1. No category

From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
1993 81: 2735-2743
A novel syndrome of severe neutrophil dysfunction:
unresponsiveness confined to chemotaxin-induced functions
D Roos, TW Kuijpers, F Mascart-Lemone, L Koenderman, M de Boer, R van Zwieten and AJ
Verhoeven
Updated information and services can be found at:
http://www.bloodjournal.org/content/81/10/2735.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.
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
A Novel Syndrome of Severe Neutrophil Dysfunction: Unresponsiveness
Confined to Chemotaxin-Induced Functions
By Dirk Roos, Taco W. Kuijpers, Francoise Mascart-Lemone, Leo Koenderman, Martin de Boer,
Rob van Zwieten, and Arthur J. Verhoeven
We have identified a patient with a number of neutrophil
dysfunctions. The patient was a female baby who lived for
8 months. During her life, she developed severe bacterial
infections and showed omphalitis, impaired wound healing,
and a pronouncedleukocytosis. She was not a patient with
leukocyte adhesion deficiency, because all leukocyte CD18
complex proteins were expressed at normal levels. Yet,
neutrophil polarizationand chemotaxisto platelet-activating
factor, leukotriene 84, or formyl-methionyl-leucyl-phenylalanine (FMLP) were completely absent. We found a strong
defect in actin polymerization in response t o chemotactic
stimuli, but only a retarded or even normal reaction with
other stimuli. This indicates that the cellular dysfunctions
were not due t o an intrinsic defect in actin metabolism.
Instead, the regulation of actin polymerization with chemotactic stimuli seemed to be defective. We concentrated
on FMLP-induced responses in the patient's neutrophils.
Functions dependent on activation of complement receptor
type 3, such as aggregation or adherence to endothelial
cells, were normally induced. Binding t o serum-coated
coverslips was normal in cell number; however, spreading
was not observed. Exocytosis from the specific granules
was readily induced. In contrast, FMLP failed to induce a
respiratory burst activity or degranulation of the azurophil
granules. FMLP induced a normal increase in free intracellular Ca2+, but a decreased formation of diglycerides (especially the 1-0-alkyl.2-acyl compounds). Thus, w e have
described a patient whose neutrophils show a severe defect
in functional activation via chemotaxin receptors, resulting
in a selective absence of NADPH oxidase activity, exocytosis from the azurophil granules, and actin polymerization.
Our findings show that actin polymerization for neutrophil
spreading and locomotion is regulated differently from that
for phagocytosis. Also, the release of azurophil and specific
granule contents is clearly shown to be regulated in a different way.
0 1993 by The American Society of Hematology.
I
granules, and increase in intracellular Ca2+)were not significantly affected. Together, these data suggest a severe neutrophil dysfunction at the level of intracellular signalling.
"AMMATION
involves a complex series of events,
including vasodilatation, increased vascular permeability,
and exudation of fluids and plasma proteins. These processes
are followed or coincide with an influx of inflammatory cells.
Several inflammatory mediators are generated at the site of
the lesion, eg, the bacterial tripeptide formyl-methionyl-leucyl-phenylalanine (FMLP), the complement fragment C5a,
the lipid mediators leukotnene B4 (LTB4) and platelet-activating factor (PAF), and the cytokines interleukin-8 (IL-8)
and monocyte chemotactic protein-1 (MCP- 1). These mediators have chemotactic activity, ie, they bind to specific
receptors on the surface of neutrophils and monocytes and
thus induce these cells to move to the inflammatory site.
After a coordinated repertoire of adherence to vascular endothelium, diapedesis, and subsequent migration, the neutrophilic granulocytes are the initial cells found at areas of
tissue damage.'-6
The signal transduction induced by binding of chemotactic
stimuli to neutrophil receptors is only partly understood. After
agonist binding, these receptors couple with guanosine triphosphate (GTP)-binding proteins (G-protein~),~,'
which may
then activate phospholipases and protein
In some
of the intracellular pathways, diglycerides and arachidonic
acid metabolites are generated and calcium ions are liberated,
which aids in the activation of these e n z y m e ~ . ~ - Sort'
~ ~ '1ng
~~''
out which intracellular pathway leads to the activation of
each neutrophil function induced by chemoattractants is now
in full progress. Cells deficient in one or more of these pathways would provide a powerful tool for such studies.
We have investigated the neutrophil responses of a patient
who shortly after birth developed several bacterial infections,
impaired wound healing, and a marked leukocytosis. Some
neutrophil functions were almost absent (actin polymerization, chemotaxis, respiratory burst, and release from the
azurophil granules), whei eas other responses (phagocytosis,
intracellular killing, release of proteins from the specific
Blood, Vol
81,No 10 (May 15).1993:pp 2735-2743
CLINICAL HISTORY
The patient (female) was the first child from nonconsanguineous
healthy parents, both originating from India. From the second day
oflife, she developed periods clinically suspect for sepsis. She presented
an umbilical hernia with chronic omphalitis (cultures positive for
Staphylococcus aureus). She suffered from otitis media (cultures positive for S aureus) and from buccal candidiasis. Poor response to
antimicrobial agents was noted, except for the oral candidiasis, which
remitted. In vitro investigations shortly after birth were indicative
for a diagnosis of severe combined immunodeficiency disease (SCID),
but the lymphocyte population progressively normalized. At 4 months
of age, the diagnosis of SCID was ruled out on a normal lymphocyte
count, on the basis of normal positive cutaneous hypersensitivity
responses to candidin and pokeweed mitogen (PWM), and on positive
in vitro proliferation to phytohemagglutinin (PHA) and OKT3.
The patient's neutrophil counts were very high, fluctuating between
15 and 60 X 109/L. In contrast, neutrophils were not observed on
direct examination of material collected from the umbilical lesion.
