From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
An Inherited Defect of Neutrophil Motility and Microfilamentous Cytoskeleton
Associated With Abnormalities in 47-Kd and 89-Kd Proteins
By Thomas D. Coates, Joseph C. Torkildson, Martine Torres, Joseph A. Church, and Thomas H. Howard
A 2-month-old male Tongan infant presented with fever,
severe skin and mucosal infections, hepatosplenomegaly,
thrombocytopenia, and normal neutrophil counts. While
polymorphonuclear neutrophil (PMN) morphology was normal, several neutrophil motile functions were found t o be
altered in the patient. Furthermore, t w o siblings had died in
infancy with a similar clinical picture, raising the possibility of
an inherited neutrophil defect. Random migration and chemotaxis, assessed by the under agarose method, were profoundly impaired. Actin polymerization, as measured by flow
cytometry of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phallacidin (NBD-phallacidin)-stained PMNs, showed lower basal
F-actin and a 1.75-fold increase in response to
mol/L
formyl-methionyl-leucyl-phenylalanine(FMLP) compared with
a 4.51-fold increase in control. Microscopic examination of
NBD-phallacidin-stained PMN spread on glass showed decreased area of spreading and F-actin-rich filamentous projections distinct from control. The early phase of FMLPinduced right angle light scattering was absent, similar t o the
effect caused by cytochalasin-B (CB), an inhibitor of actin
polymerization. Accordingly, FMLP induced secretion of
elastase without the addition of CB. Staphylococcus aureus
killing was 50% of control whereas superoxide production in
response t o FMLP and surface expression of C D l l b were
greater than twice normal. Partial defects in actin polymerization and scatter were seen in the parents and release of
elastase, in the absence of CB, was also increased in both
parents. Sodium dodecyl sulfate-polyacrylamide electrophoresis of whole cell proteins from the patient showed a
marked decrease in an 89-Kd protein (8% of control) and a
marked increase in a 47-Kd protein (4.2-fold). Both mother
and father had decreased 89-Kd (77% and 42% of control)
and increased 47-Kd proteins (2- and 3.4-fold), although
neither had recurrent infections or chemotactic defects.
These studies describe a new inherited actin dysfunction
syndrome associated with severe propensity t o fungal infection and draw attention t o the proteins of apparent molecular weights of 89 Kd and 47 Kd, which may be of great
importance in the regulation of actin polymerization in
human PMNs.
0 1991by The American Society of Hematology.
C
reports of patients with defective microfilamentous cytoskeleta1 dynamics in PMNs.~,~
Cytochalasins are alkaloids that
can block the polymerization of monomeric actin (G-actin)
to polymeric F-actin, sever actin filaments, and inhibit PMN
locomotion, chemotaxis, phagocytosis,’”,” and dramatically
alter cell shape. In addition, disruption of the F-actin-rich,
subcortical cytoskeleton in cytochalasin-treated PMNs or in
genetically defective PMNs enhances secretion and superoxide release.3f6Evidence suggests that this enhancement
may be due to facilitation of granule-plasma membrane
fusion3 or to direct effects of the cytochalasins on the
interactions of receptors and NADPH oxidase components
with the microfilamentous
Elucidation of the biochemical mechanisms for regulation of the microfilamentous cytoskeletal organization and
actin polymerization and the consequences of such regulation for the function of nonmuscle cells such as neutrophils
is the focus of intense current research.” In recent years,
investigators have identified and characterized a variety of
proteins that are present in small quantities in phagocytes
and regulate actin polymerization in vitro. These include
profilin, gelsolin, actin-binding protein, and related protein~.’~.’~
Growing evidence suggests that these proteins also
play a role in regulating cytoskeletal organization in
PMNs.’~,’~
However, much is yet to be learned about the
role of these proteins in regulating actin polymerization in
PMNs. The description of patients with motility-deficient
PMNs and the biochemical elucidation of such defects are
critical to continued growth in our understanding of the
role of these proteins and of the cytoskeleton in normal and
abnormal PMN function.
This report describes a patient with severe recurrent
infections, defects in PMN motile behaviors, and defective
actin polymerization and microfilamentous cytoskeletal
organization in basal and activated PMNs. The abnormalities in PMN function and microfilamentous cytoskeletal
IRCULATING polymorphonuclear neutrophils
(PMNs) sense bacteria in tissue, adhere to endothelium, and then move toward, ingest, and kill the bacteria.
This mobilization of PMNs to sites of infection requires
intact surface receptors for bacteria-derived attractants,
surface adhesion molecules, and signal transduction events
that convert the surface stimulus into biochemical responses that produce the force for motility and the microbicidal agents for killing. Severe defects in neutrophil adherence, motility, or intracellular killing result in recurrent,
often life-threatening bacterial infections in humans.’--’
Stimulus-induced reorganization of the microfilamentous
cytoskeleton and its basic structural element, filamentous
actin (F-actin), is critical for most motile behaviors of
PMNs including shape change, locomotion, chemotaxis,
phagocytosis, secretion, and may influence other functions
such as superoxide (0;)production?6 Evidence that links
the integrity and dynamics of microfilamentous cytoskeleton to neutrophil motility derives primarily from studies of
the effect of cytochalasins on neutrophil motility’” and case
From the Department of Pediatrics, Universiiy of Southem California School of Medicine, Childrens Hospital Los Angeles; and the
Department of Pediatrics, University of Alabama at Birmingham.
Submitted October 1, 1990; accepted April 26, 1991.
Supported by Grants AI23547 to T.D.C. and AI25214 to T.H.H.
from the National Institutes of Health. T.H.H. is the recipient of an
Established Investigator award fromthe American Heart Association.
Address reprint requests to T.D. Coates, MD, Division of HematologyOncologv, Childrens Hospital Los Angeles, 4650 Sunset Blvd, Los
Angeles CA 90027.
