL
234
Fig 2. NBT reduction by neutrophils and monocytesafter PMA activation (see Materials and Methods).
la) NBT-positiveneutrophilsfrom P11s; (b) NBT-positive neutrophils
from B-l13; (c) NBT-positive neutrophils from a patient with typical
X91" variant CGD in which greater
than 95% of cells are weakly NBTpositive; and Id) shown for comparison are neutrophils from a normal
donor (original magnification x
630).
histochemically by NBT dye reduction in response to PMA stimulation, as previously de~cribed.".'~Colonies of >25 cells were counted
and scored as positive (>90% of cells within the colony stained
blue with reduced NBT), mixed (10% to 90% of cells reducing
NBT), or negative (<lo% of cells reducing NBT). In addition,
colonies were plucked, cytocentrifuged onto glass slides, stained
with Wright-Giemsa, and examined microscopically for cell morphology.
Southern blot analysis. High molecular-weight genomic DNA
was extracted from whole peripheral blood, digested with restriction
endonuclease Tag I, electrophoresed in 0.8% agarose, and transferred
to nitrocellulose filters by standard rnethod~.'~
Filters were hybridized with labeled3' probe 754, a 2.3-kb HindIII fragment closely
linked to the X chromosome CCD locus.3s In addition, DNA was
analyzed (by Collaborative Research, Inc, Waltham, MA) for restriction fragment-length polymorphisms (RFLPs) detected by the highly
polymorphic, non-CCD-liked probe S-232, which maps near the
x chromosome c e n t r ~ m e r e . ~ ~
RESULTS
NBT reductionbycirculatingleukocytes.
Although the
large majority of cells remained negative, all three patients
showed a small subset (5%to 10%) of neutrophils and monocytes that were capable of reducing NBT after stimulation
with PMA (Table 1). Not only did the percentage of NBTpositive cells approximate the patients' intact cell 0; production, but they stained as intensely as cells from normal
donors, unlike the uniformly weakly-staining cells from patients with the more common form of variant X-linked CGD
(Fig 2).
Neutrophil respiratory burst activity. Intact neutrophil
respiratory burst activity fiom patients P-113, B-112, and B113 is summarized in Table 1. Neutrophils from all three
patients had 0; production, as determined by PMA-induced
cytochrome c reduction, that was consistently 5% to 15% of
normal (P-113, 12%; B-&, 8%; B-&, 6%). Lag times after
PMA activation were similar for patient (P-113, 48 sec; BTI,,54 sec; B-1113, 53 sec; n = 2) and control neutrophils
(54 ? 8 sec; n = 8).
In two of the patients (P-113 and B-112),neutrophil oxidase
activity in response to a particulate agonist (opsonized S
aureus) was also examined. Neutrophil O2 consumption in
P-11, (12%) and B-112 (1 1%) closely approximated the neutrophil PMA-induced 0; rates and the proportion of NBTpositive cells, when these are expressed as a percentage of
normal. Although neutrophils from P-113 and B-112responded
to soluble (PMA) and particulate agonists (opsonized S
aureus), for reasons that remain unclear, we were unable to
detect 01 production after Fh4LP stimulation (with or without dihydrocytochalasin B pretreatment). Studies on neutrophils from P-113 and other members of his family (P& P12,P-I&, P-112) showed normal surface expression of FMLP
receptors (data not shown). One possible explanation is that
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235
NEW X-LINKED VARIANT OFCYTO-&DEFICIENTCGD
0.0
0.4
1.60.8
1.2
6
Cytosol (x 10 cell equivalents)
4r
/
Fig 3. Superoxide productionin the cell-free sctivation system.(A) control nnd paient OF production
from neutrophil cytosol using membranes from normal donors. (B) control and patient 0; production
from neutrophil membranes usingcytosol from normal donors.
m
Control 1
2
v Control
0 B-II-3
v P-n-3
0
with oxidase activity of only 5 % to 15% of normal, FMLP
stimulation that is typically 20% to 25% of maximal PMA
rates may be below the level of detection of ferricytochrome
c reduction. Together the 0; production and O2consumption
results show that the neutrophil oxidase from these patients
respond comparably to both soluble (PMA) and particulate
stimuli (opsonized S aureus).