From the Central Laboratory of the Netherlands Red Cross Blood
Transfusion Service and Laboratory for Experimental and Clinical
Immunology, University ofiinsterdam, Amsterdam, The Netherlands;
and the Laboratory of Immunology, Brugmann Hospital, Free University of Brussels, Brussels, Belgium.
Submitted February 19, 1992; accepted December 28. 1992.
Address reprint requests to Dirk Roos, PhD, % Publication Secretariat, Central Laboratory of the Netherlands Red Cross Blood
Transfusion Service, PO Box 9190, 1006 A D Amsterdam, The Netherlands.
The publication costs ofthis 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 1993 by The American Society of Hematology.
0006-4971/93/81 IO-0024$3.00/0
2735
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
ROOS ET AL
2736
This led to analysis of neutrophil chemotaxis. In vitro analysis of
neutrophil chemotaxis under agarose performed in autologous and
allogeneic serum (activated with zymosan) showed on two separate
occasions a total absence of migration of the patient’s neutrophils.
In contrast, phagocytosis of serum-opsonized yeast and S aureus was
normal (microscopicassay), as was the intracellularkilling o f S aureus.
As the patient’s neutrophil count remained high with an absence
of in vitro neutrophil migration, blood was sent to Amsterdam for
further investigations (May-July 1989). Approval was obtained from
the Institutional Review Board for these studies. Patients and volunteers were informed that blood samples were obtained for research
purposes, and that their privacy would be protected.
Several B-cell functions (clustering, proliferation, and Ig production) as well as T-cell functions (proliferation, homotypic aggregation,
cytotoxicity,and T-cell receptor (TCR)/CD3 modulation) were indeed
normal (C.J. Jochems, C.J.M. van Noesel, K.C. Kuijpers, R.A.W.
van Lier, T.W. Kuijpers, unpublished observations). The apparent
phagocyte dysfunction is described in the present study.
At 5 months of age, she developed an internal fistulation from the
cecum extending along the rightside musculus iliopsoas. The ileum
was surgically exteriorized, and daily transfusions of leukocytes were
administered. No cicatriciation was observed and the surgical wound
became necrotic. Because of unremitting clinical deterioration with
periods of septicemia (gram-negative bacteria), it was impossible to
try a bone marrow transplantation. The child died at 8 months of
age.
MATERIALS AND METHODS
Reagents. FMLP, PAF, cytochalasin B (Cyto B), phorbol myristate acetate (PMA), and fluorescein isothiocyanate (FIX)-labeled
phalloidin were purchased from Sigma Chemical Co (St Louis, MO).
Indo-1/AM and NBD-phallacidin were obtained from Molecular
Probes (Junction City, OR). Except for the phallacidin and phalloidin,
the agents were dissolved in dimethylsulfoxide(DMSO) at 1,000 times
the final concentration, and were stored at -20°C. Serum-treated
zymosan (STZ) was prepared according to Goldstein et al.I2RANTES
was a kind gift of Dr T.J. Schall (Genentech Inc, South San Francisco,
CA). The basal incubation medium for cell suspensions contained
132 mmol/L NaC1, 1 mmol/L MgS04, 1 mmol/L CaCI2, 6 mmol/
L KCI, 1.2 mmol/L KHzPOl, 20 mmol/L HEPES, 5.5 mmol/L glucose, and 0.5% (wt/vol) human serum albumin (HSA), pH 7.4.
Antibodies. The following monoclonal antibodies (MoAbs) were
used: CDI laCLB-LFA1/2,I3 CDI Ib CLB-B2.l2,l4 CDI I C (LeuM5;
Becton Dickinson, Mountain View, CA), CD18 CLB-LFA1/l,l5
CD32 IV.3,I6W6/32 (HLA class I), and 7D5 (directed to the small
22-Kd subunit of cytochrome bSssl’).
Leukocyte isolation. Blood was obtained from the patient and
healthy volunteers by venapuncture, and was anticoagulated with
0.4% (wt/vol) trisodium citrate (pH 7.4). Granulocytes were purified
as described before’* and were resuspended in incubation medium
at a final concentration of IO6 cells/mL. Purity of the granulocytes
was more than 9896, with greater than 95% neutrophils. Lymphocytes
and monocytes were purified from the mononuclear cell fraction by
countercurrent elutriation’’ (cell punty was more than 90%).
Determination ofsurface antigen expression. In the experiments
for surface antigen expression,19 lymphocytes or neutrophils were
incubated with primary antibody for 30 minutes at 4°C. Thereafter,
the cells were washed twice in excess of ice-cold phosphate-buffered
saline (PBS), and the procedure was repeated with FITC-labeled goatantimouse-Ig for another 30 minutes at 4°C. The cells were analyzed
with a FACScan (Becton Dickinson, San Jose, CA). Data were collected from 10,000 cells.
Measurement of oxygen consumption. Oxygen consumption was
measured at 37°C with an oxygen electrode, as described before.”
The results are expressed as maximal rates of O2 consumption obtained after activation.
Determination of degranulation. Degranulation was measured
as described b e f ~ r e . ’The
~ ~ ’activity
~
of vitamin B12-binding protein,
P-glucuronidase, and lactate dehydrogenase were measured in cellfree supernatants and expressed as percentage of the values found in
Triton X-100 (0.5%,vol/vol) lysed cells.