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 1991 by The American Socieiy of Hematology,
0006-4971I9117805-0010$3.OOIO
1338
Blood, Vol78, No 5 (September l), 1991:pp 1338-1346
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
INHERITED PMN MOBILITY DEFECT RELATED TO F-ACTIN
organization are associated with decreased amount of an
89-Kd protein and increased amount of a 47-Kd protein
(apparent molecular weights, MW) in the PMNs. Clinical,
functional, and biochemical studies in the patient and the
parents show that this is a novel inherited disorder of
neutrophil motility and suggest that either or both the
89-Kd and the 47-Kd proteins play an important role in
regulating microfilamentous cytoskeletal organization and
motile functions of human PMNs.
1339
The cell area was determined by video image analysis. Surface
C D l l b was measured by flow cytometry (FACScan; BectonDickinson, Mountain View, CA) using MO-1 antibody (Coulter,
Hialeah, FL). Monocyte contamination was excluded by double
staining with Leu-M3 (Becton-Dickinson).
F-actin quantitution. PMNs were treated with DMSO or stimulus for the indicated times at 37°C or 25"C, fixed with formalin
(3.7%) for 15 minutes, and stained with a cocktail containing
lysophosphatidylcholine (100 pg/mL) and NBD-phallacidin
(3.3 x lo-' mol/L) as previously described." Fluorescence was
quantitated by flow cytometry. To compare the F-actin content
MATERIALS AND METHODS
measured on different days in family members, all values were
Cellpreparation. Heparinized venous blood was obtained from
normalized to the maximum F-actin content from the control cells
the patient, family, healthy adults, and age-matched donors in
used on the same day.
accordance with the Helsinki declaration and with approval of the
Sodium dodecyl sulfate-polyaciylamidegel electrophoresis (SDSHuman Experimentation Committee of Childrens Hospital Los
PAGE) and gel scanning. PMNs were treated on ice for 10
Angeles. PMN were prepared by dextran sedimentation and
minutes with 5 mmol/L diisopropyl fluorophosphate (DFP)29and
Ficoll-Hypaque centrifugation (Winthrop Pharmaceuticals, New
whole cell proteins were solubilized by boiling in 2% SDS, 20%
York, Ny; Sigma Chemicals, St Louis, MO).'* Erythrocytes were
glycerol, 10% i3-mercaptoethanol, 6 mmol/L DFP, 0.125 mol/L
removed by hypotonic lysis.
Tris-HC1 pH 6.8. Samples were kept frozen at -70°C until use.
Reagents. Stock solutions of formyl-methionyl-leucyl-phenylala- After thawing, the samples were sonicated and the protein mncennine (FMLP; Sigma Chemical, St Louis, MO) were prepared at
tration was determined using a modification of the method of
lo-' mol/L in 1% dimethyl sulfoxide (DMSO) and kept frozen.
Low$" after precipitation with 10 vol of 10% perchloric acid/l%
Medium 199 2X containing Earle's modified salts and supplephosphotungstic acid?' Bovine serum albumin was used as the
mented with 20% heat-inactivated fetal bovine serum, penicillin
standard. Equal loads of whole cell proteins were analyzed on 5%
(100 UlmL), and streptomycin (100 pg/mL) (GIBCO, Grand
to 15% polyacrylamide gradients as described by Laemmli.'* To
Island, NY) was used to prepare the agarose gel. Medium 199 lX,
quantify the relative amounts of protein on the one-dimensional
supplemented with 10% fetal bovine serum and 1% penicillinCoomassie blue-stained gels, the intensities of the 47-Kd, 89-Kd,
streptomycin, was used for cell suspension. Electrophoresis-grade
and actin bands were measured in optical density (OD) units by
agarose, 15 mg/mL (J.T. Baker Chemical Co, Phillipsburg, NJ) was
laser scanning densitometry (Ultrascan; LKB Biotechnology, Uppdissolved in sterile distilled water by heating. N-(7-nitrobenz-2-oxasala, Sweden) as previously described."
1,3-diazol-4-yl)phallacidin(NBD-phallacidin) and MeO-Suc-AlaData analysk. Comparisons of group means were tested by
Ala-Pro-Val-methylmumarin amide were obtained from Molecuanalysis of variance or the Student's t-test. The results presented
lar Probes Inc (Junction City, OR). Reagents for electrophoresis
are representative of at least three experiments. Except where
were from BioRad (Richmond, CA). All other reagents were from
otherwise noted, the specific data points represent the mean and
Sigma or as indicated.
standard deviation of a triplicate determination on a single donor.
Neutrophilfunctions. Chemotaxis was assayed using a modification of the under agarose technique described by Nelson et a1.'9-2'
The leading front (LF) and random migration (RM) were meaRESULTS
sured from the cell migration pattern using a video imaging system
Clinical data. A 2-month-old male Tongan infant was
as described?','' Adherence to nylon-wool fibers was measured as
referred to Childrens Hospital Los Angeles with recurrent
described by McGregor et al?' The respiratory burst was assessed
fevers of several weeks duration. Physical examination
by reduction of nitroblue tetrazolium (NBT) of PMN adherent to
showed an ulcer of the hard palate, hepatosplenomegaly,
endotoxin-coated glass slides prepared with lipopolysaccharide B
bruising, and petechiae. During the ensuing 3-month hospi(Escherichia coli 0.26:B6; Difco, Detroit, MI)?3 The release of
superoxide anion (02J
was measured in an end-point assay using
talization, he developed recurrent pulmonary infiltrates,
the superoxide dismutase-inhibitable ferricytochrome c reduction
progression of the hard palate lesion, and a subsequent
technique. The results were expressed as nanomoles/5 min/lO7cells
lingual ulcer. Culture of the lingual ulcer grew Candida
using an extinction coefficient of 21.1 mmol/L-' cm-1.24Phagocytotropicalis. Repeated blood cultures were negative. Episodes
sis of 2-pm fluorescent latex beads was measured in suspended
of pulmonary infiltrates resolved on broad-spectrum antibicells in buffer containing 10% serum. After a 15-minute incubation
otics. The oral ulcers resolved slowly on amphotericin-B.