Levels of neutrophil cytochrome b558 from each patient
also corresponded (P-113, 6%;B-I12, 9%; B-I13, 7%) to the
amount of intact cell respiratory burst activity. Spectroscopy
of an enriched-membrane preparation from neutrophils of P113 confirmed the presence of a small amount of cytochrome
(9% of control).
The cytosolic and membrane oxidase activity for neutrophils from patients P-11, and B-II, is shown in Fig 3 . Cytosol
from both patients, when reconstituted with normal membranes, was capable of 0; production comparable with control cytosol. In contrast, oxidase activity of neutrophil membranes from both patients, when reconstituted with normal
cytosol, was detectable, but significantly reduced compared
0
2
4
5 6
8
Membranes (x 10 cell equivalents)
10
with normal membranes. The membrane oxidase activity that
was present closely approximated the percentage of oxidase
activity observed with intact cells from each patient.Overall,
these results are consistent with the diagnosis of cytochrome
b-deficient variant CGD and suggest that the residual respiratory burst (5% to 15% of normal) in P a 3 , B-& and B-I13
was caused by a small subset of circulating neutrophils (and
monocytes) that appeared to have a normal oxidase activity.
Flow cytometry. How-cytometric analysis of DHRloaded neutrophils also confirmed that 5% to 6% of the
neutrophils underwent a normalrespiratory burst after PMA.
Representative histograms illustrating the subset of DHRpositive cells from P-11, and B-I12 are shown by the arrows
in Fig 4, A and B, respectively.
To confirm these results, experiments (n = 3 ) were performed in which purified neutrophils from a normal donor
were mixed (0% to 6%) with neutrophils (94% to 100%)
from an X91" CGD patient with undetectable oxidase activity. This cell suspension was then activated with PMA under
identical conditions as described in Materials and Methods.
From www.bloodjournal.org by guest on November 10, 2014. For personal use only.
236
WOODMAN ET AL
100
50
0
103
102
101
100
104
101
100
102
103
104
100
50
0
100
101
102
104
103
103
102
101
100
104
Fluorescence Intensity
Fig 4. Flow-cytometrichistogramsof neutrophil respiratoryburst
activity. Shaded region represents mean fluorescence (MW)of DHRloaded neutrophilsactivated with PMA as describedin Materials and
Methods, whereas unshaded region depicts resting neutrophils. IA)
MW of neutrophils from P-lls; (B) MW of neutrophils from B-l13; IC)
neutrophilsfrom an X9l"CGD patient with no measurable 0; production; and (D) MW of neutrophilsfrom a normal donor.
The mean fluorescence value of the oxidant-producing cells
from these mixing experiments equalled the results that were
obtained with neutrophils from P-&, B-& and B-113 (data
not shown). More importantly, the calculated percentage of
DHR-positive cells was within1.O% of the number of normal
neutrophils that were added to the cell suspension for flow
cytometry.
Even in the presence of excess catalase (1,100 UlmL), a
small increase in mean fluorescence intensity was also observed in the population of cells that corresponded to the
patient's NBT-negative cells. It was determined that this was
secondary to released extracellular oxidants produced by the
respiratory burst-competent cells, probably permeating the
membranes of the respiratory burst-incompetent cells and
causing oxidation of the DHR label present in these cells.
This change in mean fluorescence was not observed when
X91" CGD neutrophils were incubated alone with PMA.
NBT reductionaftermethylprednisolone.