Phagocytosis assays. Phagocytosis was measured with four different methods: a turbidimetric assay?’ microscopic inspection,” an
enzymatic assay (see below), and at the ultrastructural level by electron
microscopy. In the turbidimetric assay, the increase in light transmittance at 400 nm was measured. In the absence of extracellular
Ca2’, this assay measures exclusively light transmittance changes due
to phagocytosis, because cell aggregation is prevented under these
conditions.20
Bacterial killing. The assay to determine the capacity of neutrophils to kill Escherichia coli uses the E coli mutant ML35. This organism lacks the lactose permease, but constitutively forms the cytoplasmic enzyme ,&galactosidase. The kinetics of the P-galactosidase
assay give a direct indication for intracellular uptake (k, ), perforation
of the bacterial envelope (k2),and the inactivation of P-galactosidase
(k3),respectively. Perforation of the bacterial envelope correlates with
the loss of colony-forming capacity of the bacteria.”
Turbidimetric assay jtlr neutrophil aggregation. Neutrophil aggregation was determined by measuring the light absorbance at 400
nm.” Experiments were performed under continuous stirring at 37°C
in siliconized cuvettes. After 5 minutes of preincubation, the stimulus
(dissolved in DMSO) was added to the cell suspension and the change
in light transmittance was recorded. DMSO content never reached
values above 0.1% (vol/vol).
Adherence to endothelial cells. Adherence of neutrophils to
monolayers of endothelial cells was determined as described by Hakkert et aLz3
Neutrophil chemoiaxis. Chemotaxis was measured with the
leading front method of Zigmond and H i r s ~ h . *Neutrophils
~
(2 X
IO5 in 0.2 mL of incubation medium) were incubated for 70 minutes
at 37°C on top of cellulose ester filters with a thickness of 1 I O to 150
pm and a pore size of 3 pm (Millipore S.A., Molsheim, France; type
SS). Afterwards, the filters were fixed in absolute ethanol and stained
with Hams hematoxylin. Cell penetration was measured as the distance between the top of the filter and the plane in which two to
more nuclei of the fastest neutrophils were in focus. This procedure
was performed at five different places in the filter with five filters per
test. The mean of these readings was taken as the chemotactic response. The variance of this test is 9%.
Neutrophil polarization. Polarization was measured according to
Ciancolo and Snyderman.” Neutrophils (2 X 106/mL)in Earle’s medium in siliconized tubes were incubated for 5 minutes at 37°C with
lo-’ mol/L FMLP, IO-’ mol/L PAF, or IO-’ mol/L LTB4, fixed
with 2% (vol/vol) paraformaldehyde, and centrifuged. The preparations were stained with May-Grunwald-Giemsa and scored microscopically for round or polarized appearance (200 cells counted per
coverslip).
Cell spreading. Neutrophils (5 X 1O5/mL)were placed on top of
coverslips coated with fibronectin or heat-treated serum. Adhesion
was induced by addition of the same amount (50 pL) of incubation
medium containing FMLP ( 1 pmol/L). After 15 minutes, the coverslipswere washed three times with PBS and the adherent neutrophils
were fixed with PFA (1% [wt/vol] in PBS). Spreading was visualized
by staining of F-actin with phalloidin-FITC after permeabilization
of the adherent cells with lyso-PC (0.2 mg/mL).
Actin polymerization. Actin polimerization in intact neutrophils
or lymphocytes was measured by FACS analysis of NBD-phallacidinstained cells as described by Howard and Meye?6 Results were scored
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
2737
A NOVEL SYNDROME OF NEUTROPHIL DYSFUNCTION
Table 1. Expression of Surface Proteins on Neutrophils
Control
CD1 l a
CDllb
CDl l c
CD18
W6/32 (HLA)
7D5 (cyto B)
CD32
Patient
Control
10
78
303
32
251
235
72
136
10
41
185
120
138
159
68
166
Values are the mean fluorescence intensity
e
0-251
0.20
0
0
a
0.16
W
Y
!f
B
2
0.1 0
0.05
4
as percentage of cells with a fluorescence level above the unstimulated
control (10,000 cells measured).
Measurement of cyfosolicfreeCd’. Cytosolic free Ca2+([CaZ+],)
was measured as described before.” In short, prewarmed neutrophils
(5 minutes at 3 7 T , 25 X IO6 cells/mL in incubation medium) were
incubated with 0.5 pmol/L indo-I/AM for 40 minutes at 37°C. After
two washes, the cells were resuspended to 2 X lo6 cells/mL in incubation medium and kept at room temperature. Fluorescence measurements were performed at 37°C under continuous stimng in a
spectrofluorometer (model RF-540 Shimadzu Corporation, Kyoto,
Japan). Excitation and emission wavelengths were 340 nm and 390
nm, respectively. Calibration of indo- 1 fluorescencewas determined
by saturation of trapped indo-1 with Ca2+after permeabilization of
the cells with digitonin (5 pmol/L), followed by quenching with MnZ+
(0.5 mmol/L).’* A kd of 250 nmol/L was used for the indo-1/Ca2+
complex for the calculation of [Ca2+],.29
Measurement ofdiglycerides. The determination of I -O-alkyl,2acyl glycerides (EAG) and di-acyl glycerides (DAG) was exactly as
described by Tyagi et
In this assay, the total diglycerides are
extracted and converted to 32P-phosphatidicacid (PA) by the method
of Preiss et a13’ (30°C for 90 minutes). Subsequently, lipase from
Rhizopus arrhizus was added to one part of the samples, which selectively degrades the 1-acyl-containing species (DAG) leaving the
ether lipids (EAG) intact. The lipids were separated by thin-layer
chromatography (TLC) and visualized by autoradiography. The radioactive spots corresponding to a mixture of 1-alkyl and I-acyl PA
(representing total diglycerides)or only I-alkyl PA (reflecting EAG)
were scraped from the plates and counted.
RESULTS
As the clinical history shows, the patient presented with
persistent neutrophilia (>15 X 109/L) and omphalitis from
the first day of life. She showed impaired wound healing and
developed severe bacterial infections in the first 2 months.
