PHAGOCYTES

Natural history and early diagnosis of LAD-1/variant syndrome

Taco W. Kuijpers,1,2 Robin van Bruggen,1,2 Nanne Kamerbeek,2 Anton T. J. Tool,2 Gonul Hicsonmez,3 Aytemiz Gurgey,3
Axel Karow,4 Arthur J. Verhoeven,2 Karl Seeger,5 Özden Sanal,6 Charlotte Niemeyer,4 and Dirk Roos2
1Emma Children’s Hospital, Academic Medical Center (AMC), University of Amsterdam, The Netherlands; 2Department of Blood Cell Research, Sanquin

Research and Landsteiner Laboratory, AMC, University of Amsterdam, The Netherlands; 3Pediatric Hematology Unit, Hacettepe University, Ankara, Turkey;
4Department of Pediatrics and Adolescent Medicine, Division of Pediatric Hematology and Oncology, University of Freiburg, Germany; 5Department of Pediatric

Oncology/Hematology, Otto-Heubner-Center for Pediatric and Adolescent Medicine, Charité-Universitätsmedizin, Berlin, Germany; 6Pediatric Immunology Unit,
Hacettepe University, Ankara, Turkey

The syndrome of leukocyte adhesion defi- as a severe bleeding tendency. This is regulating molecules, were normally
ciency (LAD) combined with a severe compatible with 2 major blood cell types present. Moreover, Rap1 activation was
Glanzmann-type bleeding disorder has contributing to the clinical symptoms (ie, intact in patients’ blood cells. Defining

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been recognized as a separate disease
entity. The variability in clinical and cell
biological terms has remained largely un-
clear. We present data on 9 cases from 7
unrelated families, with 3 patients being
actively followed for more than 12 years.
The disease entity, designated LAD-1/
variant syndrome, presents early in life
and consists of nonpussing infections
from bacterial and fungal origin, as well
granulocytes and platelets). In granulo-
cytes of the patients, we found adhesion
and chemotaxis defects, as well as a
defect in NADPH oxidase activity trig-
gered by unopsonized zymosan. This last
test can be used as a screening test for
the syndrome. Many proteins and genes
involved in adhesion and signaling, in-
cluding small GTPases such as Rap1 and
Rap2 as well as the major Rap activity-
the primary defect awaits genetic linkage
analysis, which may be greatly helped by
a more precise understanding and aware-
ness of the disease combined with the
early identification of affected patients.
(Blood. 2007;109:3529-3537)
© 2007 by The American Society of Hematology

Introduction
The leukocyte adhesion deficiency 1 (LAD-1)/variant syndrome as
described in 1997 consists of 2 major hallmarks: a moderate
LAD-1–like syndrome and a severe Glanzmann-like bleeding
tendency.1 To date, 3 similar patients have been reported in
addition.2-4 The complex of clinical features is predominantly
related to a neutrophil defect and platelet dysfunction.
Leukocytes and platelets in these patients have a normal level of
integrin expression, but these integrins do not function properly. In vitro,
the absence of chemotaxis toward all chemoattractant stimuli tested and
the lack of adhesion to substrates (in contrast to lymphocytes) were
noted, but were not due to the absence of any of the 7-transmembrane
spanning G-protein–coupled receptors (GPCRs) involved in cell activa-
tion, nor to a defect in any of the known signaling cascades transducing
the signals via mitogen-activated protein kinase (p38MAPK, ERK1,
and ERK2), phospholipase-C (PLC), or phosphatidyl-inositol-3 kinase
(PI3K) to effector functions. Mobilization of intracellular calcium,
polymerization of F-actin, release of proteins from azurophil and
specific granules, and the induction of NADPH-oxidase activity in
neutrophils were found normal. The same is true for the platelets in the
patients, because effector functions induced by the platelets were intact
as long as the ␤3 integrin activation was not a determinant step in the
process1 (T.W.K., unpublished data, March and May 2004, February
and October 2005, and January 2006).
A general integrin-associated defect or intracellular signaling
defect has not yet been identified in these patients. Of interest, Alon
and colleagues have suggested that the syndrome is caused by an
aberrant activation of Rap1 in these patients (Alon et al4 and
Kinashi et al5). The small GTPase Rap1 belongs to the large Ras
family of GTPases and is involved in several aspects of cell
adhesion, including integrin-mediated cell adhesion and cadherin-
mediated cell junction formation. To date, various effector proteins
for Rap1 have been identified, providing a clear link between Rap1
and actin dynamics. Furthermore, evidence is accumulating that
Rap1 functions in spatial and temporal control of cell polarity.6-9
Similar to all other small GTPases, Rap1 binds either GDP or GTP,
and the change between these 2 states represents a molecular
switch (ie, the GTP-bound form is “on” and the GDP-bound form is
“off”).6,10,11 The GDP/GTP exchange is regulated by tissue- and
cell-specific guanine nucleotide exchange factors (GEFs) and
GTPase-activating proteins (GAPs).12-16
Over the last years, we have obtained many data on these
pathways that do not favor a defect in Rap activation in the
leukocytes of several patients with LAD-1/variant syndrome. In
this paper, we describe in more detail our present findings on Rap-1
activity. Although we cannot exclude the existence of several
genetic defects resulting in a similar phenotype, every claim about
the diagnosis of a so-called LAD-variant syndrome will be
determined by the sensitivity and reproducibility of a test with
which the syndrome can be diagnosed. Instead of using monoclonal
antibodies (MoAbs) to detect activation epitopes on integrin