One possible
explanation for the presence of NBT-negative cells could be
4
8.1
the presence of an unstable
oxidase complex that decayed
4
1.4 bone
during the final stage of neutrophil maturation in the
marrow storage po01.~Although such a process hasnot
been previously reported, it could theoretically result in a
progressive loss of respiratory burst activity. To exclude
7.3 this
possibility, each patient had serial NBT tests performed at
selected times up to 4 hours after the intravenous administration of 20 mg methylprednisolone, which was given to accel5.4
erate premature release of neutrophils from bone marrow
reser~es.~'
Despite a twofold or more increase in absolute
neutrophil counts, the percentage of NBT-positive neutrophils remained stable for each patient at -5% (Table 2).
This finding suggests that the proportion of cells capable of
oxidase activity remains unchanged during myeloid storage
pool maturation and that the subset of NBT-positive cells is
in fact a distinct clone of cells.
NBT reduction by progenitor cells. To examine the level
of clonal origin of the subsets, intact colonies grown from
peripheral blood CFU-GM were also evaluated for respiratory burst activity by NBT staining in their methylcellulose
semisolid culture medium. This technique has previously
been used to identify a CGD carrier
As illustrated in
Fig 5 and quantitated in Fig 6, colonies from patient P-II?
showed striking clonal expression of residual oxidase activity. A minority of the colonies were virtually all NBT-positive cells (Fig 5A), similar to colonies from normal controls
(E); whereas the remaining colonies resembled classic CGD
cells in their complete absence of NBT reduction (B). However, cultures from both patients in family B (B-& and B113) showed a heterogenous pattern, in which most colonies
contained both NBT-positive and NBT-negative cells in varying proportions (C and D), in addition to negative colonies,
but no completely positive ones. Microscopic examination
of cells plucked from parallel cultures showed similar cell
maturation, morphology, and granulocyte-macrophage ratios
in colonies derived from patient and normalperipheral blood
progenitors. The patterns of distribution of NBT reduction
inbothof these kindreds differed from the homogeneous
pattern previously observed in other patients withvariant
CGD in which cells are uniformly, faintly NBT-positive in
all
Effects of cytokines on respiratory burst activity. Superoxide production by neutrophils from P-I13was also measured after prolongedin vitro incubation ( l and 2 hours)
with each of the following cytokines: recombinant G-CSF
(1,000 U/mL) alone, GM-CSF (1,000 U/mL) alone, and
rhIFNy (250 U/mL) alone. Using SOD-inhibitable reduction of femcytochrome c, none of these cytokines had any
appreciable effect on neutrophil 0; production after activation
with
PMA
(200 ng/mL), FMLP
mol/L), or
Table 2. Effect of Corticosteroid Challenge
on % of NBT-Positive Neutrophils
Time After IV
Corticosteroid
Administered
(hr)
P-l13
8-11*
8-113
0
1
2
4
0
1
2
4
0
1
2
4
Neutrophils
(x10~/mm~)
NET' Neutrophils
(%H
3.0
2.9
5
2.4
4.0
5.7
4
5
3
5
3.8
3.4
6.4
3
6
6
6
5
Patients were administered 20 mg of methylprednisolone intraveNET slide tests
nously over 10 minutes. Complete blood count and
were performed serially at the time points indicated.
Abbreviation: IV, intravenous.
t Neutrophils were activated with PMA 200 ng/mL.
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VARIANT NEW X-LINKED
237
CGDOF CYTO-B-DEFICIENT
Fig 5. NET reduction by CFUgrown in semisolid medium
from peripheral blood progenitor cells from the indicated donors: patient P-l13 (A and B); patient B-IIZIC and D); and normal
(E). Colonies were cultured, NET
tested as described in Materials
and Methods, and photographed
through a 6.3~ objective on
a h i s s inverted microscope
(Zeiss, New York, NY). NET reduction in granulocyte-macrophage colonies grown in vitro
from peripheral blood progenitor cellsof a normal donor (E)
and P-I13patient (A,B) are shown.