Because the patient was suspected to suffer from leukocyte
adhesion deficiency (LAD), leukocyte adhesion molecules
were measured. However, deficiencies in these proteins were
not found. In particular, CD1 la, CD18, and intercellular
adhesion molecule-1 (ICAM-1) were normally present on the
lymphocytes (not shown), and CD 1 1a, CD 1 1b, CD I 1c, and
CD18 were present on the neutrophils in normal amounts
(Table 1). Thus, the patient did not suffer from the LAD
syndrome.
We then measured several neutrophil functions that depend on a normal function of adhesion proteins. Homotypic
aggregation induced by PMA or FMLP was normal, except
that the usual desaggregation, with its onset about 3 to 5
minutes after FMLP addition, was not observed with the
0
5
0
10
16
TIME (mid
Fig 1. Aggregation of neutrophils. Neutrophil aggregation was
turbidimetrically determined. Neutrophils (5 X 1Oe/mL in incubation
medium) were preincubated for 5 minutes at 37°C before the addition of FMLP (final concentration, 1 pmol/L). Patient neutrophils
(0)responded with a long-lasting aggregation, in contrast to the
normally observed transient response (B). Addition of cyto B at t =
5 minutes after FMLP caused a reaggregation of control cells to
about twice the maximal value found with FMLP alone, but had no
effect on patient cells.
patient’s neutrophils (Fig 1). Cyto B, which reverses the desaggregation after FMLP in normal cells, had no effect on the
patient’s cells.
Adherence of neutrophils to endothelial cell monolayers,
induced by IL-lP pretreatment of the endothelial cells for 4
hours or by FMLP treatment of the neutrophils, was normal
(not shown). In contrast, migration ofthe patient’s neutrophils
either spontaneously or in a gradient of activated serum or
casein was totally absent when measured at four different
occasions (Table 2). Also, when FMLP, PAF, or LTB4 was
tested as the chemotactic agent, no migration was measured
(not quantified). Neutrophil spreading on glass, fibronectincoated or inactivated serum-coated coverslips was totally ab-
Table 2. Chemotaxis and Polarization
Addition
Patient
Control
Normal Range
Chemotaxis
None
Casein (1 mg/mL)
56°C serum (5%, vol/vol)
Fresh serum (5%. vol/vol)
0
0
0
0
30
84
68
121
19-43
>54
26-74
62-1 18
Polarization
None
FMLP (10 nmol/L)
PAF (10 nmol/L)
LTB4 (1 0 nmol/L)
0
0
0
0
5
82
32
30
1-6
72-86
-
Values for chemotaxis in micrometers travelled by the leading front
cells in 70 minutes. Values for polarization in percentage of cells examined
(n = 200).
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
ROOS ET AL
2738
100
-aR
100
A
A
80
80
60
60
i?
J
w
0
c
Z
w
0
B
40
5
3LL
20
0
0
2
4
6
8
0
10
2
-aR
6
8
10
8
10
TIME (mid
TIME (mid
100
4
100
C
80
80
60
60
B
0
B
K
40
40
3
LL
20
20
D
2J
w
0
c
0
0
0
0
2
4
6
8
10
TIME (mid
0
2
4
6
TIME (mid
Fig 2. Actin polymerizationof neutrophils. Neutrophils (107/mL in incubation medium) were activated for the time indicated and instantaneously fixed, permeabilized, and incubated with NBD-phallacidin,essentially as described.** (e)Control cells; (0)patient cells. Stimulation
with (A) FMLP (1 pmol/L): (B) PMA (100 ng/mL): (C)A23187 (1 pmol/L); (D) PMA (I00 ng/mL) -t A23187 (1 pmol/L).
sent, with or without FMLP. Control neutrophils all reacted
with a shape change after the addition of FMLP, and about
50% of these cells spread on the coverslips within 30 seconds.
Moreover, staining of the control cells with phallatoxin-FITC
showed a large number of condensed foci of F-actin in the
periphery of these cells and also some in the pseudopods.
This phenomenon was virtually absent in the patient’s neutrophils.
Therefore, we quantified neutrophil polarization and polymerization of G-actin into F-actin induced by several stim-
uli. Polarization induced by either FMLP, LTB4, or PAF
was totally absent in the patient’s cells, which were tested on
two different occasions (Table 2). The patient’s serum had
no effect on the polarization of normal neutrophils. Actin
polymerization with FMLP was also strongly deficient in the
patient’s neutrophils (tested twice), but F-actin formation in
response to PMA, A23 187, or the combination of PMA and
A23 187 was only retarded or even normal (Fig 2). Thus, the
patient did not suffer from a generalized actin polymerization
defect. Sodium dodecyl sulfate-polyacrylamide gel electro-
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
A NOVEL SYNDROME OF NEUTROPHIL DYSFUNCTION
2739
100
100
80
B
80
L.
*
Y
60
60
W
2
1
40
40
20
20
0
0
K
w
0
5
10
15
20
25
30
0
5
TIME (mid
10
15
TIME
20
25
30
(mid
Fig 3. Degranulation of neutrophils. Neutrophils (5 X 106/mL in incubation medium) were preincubated for 5 minutes at 37°C before
the addition of the priming agent cyto B (5 pg/mL) or a similar amount of vehicle DMSO (0.1 %, vol/vol). After 5 minutes, the cells were
activated with FMLP (1 pmol/L). Samples were taken at the indicated times, and vitamin B,,-binding protein (A) or j3-glucuronidase (B) was
determined in the cell-free supernatants. Open symbols, patient’s neutrophils; closed symbols, control neutrophils. (0,0 ) no additions; (A,
A) FMLP; (0,w) cyto B
FMLP.
+
phoresis (SDS-PAGE) of Triton X- 100 insoluble material
showed no differences between control and patient neutrophils.