with end-over-end agitation, the number of beads per 100 cells was
The pregnancy and delivery were uncomplicated with no
counted.2s Killing of Staphylococcus aureus 502A (American Type
history of maternal drug ingestion. There was no history of
Culture Collection [ATCC], Rockville, MD) was measured as
described.% Right angle light scattering was measured in an SLM
delayed separation of the umbilical cord. The child has
8000 fluorescence spectrophotometer (SLM Instruments, Urbana,
seven siblings, two of whom died at 3 and 4 months of age as
IL) equipped with a thermostated stirred cuvette as described."
the result of complications of recurrent oral, perirectal, and
Continuous elastase release was monitored using MeO-Suc-Alapulmonary infections. The deceased infants also had bruisPro-Val-methylcoumarin amide." The results were expressed as
ing similar to that of the patient. The surviving siblings have
rate of change of fluorescence at 1minute after stimulation divided
no significant medical problems nor is there a history of
by the basal rate of change of fluorescence. The rate of spreading
severe recurrent infection on either side of the family.
was determined by allowing PMN at 1 x 106 per mL in Krebs
There are no family records that permit determination of
Ringers phosphate glucose (KRPG) to settle at 37°C on glass
the exact blood relation between the mother and father.
coverslips. The slides were fixed and stained with NBD-phallacidin
at 5 and 15 minutes and viewed by phase/fluorescence microscopy.
However, the parents were not brother and sister and it is
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
COATES ET AL
1340
likely that the maternal and paternal great-grandparents
were closer than fourth cousins.
The patient’s hemogram showed a white blood cell
(WBC) count of 16,000/mm3with a normal differential and
a platelet count of 64,000/mm3.On Wright’s stain peripheral blood smear, the nuclear morphology of the PMNs was
normal and the cytoplasm contained neutral granules but
no giant granules. The hemoglobin was 9 gldL and there
was mild elevation of the liver enzymes. Serum Igs, Ig
subclasses, response to immunization with tetanus, T-cell
numbers, and phytohemagglutinin (PHA) stimulation were
normal. Bone marrow aspiration showed normal granulocyte morphology and maturation as well as normal megakaryocyte number and morphology. Of note, the patient
had a normal response to transfused platelets, suggesting
that the thrombocytopenia was not due to increased platelet destruction. Examination of transmission electron micrographs of neutrophils fixed in suspension was normal and
demonstrated normal specific granule morphology. Bone
marrow chromosomes as well as peripheral lymphocyte
chromosomes were normal. Subsequent evaluation of neutrophil function (detailed below) showed a profound defect
in chemotaxis, normal or increased superoxide production,
and increased expression of CDllb.
During the hospitalization, the palatal ulcer worsened
and cultures of a lingual ulcer grew Candida and Aspergillus
nigrans. The child was treated with antibiotics and amphotericin-B with slow resolution of the infectious problems. At
7 months of age, the child underwent allogeneic bone
marrow transplantation from an HLA-identical sibling with
resultant correction of the thrombocytopenia and neutrophil chemotactic defect.
Neutrophil motility and relatedfunctions. Direct observation with phase microscopy of the patient’s PMNs in a
gradient of FMLP (Fig 1B) showed occasional attempts at
pseudopod formation, and frequent development of thin,
filamentous projections at the cell surface while control
cells formed broad pseudopods. However, the patient’s
PMNs did not locomote or orient in the FMLP gradient,
when compared with control PMNs. In quantitative motility
assays, random migration and chemotaxis toward FMLP
were markedly impaired when compared with normal
2.0
1.5
1
1.0
0.5
0.0
A
C
PT F M
Fig 2. Motility of control, patient, and parent PMNs. Random
migration (B) and chemotaxis as measured by leading front (0)
in
response to 10.’ mol/L FMLP in 30 adult controls (A), two agematched infant controls (C), patient (PT; n = 3). father (F; n = 3). and
mother (M; n = 3).
adults or to age-matched controls (Fig 2). Results were
similar with leukotriene B, as the chemoattractant (data not
shown). Control PMNs exposed to the patient’s serum
exhibited normal chemotaxis while exposure of patient’s
PMN to normal serum did not improve the defective
chemotaxis or random migration, indicating a defect intrinsic to the patient’s PMNs. Direct observation of vital
materna1 and paternal PMNs showed no qualitative morphologic abnormality and quantitative studies of chemotaxis
and random motility were normal.
The abnormalities in PMN motility were associated with
an altered FMLP-induced right angle light scatter. An early
decrease in right angle light scatter is observed after FMLP
stimulation of normal PMNs and correlates with shape
change and with actin polymerizati~n.~’
When compared
with controls, the expected early decrease, measured at
37”C, was significantly diminished in the patient’s PMNs
(Fig 3). The diminished right angle light scatter response
could relate to either an inability of the cells to change
shape or to a defect in FMLP-induced actin polymerization.
The early response in right angle light scatter after stimula-
Fig 1. Morphology of PMNs
during movement in a gradient
of FMLP. Normal cells viewed by
phase microscopy at 400x magnification (A) orient and move in
the gradient whereas the patient‘s cells (E) remain round and
produce hairlike pseudopods.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
INHERITED PMN MOBILITY DEFECT RELATED TO F-ACTIN
105
bl
e,
100
4
4
d
u
:
95
.r(
4
e,
m
d
90
p1
K
85
80
0
10
20
Seconds
Fig 3. Shape change in control and family PMNs. Early changes in
right angle scatter after stimulation with lo-’ mol/L FMLP in control
(C), father (F), mother (MI, and patient (PT) PMNs. The stimulus was
added at 0 seconds. Each line is the mean of three replicates on a
representativeday.
tion of PMNs from both parents was diminished and was
intermediate between that of the patient and control cells
(Fig 3). This observation suggests that the defective motile
behavior of the patient’s PMNs is inherited.
Other motile-related functions such as spreading, phagocytosis, and killing were also defective in the patient’s PMN.