Coloniesfrom normal progenitor
cells show uniform heavy deposits of reduced NET dye (El. Colonies from P-1I3 progenitors show
either similar, uniformly active
NBT reduction (A) or complete
absence of activity (B). Colonies
from B-1I2 showed a mixed pattern of NET-positive and NBTnegative cells (CD).
GM
D
E
Fh4LP (IO' mol/L) plus DHCB ( I O pmol/L) pretreatment
(data not shown). P-l13, B-IL, and B-I13 have also received
prolonged IFNy therapy, and although they have had a clinical response, the percentage of NBT-positive neutrophils,
0; production, and cytochrome hssslevels have not changed.
Although some selected patients with X-linked variant CGD
may have enhanced 0;production after IFNy administration? the results support other studies suggesting that this
is probably not the mechanism of action of IFNy in most
CGD patients.3'
Cytogenetic studies. Another potential explanation for
the subset of NBT-positive phagocytic cells in these patients
is the inheritance of an X chromosome mosaicism. For example, a variant Klinefelter's syndrome (46, XY/47, XXY)"
or an unexpected 46, XX karyotype4' could give rise to a
normal male phenotype and the inheritance of either two
maternal X chromosomes or one maternal and one paternal
X chromosome. Through random inactivation of X chromosomes, clonal expression of X-linked genes could occur in
the patient's neutrophils, leading to two populations of cells
with different respiratory burst capabilities.
Cytogenetic analysis of peripheral blood lymphocytes (P113, B-I12, and B-11.0 and cultured fibroblasts (P-I13) showed
normal male karyotypes (46, XU) in each case. Because
cytogenetic analysis was limited to nonmyeloid cells, we
also examined two informative X chromosome RFLPs in
family P. This procedure was performed to exclude pseudodiploidy of a mosaic state limited to myeloid cells that could
result in random inactivation of X chromosomes in the patient's (P-I13) neutrophils. Southern blots of genomic DNA
from peripheral blood (ie, primarily granulocytes) showed
only one allele for each polymorphism in the P-I13. The
distribution of alleles for the X-CGD-linked probe 754:*
for members of family P is shown in Fig 7. Longer exposure
films of this filter, as well as Southern blots hybridized to the
non-CGD-linked probe S-232,.'9also showed no evidence of
any additional chromosome-derived DNA other thanthat
from the single maternal chromosome. Thus, neutrophils
from patient P-I13 contained X chromosome DNA derived
only from the maternal X chromosome bearing the CGD
mutation, with no detectable DNA from either the paternal
or other maternal X chromosome.
DISCUSSION
In this study, we describe the respiratory burst characteristics of three patients from two kindreds with a unique phenotype of variant CGD. Phagocytic cells from all three patients
showed neutrophil 0; production, O2 consumption, and cytochrome hsss levels that were all 4 % to 15% of normal.
Studies with the cell-free activation system confirmed the
presence of a respiratory burst defect in the membrane fraction of the patients' neutrophils. Furthermore, the cell-free
membrane activity was comparable with the spectral levels
of cytochrome h and the intact neutrophil respiratory burst
activity. An X-linked inheritance was established in both
kindreds based on the identification of female relatives who
were camers for cytochrome b-deficient CGD.
The most novel aspect of these CGD patients is the presence of a small subset of circulating neutrophils and monocytes (5% to 10%)that are comparable with normal cells in
their respiratory burst activity as shown by NBT reduction
and flow cytometric analysis. The proportion of cells capable
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WOODMAN ET AL
238
O0
m loo
W
Z
deficiency of the gp91-phox subunit of cytochrome b.' Occasionally, patients with diminished but detectable, levels of
0;production and cytochrome b have been reported and are
referred to as variant X-linked CGD (X91-).'~'8~24~4'~4648
The
characteristics of the previously reported cases of variant
X91- CGD, as well as the three patients described in this
study, are summarized in Table 3. In almost all cases of Xlinked variant CGD described so far, NBT staining has
shown a homogeneous population of weakly positive neutrophils and monocytes. Thus, all of the phagocytic cells in
these patients collectively contributed to the residual respiratory burst activity. In a subset of patients with variant X91CGD, abnormal oxidase kinetics have been described: the
Michaelis-Menten constant (Km) for NADPH has been reported several-fold higher (10 to 100) than normal with the
AlVmax varying between 5% and 70% of normal.lR.2'.22
though the exact relationship between the oxidase kinetics
and levels of cytochrome b remains uncertain, studies from
these variant patients do suggest that minimal amounts of
cytochrome b may be adequate for some electron transport.'