Phagocytosis of serum-opsonized E coli, S uureus, Succheromyces cerevisiae, or zymosan was normal both in kinetics and extent as measured in a variety of ways (microscopically, enzymatically, turbidimetrically, or by electron
microscopy [not shown]). Intracellular killing of E coli, measured by perforation of the bacteria (see Materials and Methods), was also normal. Degradation of bacterial P-galactosidase by the patient’s neutrophils was slightly depressed
(patient k3 = 0.033; control k3 = 0.054; normal range, 0.040
to 0.160).
We concentrated our further investigations on some
FMLP-induced functions and signals. Exocytosis of vitamin
Blz-binding protein from the specific granules was induced
by FMLP in an enhanced fashion (Fig 3A). In the presence
of cyto B, FMLP induced similar amounts of vitamin BIZbinding protein from the patient’s cells and from normal
cells. This was corroborated by a normal upregulation of
CD1 l b and CD18 on the patient’s neutrophils after addition
of PAF ( 1 pmol/L) or FMLP ( 1 pmol/L) (not shown). In
contrast, FMLP failed to induce release of ,&glucuronidase
from the azurophil granules of the patient’s neutrophils (Fig
3B).
The patient’s neutrophils also showed a strongly decreased
respiratory burst after the addition of FMLP (Table 3). Priming of the cells by either cyto B, PAF, or PMA did not augment
this response. The reaction of the patient’s cells to PMA was
normal. The STZ-induced oxygen consumption was of short
duration, but could be restored by PMA.
Considering a defective or altered formation of secondary
messengers in the patient’s cells, we measured free intracellular calcium and formation of diglycerides. FMLP induced
a normal, fast increase in [Ca2’]i, followed by a depressed
second wave of intracellular CaZi due to influx of extracellular
Ca2+(Fig 4). We found subnormal formation of diglycerides
(DG) after FMLP addition, especially the 1 -O-alkyl,2-acyl
compounds (Table 4).
In contrast to neutrophils, T-cell clones from the patient
were able to respond to a chemotaxin called RANTES. This
chemotactic substance has recently been characterized to act
on the subset of CD4’ T lymphocytes only.32In accordance
with the published data on lymphocyte chemotaxis, a rapid
Table 3. Oxygen Consumption
Patient
Control
Normal Range
STZ (1 mg/mL)
PMA (1 00 ng/mL)
5.5
6.2
9.4
4.5
6.1-11.7
3.8-8.2
PMA (1 00 ng/mL)
FMLP (1 pmol/L)
Cyto B/FMLP
PAF (1 pmol/L)/FMLP
3.7
0.3
0.3
0.4
5.1
3.1
4.4
6.3
3.8-8.2
1.6-6.4
6.1-8.5
5.4-1 0.3
PMA (1 00 ng/mL)
FMLP (1 pmol/L)
Cyto B/FMLP
PAF (1 pmol/L)/FMLP
PMA (2 ng/mL)/FMLP
3.8
0.3
0.4
0.5
1.8+ 1.8
NT
3.3
5.8
7.1
3.8 + 8.0
3.8-8.2
1.6-6.4
6.1-8.5
5.4-10.3
Addition
Values in nanomoles O2 consumed per minute by
Abbreviation: NT, not tested.
-
lo6neutrophils.
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
2740
-5
-
ROOS ET AL
1000
500
-.-
+
N
m
u
u
100
0
2
4
6
0
2
4
6
1000
-
500
.-
+
N
m
2
100
TIME (min 1
Fig 4. Changes in intracellular free Ca +.Neutrophilswere loaded
with indo-1/AM and brought to 2 X 106/mL in incubation medium.
The cells were stimulated with 1 pmol/L FMLP (A) or 1 pmol/L PAF
(B). Trace a, control cells; trace b, patient cells. Resting values of
[Ca2+Ii did not differ between control and patient cells (mean, 88
nmol/L and 1 0 0 nmol/L, respectively).
actin polymerization response was observed upon stimulation
with RANTES in the patient’s CD4+ T-cell clones, in contrast
to the CD8’ T-cell clones (Fig 5A and B). The same was true
with control CD4+ and CD8’ T-cell clones. No actin polymerization (nor any change in [Ca2+li)was observed in these
T cells either with IL-8, C5a, PAF, or FMLP. In addition,
both the patient’s as well as four control Epstein-Barr virus
(EBV)-transformed B-cell lines did not react to any of these
chemoattractants. Although the EBV-transformed B cells reacted with NADPH oxidase activity upon addition of PMA,
no responses could be elicited either in the control EBVtransformed B-cell lines or in patient’s cells upon cross-linking
of surface IgM or HLA-DR, in contrast to what has been
reported.33
DISCUSSION
The patient’s neutrophils displayed an almost complete
deficiency in several chemotaxin-mediated responses. Most
notable was the lack of actin polymerization and, as a consequence, the defects in polarization, spreading, and locomotion. However, the patient did not suffer from a true form
of neutrophil actin dysfunction
First, agents that
bypass receptors for chemotaxins induced a slow or even
normal response ofthis parameter. Second, the patient’s cells
were perfectly able to phagocytize opsonized bacteria and
even larger particles (STZ), in contrast to NAD neutrop h i l ~ , ’ ~or, ~normal
~
neutrophils that have been pretreated
with Cyto B to prevent actin polymeri~ation.~’
Therefore,
actin metabolism after engagement of Fcy or complement
receptors was normal. Finally, we indeed found a similar
enhanced release of specific granule contents from FMLPtreated neutrophils, but, in contrast to one NAD patient
studied in this re~pect,’~
a total absence of degranulation of
the azurophil granules and a lack of respiratory burst (measured as oxygen consumption). Treatment of the patient’s
neutrophils with either Cyto B or PAF did not correct these
defects.