The degree of PMN spreading on glass after 15 minutes at
37”C, as measured by cell area, was significantly less for
patient’s cells (1,590 f 762 U) than for control cells
(3,238 f 628 U). Despite abnormal spreading, adherence
to nylon wool fibers was normal (patient; 79% 2 9.2% v
control; 97% 2 1.4%, P = .OS). The patient’s PMNs phagocytosed significantly fewer 2-pm latex beads than did
control cells (40 beads/200 patient PMNs v 120 beadd200
control PMNs) and, after a 2-hour incubation, the patient’s
PMNs killed fewer S aureus than control cells (patient
43% f .35% v control 71% f 11.3% of the inoculum). The
respiratory burst, as assessed by reduction of NBT, was
normal in the patient’s PMNs, suggesting that the impaired
killing correlated with the defective phagocytosis. These
data indicate that the patient’s PMNs have an intrinsic
inherited defect in functions that require normal microfilamentous cytoskeleton.
Microfilamentous cytoskeleton and actin polymerization.
Microscopic observation of patient and control PMNs that
had spread on glass coverslips for 15 minutes at 37°C
showed striking morphologic changes (Fig 4, B and D).
Phase contrast microscopy and reflectance interference
microscopy demonstrated that few cells spread significantly
and that 20% of the patient’s PMNs expressed fine, hairlike
projections from their surface when the cells attempted to
spread on a surface (Fig 4D). These thin filamentous
1341
projections were barely visible by phase microscopy (Fig
1B); however, by reflectance interference microscopy, the
projections appeared as thin dark lines, suggesting close
adhesion of the projections to the glass substratum (Fig
4D).94These filamentous thin projections were not seen on
electron micrographs of cells fixed in suspension (data not
shown). Fluorescence microscopy of cells stained with
NBD-phallacidin, a specific probe for F-actin:’ showed that
the thin filamentous projections were F-actin-rich as evidenced by the intense fluorescence (Fig 4B). The patient’s
PMNs stained with NBD-phallacidin also lack the punctate
sites of localized F-actin concentration that were observed
in normal PMNs adherent to glass in the absence of protein
(Fig 4A).16s36
The basal F-actin content and FMLP-induced actin
polymerization response of PMNs from the patient were
determined by flow cytometric analysis of NBD-phallacidinstained PMNs.4,’ Maximum FMLP-induced actin polymerization occurs after 15 seconds of incubation and is defined
as the maximum F-actin content. The results with the
patient’s PMN were expressed as percent of the maximum
F-actin content obtained with control cells. As shown in Fig
5A, both basal F-actin content and increase in F-actin
content after FMLP stimulation were markedly decreased
in the patient’s PMNs as compared with control PMNs
(P < .001; Fig 5A). The F-actin content and polymerization of two age-matched controls with decreased chemotaxis did not differ from adult controls. The maximum extent
of FMLP-induced actin polymerization in PMNs of both
Fig 4. Spreading of controland patient PMNs on glass. PMNs were
fixed after spreading on glass for 15 minutes at 37°C. Control PMNs
are seen on the left and patient’s PMNs are seen on the right. Patient‘s
PMNs (e) fail to demonstrate punctate regions of F-actin as seen in
normal control (A) and have thin F-actin-rich filamentous projections
that are not present in normal PMNs. These filaments are closely
adherent to the glass and appear as black lines by reflectanceinterference microscopy (D).
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
COATES ET AL
1
A
o
IO-’
[FMLP]
0 10 20 30 40 50
seconds
Fig 5. F-actin content in PMNs after stimulation with FMLP. (A)
F-actin content in control (A), mother (A),and patient ( 0 )after
stimulation with lo-’ mol/L FMLP at 37°C. ( 8 )More detailed kinetics
of F-actin change after stimulation with lo-’ mol/L FMLP at 25°C in
control (A), father (W), and mother (A).All points are statistically
different from controls (P < .01) except for the father at 0 and 5
seconds. All points are normalizedto the maximal F-actin content of
the simultaneous control done on the same day under identical
conditions.
parents, measured at 25”C,was decreased to levels between
patient and control values (Fig 5B). The abnormality was
more marked in maternal than in paternal PMNs and was
present both at 37°C (Fig 5A) and 25°C (Fig 5B). Furthermore, maternal PMNs had an abnormally low basal F-actin
content while the paternal PMNs had a low, but not
statistically significant, basal F-actin content. These data
show that microfilamentous cytoskeletal structure and dynamics are abnormal in the patient’s cells and suggest that
the cytoskeletal alterations are causally related to the
defective motility and right angle light scatter. In addition,
the intermediate defects in the mother’s and father’s
FMLP-induced actin polymerization correlate with the
decreased right angle light scatter responses and suggest
that the motility and cytoskeletal defects are inherited.
Cytochalasin-like abnormalities. An abnormality in microfilamentous cytoskeletal organization was further suggested by the fact that secretory and 0,- responses of
patient’s PMNs mimic the response of cytochalasin-treated
neutrophils. While cytochalasins inhibit all motile neutrophil functions including locomotion, chemotaxis, phagocytosis, and shape change, they enhance and facilitate agoniststimulated granule and 0,- secretion. Neutrophils exhibit
little FMLP-activated 0,- production and secrete little or
no secondary granule constituents such as vitamin B,,
binding protein, unless pretreated with cytochalasin^.^*^'
Similarly, in the absence of cytochalasins, FMLP causes no
secretion of primary granule constituents such as e l a ~ t a s e . ~ . ~ ’
As shown in Table 1, FMLP stimulation of the patient’s
PMNs induced the release of large amounts of vitamin B,,
binding protein and the production of significant amount of
0,- without prior exposure to cytochalasins. Furthermore,
surface expression of CD1lb, a membrane-associated secondary granule component, was upregulated in the patient’s basal PMNs, ie, in the absence of stimulus or
cytochalasin B (CB). These findings suggest that the secretion of secondary granules is facilitated in the patient’s cells
without addition of CB. Surprisingly, stimulation of the
patient’s PMNs with FMLP induced significant release of
the primary granule constituent elastase, in the absence of
added CB (Table 1). The data on secretion and 0,production suggest that the cytoskeletal defect in the
patient’s PMNs mimics the effects observed in cytochalasintreated cells. Interestingly, PMNs from both parents also
exhibited FMLP-induced elastase release without exposure
to cytochalasins (Table 1).