In contrast with the typical X91- CGD variants described
above, the patients in this report each have a subset of NBTpositive phagocytic cells, the percentage of which is directly
proportional to the measured respiratory burst activity (5%
to 15%). Only one other patient has been reported with a
similar, albeit less completely characterized, X-linked variant of CGD that resembles the three patients described in
this study." This patient also had a small subset of NBTpositive cells (1 6%)that corresponded to the measured intact
cell respiratory burst activity (12% to 30%). Although a
population of phagocytic cells with near-normal respiratory
burst activity was suspected, more detailed studies to confirm
1 P-11-3
1 "-2
0
-l
0
0
U,
0
+
z
W
0
c
W
a
Negative
All Cells
Positive
Mixed All Cells
Fig 6. Distribution of NBT reduction in granulocyte-macrophage
colonies from the indicated donors. Colonies of greater
than 25 cells
were counted and scored as
positive (>90%0of cellswithin the colony
stained blue with reduced NBT; W), mixed (10%t o 90% of cells reducing NBT; B),or negative (<lo% of cells reducing NBT; 0).Each graph
represents pooled results from the following
number of experiments
for each donor: P-113, n = 4; B-II2, n = 4; B-1113,n = 2; normal, n = 6.
of NBT reduction (6% to 12%) closely corresponds to the
percentage of total normal 0;production achieved by intact
cells. Studies involving myeloid progenitor cells (peripheral
blood CFU-GM) also showed a discrete population of cells
with normal respiratory burst capabilities as determined by
NBT reduction. The inheritance of a mosaic diploid X chromosome state as an explanation for the NBT-positive cells
or the occurrence of an unstable oxidase complex as an
explanation for the NBT-negative cells (P-I13, B-I12, B-I13)
were both excluded. Together these results suggested the
presence of two distinct clones of phagocytic cells, one of
which is respiratory burst-competent.
Phagocytic cells from the majority of patients with Xlinked CGD areunable to generate 0;because of a complete
Family P
I
1 2
W 1
2
3
II
Fig 7. Southern blot of genomic DNA isolated from neutrophils
of patient P-I13 and other members of family P. Hybridization was
done with the RFLP probe 754, which is closely linked to the X chromosome CGD locus.'* Each lane contains DNA from the individual
indicated in the overlying pedigree. The lanes show (from left to
right) patient's hemizygousfather (P-l,); heterozygous CGD carriers,
mother (P-lp) and sister (P-IIJ; unaffected hemizygousbrother (P-IIJ;
and the proband (P-l13), hemizygous for the CGD-associated RFLP
allele. No trace of
the higher molecular-weightband is evident in the
proband. Blotting and hybridization were performed as described in
Materials and Methods.(31, unaffected male;(0).