The defects were confined to some, but not all, chemotaxininduced functions. These functions were normally induced
by other stimuli, thus excluding intrinsic functional defects
in the patient’s cells. The receptors for FMLP, PAF, and C5a
have been cloned; due to their seven transmembrane segments, they show strong homology to the G-protein-coupled
receptors of the rhodopsin-like superfamily of cell surface
receptor^.'^-^' However, the defect cannot be localized in the
chemotaxin receptors, because several functional responses
were normally induced via these receptors. Moreover, the
defect seems to be phagocyte restricted, because all the Tand B-cell functions that were measured either in vivo or in
vitro were normal, as exemplified in particular by the intact
ability of actin polymerization in the patient’s CD4’ T-cell
clones upon activation via the chemotaxin receptor for
RANTES (Fig 5). Although the receptor for RANTES has
not yet been cloned, the homology between RANTES and
IL-8 renders it likely that both receptors belong to the same
rhodopsin-like supergene family.
The signal transduction mechanism via the chemotaxin
receptors involves pertussis toxin (PT)-sensitive G-prot e i n s . ’ ~ ~Thus,
~ ~ ‘ one possibility would be a deficiency of a
phagocyte-specificG-protein that would be specificly involved
in the signal transduction leading to actin polymerization
(necessary for shape change, spreading, and migration), fusion
of azurophil granules with the plasma membrane, and
NADPH oxidase activation. However, the defect may also
be localized more distal from the chemotaxin receptors. A
defect in key enzymes such as protein kinase C (PK-C) or
phospholipase C (PL-C) seems unlikely because of the normal
Table 4. Diglyceride Formation
Control
Patient
Addition
DAG+EAG
EAG
DAG+EAG
EAG
None
FMLP (1 smol/L)
Cyto B/FMLP
PMA (100 ng/mL)
STZ (1 mn/mL)
124
126
273
489
529
NT
38
53
NT
NT
79
146
500
358
382
NT
16
274
NT
NT
~
Values in picomoles per 10’ cells The reaction time for the experiments
with FMLP and the combination of Cyto B/FMLP was 2 minutes The
reaction time for PMA and STZ was 5 minutes
Abbreviation NT, not tested
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
2741
A NOVEL SYNDROME OF NEUTROPHIL DYSFUNCTION
100
100
9
CD8+
CD4+
n
E
B
80
80
60
60
40
40
20
20
cn
3
W
o
+z
W
2
W
8
2
0
0
0
2
4
6
8
10
TIME (mid
0
2
4
6
a
10
TIME (mid
Fig 5. Actin polymerizationof T-cell clones. T cells (107/mL in incubation medium) were activated for the time indicated and instantaneously
fixed, permeabilized, and incubated with NBD-phallacidin, essentially as described.” ( 0 )Control cells; (0)
patient cells. Stimulation with
RANTES (1 pmol/L) of CD4+ T cells (A) or CD8+ T cells (B). Unimodalshifts in fluorescence levels wereobsewed, indicating that subpopulations
were not present in these T-cell populations.
PMA responsiveness and the (almost) unaffected increase of
[Ca2+Iiupon the addition of PAF or FMLP (Fig 4), respectively. Although formation of PA from phosphatidylcholine
correlates with NADPH oxidase activity and exocytosis from
the azurophil g r a n ~ l e s ,both
~ ~ - of
~ ~which were disturbed in
the patient’s cells, deficient PL-D activity, which catalyzes
this formation of PA, cannot explain the absence of actin
polymerization or chemotaxis; the Ca” requirements of PLD a ~ t i v i t ycontrast
~ ~ , ~ ~with the Ca2+independency of these
functional r e ~ p o n s e s . ~Still,
~ . ~other
’
enzymes involved in P L
D activation and actin polymerization (eg, tyrosine kinase^^^-^^)
may be deficient.
To summarize, we have characterized a severe neutrophil
dysfunction of the chemotaxin receptor-mediated responses.
Although we have been unable to localize the defect, our
results show that these receptors share a common, as yet
unknown, regulatory mechanism of actin polymerization,
azurophil granule release reactions, and respiratory burst activity. The importance of signalling via the chemotaxin receptors for neutrophil function and clinical condition is
clearly exemplified by the identification of this patient.
ACKNOWLEDGMENT
We thank Drs C. Cohen (HBpital des Enfants Reine Fabiole, Brussels) and A. Ferster (Brugmann Hospital, Brussels) for the clinical
contribution; Dr B. Cantineaux-Magrez (Brugmann Hospital, Brussels) for the first studies of phagocytosis and intracellular killing; and
Drs K.C. Kuijpers, C.J.M. van Noessel, G.J. Jochems, and R.A.W.
van Lier (CLB, Amsterdam) for the lymphocyte studies; A.T.J. Tool
(CLB, Amsterdam) for the diglyceride assay; Dr M. Tas (Free University, Amsterdam) for the polarization studies; and Dr J. Calafat
(Netherlands Cancer Institute, Amsterdam, The Netherlands) for EM
studies.