Electrophoretic studies of PMN proteins. Total cellular
protein from neutrophils of the patient and the parents was
analyzed by one-dimensional SDS-PAGE (1D-SDS-PAGE).
Results are shown in Fig 6 and Table 2. SDS-PAGE of the
patient’s PMNs showed a marked increase in a 47-Kd
protein while the amount of an 89-Kd protein was markedly
decreased. The decrease in 89-Kd protein was most obvious
in gels heavily loaded with total cellular protein (Fig 6A).
Table 1. Secretion-Dependent Functions
~~~
CD11b* (fold control)
Superoxide (nm/5 min/107PMN)
FMLP l o 7 mol/L
PMA 50 ng/mL
Vitamin B, binding proteint (% release)
FMLP 10 mol/L
FMLP CB
PMA 50 ng/mL
Elastase release* (fold baseline)
FMLP l o 7 mol/L
+
~
Control
Patient
Mother
Father
1.o
2.13
1.77
1.56
ND
ND
ND
ND
132 2 7.5
260 -c 4.8
15.8 1 %
23.0 k 1.6%
46.7 k 3.2%
? 0.5
(NSI
0.99
416 2 6.7
426 2 3.6
36 -t 2.3%
38 2 1 . 2 ~ ~
52 k 2.0%
5.4 f 1.0
(f < ,011
5.2 k 1.7
(f < .01)
1.7 2 .56
< .02)
(f
’Unstimulated mean channel number compared with control.
tDifferences between patient and control are significant a t f < .01. The effect of CB was not significant in the patient.
*The fold increase of the rate of change in fluorescence 1 minute after stimulation compared with baseline. The f values indicate difference from
baseline.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1343
INHERITED PMN MOBILITY DEFECT RELATED TO F-ACTIN
Fig 6. SDS-PAGE of PMN proteins from patient (A), parents
(B), and controls. Shown are
Coomassie blue-stained 5 % to
16% polyacrylamide gradient
gels of proteins of (A) patient
PMNs (lane 2 = 80 pg, lane
4
30 pg), control PMNs (lane
1 z 80 pg, lane 3 z 30 pg), and
MW markers (lane 5); (B) PMN
proteins of control PMNs (lane
1 = 20 pg, lane 4 I 40 pg) father’s PMNs (lane 2 = 20 pg, lane
5 z 40 pg), and mother‘s PMNs
(lane 3 = 20 pg, lane 6 z 40 pg)
and MW markers (lane 7). Note
the increase in the 47-Kd bands
and the decrease in 89-Kd bands
(arrowheads).
A
195
os
D
’I
D
4
!7
B
1
2
3
4
5
SDS-PAGE of the parent’s PMNs also showed decreased
amounts of the 89-Kd protein and increased amounts of the
47-Kd protein. As shown in Table 2, quantitative scans of
replicate gels demonstrated that the magnitude of the
decrease in 89-Kd protein was less marked in the father and
the mother than in the patient. Specifically, the amount of
89-Kd protein in the mother, father, and the patient was
77%, 42%, and 8% of control, respectively. The increase in
47-Kd protein was statistically significant in PMNs from the
mother, father, and patient (2.0-fold, 3.4-fold, and 4.2-fold,
respectively). Similar qualitative changes in 89-Kd and
47-Kd proteins were seen in the PMNs from a sister (74%
of control 89-Kd protein and twofold increase in 47-Kd
protein). PMNs from a brother were normal (92% of
control 89-Kd protein and 100% of control 47-Kd protein).
The observed quantitative differences in the amount of
47-Kd and 89-Kd proteins could not be accounted for by
unequal load of whole cell proteins on the gels because
similar results were obtained when the ratio of each protein
to actin (OD 47 Kd or 89 Kd/OD 43 Kd) was used for
calculation. Other proteins were equal to control when
expressed as a ratio to actin. For example, the ratio of the
protein at 71 Kd to actin was 0.087 ? 0.042 (n = 11) in
control and 0.080 ? 0.032 (n = 10) in the patient. The
results of relative differences in amounts of 47-Kd and
89-Kd proteins in PMNs from the patient and the parents
Table 2. RelativeQuantities of 47-Kd and 89-Kd Proteins in
Neutrophils
Control
Patient
Mother
Father
OD 47 Kd’
47 Kd: t
Actin Ratio
OD 89 Kd’
89 Kd: t
Actin Ratio
0.035 & 0.015
0.146 f 0.030
0.073 f 0.020
0.120 f 0.023
0.21 & 0.11
1.00 f 0.15
0.75 f 0.12
0.80 f 0.10
0.184 f 0.015
0.015 f 0.006
0.143 f 0.038
0.078 f 0.009
0.318 f 0.013
0.023 f 0.007
0.230 f 0.050
0.130 f 0.030
The amount of actin in equally loaded gels from parents and patient
was the same as control.
‘OD determined by gel scan (n = 3) on gels of equal total protein
load (micrograms).
tRatio of OD 47 Kd or 89 Kd to OD actin (ie, independent of protein
load; n = 4).
1
2
3
4
5
6
1
suggest that alterations in the amounts of 47-Kd and 89-Kd
proteins may be inherited as an autosomal recessive trait.
DISCUSSION
This communication reports the clinical and biochemical
studies of a Tongan infant who presented with severe,
recurrent infections. Functional studies of the patient’s
neutrophils showed abnormalities in locomotion, phagocytosis, shape change, and secretion that were associated with
a defect in microfilamentous cytoskeletal organization and
actin polymerization. Clinically, the patient had normal
total and differential leukocyte counts, marked thrombocytopenia with normal survival of transfused platelets, and a
propensity to infection. Morphologically, by light microscopy, the patient’s PMNs failed to form pseudopods, but
developed long, filamentous, and F-actin-rich projections
during attempts at spreading. Because several motilityrelated PMN functions were significantly depressed, the
basal F-actin content and FMLP-induced actin polymerization responses of the patient’s cells were analyzed by
NBD-phallacidin fluorescence. Both basal and stimulated
F-actin content were markedly decreased, suggesting that
the defects in motility are related to abnormalities in
microfilamentous cytoskeletal structure and dynamics.