carrier female; (W),
CGD
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EFICIENT
18
OF
NEW X-LINKED VARIANT
CGD
239
Table 3. Reported Cases of X-Linked Variant CGD
No. of
Patients
3,
3
1
1
1
3
3,
3
1
5
2
Total 20
Age of Onset
(yrs)
4. 14
12,
Visceral
Infections
3 Cases
None
None
None
None
1 Case
None
69
<l, <l, 2,8, 12 1 Case
2,3
1 Case
19
8
15
<l, 9,15
Intact Cell 0;
Production
(% normal)
Score
NET
(% positive1
5-10(strong)'
100 (weak)
100 (weak)
16
100 (weak)
80 (weak)
100 (weak)
>90 (weak)
>90 (weak)
55-155-1
3
2
12-30
9-30
8-50
13-35
6
0-24
14-17
5-10
Cytochrome b
Spectrum
(% normal)
ND
ND
88
ND
0-10
1
7-65
Oxidase Activation
in Cell-Free System
Membrane defect
ND
ND
ND
ND
ND
ND
Membrane defect
Membrane defect
Membrane defect
Oxidase
Kinetics
ND
Km defect
Km defect
Km defect
ND
ND
ND
Reference
This report
22
20
21
18, 43
23
46
24,47
48
Abbreviation: ND, not determined.
Refers to intensity ofNBT staining compared with control.
this (such as NBT reduction by CFU-GM colonies or flowcytometric analysis of circulating neutrophils) were not performed.
Many of the patients with variant X91- CGD have had a
delayed presentation and a milder clinical course with infections limited to the skin, lungs, and lymph nodes.'gr2'Presumably, this amelioration is caused by the residual respiratory
burst activity in their phagocytic cells. Although the patients
in this report have residual respiratory burst activity of 5%
to 10% of normal, they have had visceral infections similar
to that seen in patients with classical X91" CGD. It had
previously been suggested that patients with variant X-linked
CGD, characterized by a subset of phagocytic cells with
normal respiratory burst activity, would have fewer and less
severe infections.21
Rarely, X-linked carriers have been reported with only a
very small population of NBT-positive cells (510% of normal) because of extreme unbalanced X chromosome inactivation (Ly~nization)?~~~'
Although the proportion of NBTpositive cells is similar to that seen in variant X91- CGD,
these carriers are usually free of infections. This is consistent
with our experience of more than 70 CGD patients and kindreds; from these individuals, only two X-linked carriers
with extreme Lyonization (8% of cells NBT-positive) were
identified and both were asymptomatic (unpublished observations, J.T. Curnutte, April 1994). To our knowledge there
have only been two other X-linked carriers reported with
extreme Lionization (approximately 5% of their cells positive after NBT reduction) and both had mild infectious complications as a result of their reduced respiratory burst activity :7.49
The frequency and severity of infections amongst the
X91- variant patients, the extremely Lyonized X-linked carriers, and the three patients described in this report vary
considerably, even though these individuals collectively all
have comparable levels of respiratory burst activity (5% to
15% of normal). Reasons for the discrepancies between the
infectious complications and the respiratory burst capabilities in these patients are unclear. One potential explanation
is that a critical threshold exists in which infectious complications are more likely to occur when respiratory burst activity falls below 5% of normal. It is also possible that other
variables of host defense influence the clinical phenotype in
CGD patients and are not predicted by the percentage of
NBT-positive cells. In support of this, we have seen several
families where more than one sibling has inherited CGD,
each with identical amounts of respiratory burst activity and
NBT-positive cells, yet thay have an entirely different spectrum of infectious complications (unpublished observations).
This latter explanation, combined with our earlier studies
regarding the lack of effect of m y on the oxidase in the
majority of CGD patients, suggests that other aspects of
immune function warrant further investigation in CGD patient~.~'
The precise molecular defect responsible for this unique
variant of X91- CGD is currently under investigation. It is
likely that new information regarding the structure and function of the gp91-phox gene will be uncovered as a result of
these studies. Although variant forms of CGD are rare, they
continue to provide important clues to our understanding of
the oxidase complex of phagocytic cells.
ACKNOWLEDGMENT
The authors gratefully acknowledge the assistance of Dr 0. Jones
and the University of California San Diego Cytogenetics Laboratory
for performing the karyotypes; the San Diego Blood Bank for the
Kell antigen testing; Sally Teo for preparation of the manuscript;
and the General Clinical Research Center staff of Scripps Clinic and
Research Foundation for their assistance.
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