REFERENCES
1. Klebanoff SJ, Clark RA: The Neutrophil Function and Clinical
Disorders. Amsterdam, The Netherlands, Elsevier, 1978
2. Malech HL, Gallin JI: Neutrophils in human disease. N Engl
J Med 317:687, 1987
3. Snyder F: Platelet-Activating Factor. New York, NY, Plenum,
1987
4. Williams KI, Higgs GA: Eicosanoids and inflammation (review).
J Pathol 156:101, 1988
5. Baggiolini M, Walz A, Kunkel SL: Neutrophil-activating peptide/interleukin 8, a novel cytokine that activates neutrophils. J Clin
Invest 84: 1045, 1989
6. Leonard EJ, Yoshimura T: Human monocyte chemoattractant
protein-I (MCP-I). Immunol Today 11:97, 1990
7. Snyderman R, Uhling RJ: Phagocytic cells, stimulus response
coupling mechanisms, in Gallin JI, Goldstein IM, Snyderman R (eds):
Inflammation: Basic Principles and Clinical Correlates. New York,
NY, Raven, 1988, p 309
8. Rhee SG, Suh PG, Ryu SH, Lee SY: Studies of inositol phospholipid-specific phospholipase C. Science 244546, 1989
9. Nishizuka Y: The role of protein kinase C in cell surface signal
transduction and tumor production. Nature 308:693, 1984
10. Benidge MJ, Irvine R F Inositol phosphates and cell signalling.
Nature 341:197, 1989
1 I. Dennis EA, Rhee SG, Billah MM, Hannun YA: Role of phospholipases in generating lipid second messengers in signal transduction. FASEB J 5:2068, 1991
12. Goldstein IM, Roos D, Kaplan HB, Weissmann G: Complement and immunoglobulins stimulate superoxide production by hu-
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
2742
man leukocytes independently of phagocytosis. J Clin Invest 56: 1 155,
1975
13. van Noesel C, Miedema F, Brouwer M, de Rie MA, Aarden
LA, van Lier RAW Regulatory properties of LFA-I alpha and beta
chains in human T-lymphocyte activation. Nature 333350, 1988
14. van der Reyden HJ, van Rhenen DJ, Lansdorp PM, van't
Veer MB, Langenhuysen MMBC, Engelfriet CP, von dem Borne
AEGKr: A comparison of surface marker analysis and FAB classification in acute myeloid leukemia. Blood 61:443, 1983
15. Miedema F, Tetteroo PAT, Hesselink WG, Werner G, Spits
H, Melief CJM: Both Fc receptors and lymphocyte-function-associated antigen- I on human T lymphocytes are required for antibodydependent cellular cytotoxicity (killer cell activity). Eur J lmmunol
14518, 1984
16. Roosenfeld SI, Looney RJ, Leddy JP, Phipps DC, Abraham
GN, Anderson CL: Human platelet Fc receptor for immunoglobulin
G. Identification as a 40,000-molecular-weight membrane protein
shared by monocytes. J Clin Invest 76:2317, 1985
17. Nakamura M, Murakami M, Koga T, Tanaka Y, Minakami
S: Monoclonal antibody 7D5 raised against cytochrome bSSsof human
neutrophils: Immunocytochemical detection of the antigen in peripheral phagocytes of normal subjects, patients with chronic granulomatous disease and their camer mothers. Blood 69:1404, 1989
18. Roos D, de Boer M: Purification and cryopreservation of
phagocytes from human blood. Methods Enzymol 132:225, 1986
19. Kuijpers TW, Tool ATJ, van de Schoot CE, Onderwater JJM,
Roos D, Verhoeven AJ: Membrane surface antigen expression on
neutrophils: A reappraisal of the use of surface markers from neutrophil activation. Blood 78: 1105, 1991
20. Kuijpers TW, Eckmann CM, Weening RS, Roos D: A rapid
turbidimetric assay of phagocytosis and serum opsonizing capacity.
J Immunol Methods 124535, 1989
21. Hamers MN, Bot AAM, Weening RS, Sips HJ, Roos D: Kinetics and mechanism of the bactericidal action of human neutrophils
against Escherichia coli. Blood 64:635, 1984
22. Kuijpers TW, Koenderman L, Weening RS, Verhoeven AJ,
Roos D: Continuous cell activation is necessary for a stable interaction
of complement receptor type 3 with its counterstructure in the aggregation of human neutrophils. Eur J Immunol 20501, 1989
23. Hakkert BC, Rentenaar JM, van Aken WG, Roos D, van
Mourik JA: A three-dimensional model system to study the interactions between human leukocytes and endothelial cells. Eur J Immunol20:2775, 1990
24. Zigmond SH, Hirsch JG: Leukocyte locomotion and chemotaxis. New method for evaluation and demonstration of cell-derived chemotactic factor. J Exp Med 137:387, 1973
25. Cianciolo GJ, Snyderman R: Monocyte responsiveness to
demonstrate stimuli in vitro is a property of a subpopulation of human
mononuclear cells which can respond to multiple chemoattractants.