Quantitative scanning of 1D-SDS-polyacrylamide gels
showed normal actin content in the patient’s PMNs; however, proteins of apparent MW 47 and 89 Kd were profoundly abnormal. The association of a defect in motility
and actin polymerization with abnormalities in proteins of
47 and 89 Kd apparent h4W raise the possibility that these
proteins play a role in regulating the organization of the
microfilamentous cytoskeleton in PMNs.
Secretion and 0,- release, functions that are not usually
thought to require an intact microfilamentous cytoskeleton,
were also found to be abnormal in the patient’s PMNs. On
stimulation with FMLP, normal PMNs do not release
primary granule constituents and exhibit limited 0,- production or specific granule release. However, after pretreatment of normal PMNs with cytochalasins, which block actin
polymerization, FMLP induces the release of primary
granule components like elastase and causes increased
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1344
release of specific granule constituents such as vitamin B,,
binding
increased surface membrane expression
of C D l l b that is stored in the specific granules? and
enhancement of the respiratory burst, components of which
are located in specific granule^.^' Possible explanation for
this enhancement includes upregulation of surface receptors through membrane addition6and cytochalasin-induced
dissolution of the subcortical microfilaments? PMNs from
the patient were functionally similar to cytochalasin-treated
PMNs in that they exhibited increased surface CDllb,
enhanced 0,- production to FMLP, increased vitamin B,,
binding protein release that was not further enhanced by
cytochalasins, and release of elastase in the absence of
cytochalasins. Furthermore, the patient’s PMNs share morphologic features with cytochalasin-treated PMNs. Specifically, the fine hairlike projections from the patient’s adherent PMNs recall the projections produced following
cytochalasin-induced PMN “arbori~ation”.8.~~
These abnormalities in secretion and 0,production, though not as
directly related to cytoskeletal function as locomotion and
phagocytosis, support the idea that the patient’s PMNs
have an intrinsic defect that causes microfilamentous cytoskeletal dysfunctions similar to those reported in cytochalasin-treated PMNs.
Partial expression of the functional and biochemical
phenotype was observed in the patient’s PMNs. While PMN
chemotaxis, which was profoundly depressed in the patient,
was normal in both parents, FMLP-induced actin polymerization response and right angle light scatter were significantly decreased in both parents. Furthermore, PMNs from
both parents released significant amount of elastase in the
absence of cytochalasin. In general, the magnitude of the
abnormalities was greater in the mother’s than in the
father’s PMNs. Interestingly, the functional phenotype of
parents’ cells does not directly correlate with the extent of
the increase in 47-Kd or decrease in 89-Kd proteins in
parental cells. Both parents differ from control in the
relative amounts of 47-Kd and 89-Kd proteins. The father
has significantly less 89-Kd protein and more 47-Kd than
the mother, and yet the abnormalities in PMN functions are
less severe in the father’s PMNs. However, the functional
and biochemical data on PMNs, the history of affected
siblings, and the parents’ consanguinity clearly indicate that
the PMN defect is inherited and is likely autosomal
recessive.
Severe congenital deficiencies of PMN locomotion and
chemotaxis are usually associated with a marked propensity
to development of infections and include several rare
disorders such as Chediak-Higashi
specific
granule deficiency,‘ and leukocyte adhesion deficiency
(LAD).MThese were excluded in this patient based on the
normal morphology of Wright-stained PMNs, the presence
of specific granules on electron micrographs, and the
increased expression of CDllb. Another motility disorder,
named neutrophil actin dysfunction (NAD), was described
earlier by Boxer et a13 and subsequently was partially
characterized through biochemical studies on the family.’
Some features are shared by both patients, namely profound motility abnormalities, abnormal actin polymeriza-
COATES ET AL
tion, and increase in spontaneous secretion, and the parents of both patients have partial impairment of actin
assembly. However, several distinct clinical, functional, and
biochemical differences in the patients and their families
also exist. Clinically, the NAD patient had a marked
neutrophilic leukocytosis ( > 100,000/mm3),normal platelet
counts, and predominantly bacterial infections. Morphologically, at the light and electron microscopic level, PMNs
from the NAD patient formed pseudopods that were
“fork-like.” Biochemically, crude extracts of the NAD
patient’s PMNs failed to polymerize actin normally, but
SDS-PAGE of whole cells was normal. Analysis by 1D-SDSPAGE of Triton-insoluble cytoskeletons showed no abnormality in the father’s PMNs but showed increased amounts
of a 54-Kd protein in PMNs from the mother and one of the
siblings. The mother and the older sister were found to be
partially deficient for both C D l l b and CD18, the two
subunits of the glycoprotein receptor CR3, suggesting that
they were heterozygotes for LAD in association with
NAD.” However, the father had nearly normal expression
of both s u b u n i t ~ . ~In
’*~
contrast to the NAD patient, the
functional and biochemical analysis of the patient’s and
parent’s PMNs described here suggest that this patient is
homozygous for an autosomal defect that is associated with
not only an increased amount of a 47-Kd protein but also a
decrease in an 89-Kd protein. These distinctions indicate
that this patient may represent a unique disorder of PMN
motility and function which, like NAD, manifests abnormalities in the microfilamentous cytoskeleton and ligandinduced actin polymerization responses. Because the NAD
patient and this patient share an increase in proteins of
apparent MW of 47 to 54 Kd, this common finding strongly
suggests that increased amounts of the 47-Kd protein may
be linked to diminished ability of the PMN to polymerize
actin or stabilize actin in the F-actin state after polymerization.
The precise biochemical basis for this novel PMN defect
remains obscure. The fact that the patient’s cells are
deficient in chemotactic response to leukotriene B, (LTB,)
and FMLP, yet exhibit normal to increased secretion and
respiratory burst in response to FMLP argue against a
defect in the FMLP receptor or signal transduction mechanisms common to the actin polymerization and the respiratory burst responses such as G protein, PIP,, or CaZ+flux.
The presence of the abnormal amounts of two proteins is
suggestive of a role for one or both of these proteins in the
defective motility. The function of these two proteins in
regulating actin polymerization and the functional link
between them is still unknown and further studies will be
needed to delineate their relation. However, the reciprocal
relationship between decrease in 89-Kd and increase in
47-Kd proteins cannot be explained by selective proteolysis
of the 89-Kd protein because (1) the excess of 47-Kd
protein is too large relative to the total amount of 89-Kd
proteins in normal cells to account for the increase in 47-Kd
protein; (2) the cells were treated with DFP before and
during cell solubilization to inhibit proteolysis; (3) the
abnormalities were consistently observed in numerous (five)
different sample preparations; and (4) polyclonal antibod-
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1345
INHERITED PMN MOBILITY DEFECT RELATED TO F-ACTIN
ies raised to the crude 47-Kd 1D-SDS-PAGEband reacted
only with 47-Kd proteins and not with any protein of higher
molecular weight including 89-Kd on immunoblots (data
not shown). Therefore, the 47-Kd protein and 89-Kd
protein are antigenically distinct. While the 47-Kd protein
has not yet been purified, preliminary sequence data on the
first five amino acids as well as its PI indicate that it is not
analogous t o p47 of platelets (Glu-Pro-Lys-Arg-Ile)" or
p47,, of neutrophils (Gly-Asp-Thr-Phe-Ile),#
In summary, we have described an infant with recurrent
infection and a severe defect in neutrophil motile functions.
This defect appears to be inherited as a recessive trait and is
associated with impaired ability to polymerize actin, an
increase in a 47-Kd protein, and a profound decrease in an
89-Kd protein. These studies describe a new, clinically important disorder associated with recurrent infections and draw
attention to the proteins of apparent MW 47 Kd and 89 Kd that
may be of critical importance in the regulation of the microfilamentous cytoskeleton in hematopoietic cells in humans.
ACKNOWLEDGMENT
We thank Dr R.L. Baehner and Dr R. Parkman for critical
reading of the manuscript; Dr K.Weinberg and Dr C. Lenarsky for
providing the specimens; Dr C. Pryswanski for critical evaluation of
the patients electron photomicrographs; Linda Beyer, Jane Deaton,
and Tony Guerrero for excellent technical assistance.
REFERENCES
1. Boxer LA, Coates TD, Haak RA,Wolach JB, Hoffstein S,
Higgins C, Baehner R: Lactoferrin deficiency associated with
altered granulocyte function. N Engl J Med 307:404,1982
2. Anderson DC, Springer TA: Leukocyte adhesion deficiency:
An inherited defect in the Mac-1, LFA-1, and p150,95 glycoproteins. Annu Rev Med 38:175,1987
3. Boxer LA, Hedley-Whyte ET, Stossel TP: Neutrophil actin
dysfunction and abnormal neutrophil behavior. N Engl J Med
291:1093,1974
4. Howard TH,Meyer WH: Chemotacticpeptide modulation of
actin assembly and locomotion in neutrophils. J Cell Biol98:1265,
1984
5. Howard TH, Oresajo CO: The kinetics of chemotacticpeptideinduced change in F-actin content, F-actin distribution, and the
shape of neutrophils. J Cell Biol101:1078, 1985
6. Jesaitis AJ, Tolley JO, Allen RA: Receptor-cytoskeleton
interactions and membrane traffic may regulate chemoattractantinduced superoxide production in human granulocytes. J Biol
Chem 261:13662,1986
7. Zigmond SH, Hirsch J: Effects of cytochalasin B on polymorphonuclear leukocyte locomotion phagocytosis and glucolysis. Exp
Cell Res 73:383,1972
8. Howard TH, Casella J, Lin S: Correlation of the biologic
effects and binding of cytochalasins to human polymorphonuclear
leukocytes. Blood 57399,1981
9. Southwick FS, Dabiri GA, Stossel TP: Neutrophil actin
dysfunction is a genetic disorder associated with partial impairment of neutrophil actin assembly in three family members. J Clin
Invest 82:1525,1988
10. Yahara I, Harada F, Sekita S, Yoshihira K, Natori S:
Correlation between effects of 24 different cytochalasins on cellular structures and cellular events and those on actin in vitro. J Cell
Biol92:69,1982
11. MacLean-Fletcher S, Pollard TD: Mechanism of action of
cytochalasin B on actin. Cell 20:329:341,1980
12. Jesaitis AJ, Bokoch GM, Tolley JO, Allen RA: Lateral
segregation of neutrophil chemotactic receptors into actin- and
fodrin-rich plasma membrane microdomains depleted in guanyl
nucleotide regulatory proteins. J Cell Biol 107921,1988
13. Zigmond S H Introduction: Sensory adaptation and motor
activation in chemotaxis. Semin Cell Biol1:73,1990
14. Pollard TD, Cooper J A Actin and actin-bindingproteins: A
critical evaluation of mechanisms and functions. Annu Rev Biochem 55:987,1986
15. Stossel TP, Chaponnier C, Ezzell R, Hartwig J, Janmey P,
Kwiatowski S, Lind S, Smith D, Southwick F, Yin H, Zaner K
Nonmuscle actin-binding proteins. Annu Rev Cell Biol1:353, 1985
16. Southwick FS, Young CL: The actin released from profilinactin complexesis insufficientto account for the increase in F-actin
in chemoattractant-stimulated polymorphonuclear leukocytes. J
Cell BiolllOl965,1990
17. Howard TH, Chaponnier C, Yin H, Stossel TP: Gelsolinactin interaction and actin polymerization in human neutrophils. J
Cell Biol110:1983,1990
18. Boyum A Isolation of mononuclear cells and granulocytes
from human blood. Scand J Clin Lab Invest 21:77,1968
19. Nelson RD, Quie PG, Simmons R L Chemotaxis under
agarose: A new and simple method for measuring chemotaxis and