J Clin Invest 67:60, 198 I
26. Howard TH, Meyer WH: Chemotactic peptide modulation
of actin assembly and locomotion in neutrophils. J Cell Biol 98:
1265, 1984
27. Pozzan T, Lew DP, Wollheim CB, Tsien RY: Is cytosolic ionized calcium regulating neutrophil activation? Science 22: 1413, 1983
28. Bijsterbosch MK, Rigley KP, Klaus GGB: Crosslinking of
surface immunoglobulin on B lymphocytes induces both intracellular
Ca2+release and Ca2+influx: Analysis with indo-I. Biochem Biophys
Res Commun 137500, 1985
29. Grynkiewicz GM, Poenie M, Tsien RY: A new generation of
Ca2+indicators with greatly improved fluorescenceproperties. J Biol
Chem 258:3440, 1985
30. Tyagi SR, Tamura M, Burnham DN, Lambeth J D Phorbol
myristate acetate (PMA) augments chemoattractant-induced diglyc-
ROOS ET AL
eride generation in human neutrophils but inhibits phosphoinositide
hydrolysis. J Biol Chem 263:13191, 1988
31. Preiss J, Loomis CR, Bishop WR, Stein R, Niedel JE, Bell
RM: Quantitative measurement of sn-l ,2-diacylglycerols present in
platelets, hepatocytes, and ras- and cis-transformed normal rat ludney
cells. J Biol Chem 26153597, 1986
32. Schall TJ, Bacon K, Toy KJ, Goeddel DV: Selective attraction
of monocytes and T lymphocytes of the memory phenotype by cytokine RANTES. Nature 347:669, 1990
33. Furukawa K, Tengler R, Nakamura M, Urwyler A, De Weck
AL, Kanegasaki S, Maly F-E: B lymphoblasts show oxidase activity
in response to cross-linking of surface IgM and HLA-DR. Scand J
Immunol35:561, 1992
34. Boxer LA, Hedley-Whyte ET, Stossel TP: Neutrophil actin
dysfunction and abnormal neutrophil behavior. N Engl J Med 291:
1093, 1984
35. Southwick FS, Dabiri GA, Stossel TP: Neutrophil atoin dysfunction is a genetic disorder associated with partial impairment of
neutrophil actin assembly in three family members. J Clin Invest 82:
1525, 1988
36. Coates TD, Torkildson JC, Torres M, Church JA, Howard
TH: An inherited defect of neutrophil motility and microfilamentous
cytoskeleton associated with abnormalities in 47 kD and 89 kD proteins. Blood 78: 1338, I99 I
37. Southwick FS, Howard TH, Holbrook T, Anderson DC,Stossel
TP, Arnout M: The relationship between CR3 deficiency and neutrophil actin assembly. Blood 73:1973, 1989
38. Howard TH, Casella J, Lin S: Correlation of the biologic effects
and binding of cytochalasins to human polymorphonuclear leukocytes. Blood 57:399, 1981
39. Boulay F, Tardif M, Brouchon L, Vignais P: Synthesis and
use of a novel N-formyl peptide derivative to isolate a human Nformyl peptide receptor cDNA. Biochem Biophys Res Commun 168:
1103, 1990
40. Gerard NP, Gerard C: The chemotactic receptor from human
C5a anaphylatoxin. Nature 349:6 14, 199 I
41. Honda ZI, Nakamura M, Miki I, Minami M, Watanabe T,
Seyama Y , Okado H, Toh H, Ito K, Miyamoto T, Shimizu T: Cloning
by functional expression of platelet-activating factor receptor from
guinea-pig lung. Nature 349:342, 1991
42. Smith CD, Lane BC, Kusaka I, Verghese MW, Snyderman
R: Chemoattractant receptor-induced hydrolysis of phosphatidylinositol 4,5-biphosphate in human polymorphonuclear leukocyte
membranes. Requirement for a guanine nucleotide regulatory protein.
J Biol Chem 2605875, 1985
43. Goldman DW, Chang FH, Clifford LA, Goetzl EJ, Bourne
HR: Pertussis-toxin inhibition of chemotactic factor-induced calcium
mobilization and function in human polymorphonuclear leukocytes.
J Exp Med 162:145, 1985
44. Lad PM, Olson CV, Smiley PA: Association of the N-formylMet-Leu-Phe receptor in human neutrophils with a GTP-binding
protein sensitive to pertussis toxin. Proc Natl Acad Sci USA 82369,
1985
45. Agwu DE, McPhail LC, Sozzani S, Bass DA, McPhail CE:
Phosphatidic acid as a second messenger in human polymorphonuclear leukocytes. J Clin Invest 88:53 1, 199 I
46. Rossi F, Greskowiak M, Della-Bianca V, Calzetti F, Gandini
G: Phosphatidic acid and not diacylglycerol generated by phospholipase D is functionally linked to the activation ofthe NADPH oxidase
by FMLP in human neutrophils. Biochem Biophys Res Commun
168:320, 1990
47. Xie M, Jacobs LS, Dubyak GR: Regulation of phospholipase
D and primary granule secretion by P2-purinergic and chemotactic
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
A NOVEL SYNDROME OF NEUTROPHIL DYSFUNCTION
peptide-receptor agonists is induced during granulocytic differentiation of HL-60 cells. J Clin Invest 88:45, I99 l
48. Anthes JC, Eckel S, Siege1 MI, Egan RW, Billah MM: Phospholipase D in homogenates from HL-60 granulocytes: Implications
of calcium and G protein control. Biochem Biophys Res Chem 163:
657, 1989
49. Kessels GCR, Roos D, Verhoeven AJ: FMLP-induced activation of phospholipase D in human neutrophils. Dependence on
changes in cytosolic free Ca2+concentration and relation with respiratory burst activation. J Biol Chem 266:23 152, 1991
50. Naccache PH, Therrien S, Caon AC, Liao N, Gilbert C, McColl
SR: Chemoattractant-induced cytoplasmic pH changes and cytoskeletal reorganization in human neutrophils: Relationship to the
stimulated calcium transients and oxidative burst. J Immunol 142:
2438, 1989
2743
5 1. Kuijpers TW, Hoogenverf M, Roos D Neutrophil migration
across monolayers of resting and cytokine-activated endothelial cells:
The role of intracellular calcium changes and fusion of specific granules with the plasma membrane. J Immunol 148:72, 1992
52. Farrell CA, Merenda JM, Connelley PA, Showell HJ: Tyrosine
phosphorylation in human neutrophils: Evidence of common substrates involved in signalling through receptors for the chemotactic
factors C5a, fMLP, IL-8, LTB4 and PAF. J Leukoc Biol2:89a, 1991
53. Uings IJ, Thompson NT, Randall RW, Spacey GD, Bonser
RW, Hudson AT, Garland LG: Tyrosine phosphorylation is involved
in receptor coupling to phospholipase D but not phospholipase C in
the human neutrophil. Biochem J 281:597, 1992
54. Gaudry M, Caon AC, Gilbert C, Lille S, Naccache PH: Evidence for the involvement of tyrosine kinases in locomotory responses
of human neutrophils. J Leukoc Biol 51:103, 1992