spontaneous migration of human polymorphonuclear leukocytes
and monocytes.J Immunol115:1650,1975
20. Coates TD, Harman J, McGuire WA: Microcomputer based
program for video analysis for chemotaxis under agarose. Comput
Methods Programs Biomed 21:195,1985
21. Quitt M, Torres M, McGuirre W, Beyer L, Coates TD:
Neutrophilchemotacticheterogeneity to n-formyl-methionyl-leucylphenylalanine detected by the under-agarose assay. J Lab Clin
Med 115:159,1990
22. McGregor R, Spagnuolo P, Lentnek A Inhibition of granulocyte adherence by ethanol prednisone, and aspirin, measured
with an assay system. N Engl J Med 642,1974
23. Ochs HD, Igo RP: The NBT slide test: A simple screening
method for detecting chronic granulomatous disease and female
carriers. J Pediatr 83:77,1973
24. Babior BM, Kipnes RS, Curnutte JT: Biological defense
mechanism. The production by leukocytes of superoxide, a potential bacteriocidal agent. J Clin Invest 52:741,1973
25. Dunn PA, Tyrer Hw:Quantitation of neutrophil phagocytosis, using fluorescent latex beads. J Lab Clin Med 98:374,1981
26. Alexander JW, Windhorst DB, Good RA: Improved tests
for evaluation of neutrophil function in human disease. J Lab Clin
Med 72136,1968
27. Sklar LA, Omann GM, Painter RG: Relationship of actin
polymerization and depolymerization to light scattering in human
neutrophils: Dependence on receptor occupancy and intracellular
Ca++. JCellBiol101:1161,1985
28. Sklar LA, McNeil VM, Jesaitis AJ, Painter RG, Cochrane
CG: A continuous, spectroscopicanalysis of the kinetics of elastase
secretion by neutrophils: The dependence upon receptor occupancy. J Biol Chem 2575471,1982
29. Amrein PC, Stossel TP: Prevention of degradation of human
polymorphonuclear leukocyte proteins by diisopropylfluorophosphate. Blood 56:442,1980
30. Lowly OH, Rosebrough NJ, Farr AL, Randall RJ: Protein
measurement with the Folin phenol reagent. J Biol Chem 193:265,
1951
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1346
31. Brown K, Binder L Gyronemin: A novel IFAP. Cell Motil
Cytoskeleton 17:19,1990
32. Laemmli V K Cleavage of structured proteins during the
assembly of the head of bacteriophage T4. Nature 227:680,1970
33. Chaponnier C, Yin HL, Stossel T P Reversibilityof gelsolin/
actin interaction in macrophages. Evidence of calcium-dependent
and calcium-independentpathways. J Exp Med 165~97,1987
34. Bereiter-Hahn J, Fox CH, Thorell B: Quantitative reflection
contrast microscopy of living cells. J Cell Biol82767,1979
35. Howard TH, Oresajo CO: A method for quantifyingF-actin
in chemotactic peptide activated neutrophils: Study of the effect of
tBOC peptide. Cell, Motil Cytoskeleton 5545,1985
36. Southwick FS, Dabiri GA, Paschetto M, Zigmond SH:
Polymorphonuclearleukocyte adherence induces actin polymerization by a transduction pathway which differs from that used by
chemoattractants. J Cell BiollO9:1561,1989
37. Petrequin PR, Todd RF, Devall LG, Boxer LA, Curnutte
JT: Association between gelatinase release and increased plasma
membrane expression of the MO-1 glycoprotein. Blood 69505,
1987
38. Sklar LA, Oades ZG, Finney DA. Neutrophil degranulation
detected by right angle light scattering: Spectroscopic methods
suitable for simultaneous analysis of degranulation or shape
change, elastase release, and cell aggregation.J Immunol133:1483,
1984
39. Williams AJ, Cole PJ: Polymorphonuclear leucocyte membrane-stimulated oxidative metabolic activity; the effect of divalent
cations and cytochalasins.Immunology 442347,1981
COATES ET AL
40. Berger M, O’Shea J, Cross AS, Folks TM, Chused TM,
Brown EJ, Frank MM: Human neutrophils increase expression of
C3bi as well as C3b receptors upon activation. J Clin Invest
74:1566,1984
41. Borregaard N, Heiple JM, Simons ER, Clark R A Subcellular localization of the b-cytochrome component of the human
neutrophil microbicidaloxidase: Translocation during activation. J
Cell Biol97:52,1983
42. Atlas SJ, Lin S: Dihydrocytochalasin B: Biological effects
and binding to 3T3 cells. J Cell Biol76360,1978
43. Boxer LA, Smolen JE: Neutrophil granule constituents in
health and disease. Hematol Oncol Clin North Am 2:101,1988
44. Springer TA, Thompson WS, Miller LJ,Schmalstieg FC,
Anderson DC: Inherited deficiency of the Mac-1, LFA-1, p150,95
glycoprotein family and its molecular basis. J Exp Med 160:1901,
1984
45. Southwick FS, Howard TH, Holbrook T, Anderson DC,
Stossel TP, Arnaout M A The relationship between CR3 deficiency
and neutrophil actin assembly. Blood 73:1973,1989
46. Southwick FS, Holbrook T, Howard T, Springer T, Stossel T,
Arnaout M: Neutrophil actin dysfunction is associated with a
deficiency of Mol. Clin Res 34533A, 1986
47. Tyers M, Rachabinski R, Stewart M, Varricio A, Shorr R,
Haslan R, Harley C Molecular cloningand expression of the major
protein kinase C substrate of platelets. Nature 333:470,1988
48. Volpp B, Nauseef W, Donelson J, Moser D, Clark R
Cloning of the cDNA and functional expression of the 47 kilodalton cytosolic component of the human neutrophil respiratory burst
oxidase. Proc Natl Acad Sci USA 86:7195,1989
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1991 78: 1338-1346
An inherited defect of neutrophil motility and microfilamentous
cytoskeleton associated with abnormalities in 47-Kd and 89-Kd
proteins
TD Coates, JC Torkildson, M Torres, JA Church and TH Howard
Updated information and services can be found at:
http://www.bloodjournal.org/content/78/5/1338.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.