Submitted January 18, 2006; accepted July 12, 2006. Prepublished online as Blood The publication costs of this article were defrayed in part by page charge
First Edition Paper, December 21, 2006; DOI 10.1182/blood-2006-05-021402. payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.

An Inside Blood analysis of this article appears at the front of this issue. © 2007 by The American Society of Hematology

BLOOD, 15 APRIL 2007 䡠 VOLUME 109, NUMBER 8 3529

3530 KUIJPERS et al
subunits,1 we now show that the syndrome can be best defined by a
simple and robust screening test. The advantage of the screening
test with unopsonized zymosan particles derived from baker’s
yeast will mean a large diagnostic improvement. In the presence of
normal ␤2 integrin expression levels on all leukocytes, normal
up-regulation of these integrins upon activation on neutrophils, but
abnormal neutrophil chemotaxis, the zymosan test discriminates
the functional defect of such patients easily and without failure.
Now that we have identified over the years more of such
patients, a clinical picture of the syndrome can also be delineated.
To give direction to the eventual elucidation of the genetic
background of the LAD-1/variant syndrome, we describe in this
paper the natural history of 9 patients from 7 unrelated families. In
addition, we demonstrate that Rap1 activation is not the principal
defect in this syndrome. And, finally, we describe a simple and
robust screening assay to diagnose the patients.
Patients, materials, and methods
Neutrophil, lymphocyte, and platelet purification
Heparinized venous blood was collected from healthy donors and from
LAD-1/variant patients and their family members, after obtaining informed
consent. The study was approved by the AMC institutional medical ethics
committee in accordance with the standards laid down in the 1964
Declaration of Helsinki.
Granulocytes were isolated as described.17,18 Purity was always more
than 95%. In some experiments, whole leukocyte preparations were used
from which the erythrocytes were lysed by ice-cold isotonic NH4Cl
solution17 and resuspended in HEPES buffer for further experiments.18
After Percoll density gradient centrifugation of the blood, peripheral blood
mononuclear leukocytes were collected from the interface. Monocytes were
depleted by an adhesion step to plastic for 60 minutes in RPMI 1640 (Gibco,
Grand Island, NY) at 37°C. Mixed lymphocytes were resuspended in RPMI
1640 (Gibco) at a final concentration of 20 ⫻ 106 cells/mL.
Platelets were isolated from platelet-rich plasma diluted in Krebs-
Ringer solution (4 mM KCl, 107 mM NaCl, 20 mM NaHCO3, 2 mM
Na2SO4, 19 mM trisodium citrate, 0.5% [wt/vol] glucose in H2O, pH 5.0).
After centrifugation at 500g for 10 minutes, the cells were resuspended in
Krebs-Ringer solution (pH 6.0) and centrifuged again at 500g for 10 minutes.
After 2 additional washing steps, the cells were suspended in Hepes/Tyrode
buffer (2.68 mM KCl, 10 mM Hepes, 1.7 mM MgCl2, 11.9 mM NaHCO3, 5 mM
glucose in PBS) to a concentration of 200 ⫻ 106 cells/mL.
Neutrophil migration and adhesion
Chemotaxis of purified neutrophils was assessed by means of Fluoroblok
inserts (Falcon; Becton Dickinson, San Jose, CA). Cells (5 ⫻ 106/mL) were
labeled with calcein-AM (1 ␮M final concentration; Molecular Probes,
Leiden, the Netherlands) for 30 minutes at 37°C, washed twice, and
resuspended in HEPES buffer at a concentration of 2 ⫻ 106/mL. Chemoat-
tractant solution (PAF, IL-8, and C5a, all at 10 nM; Sigma, St Louis, MO) or
medium alone (0.8 mL/well) was placed in a 24-well plate, and 0.3 mL cell
suspension was delivered to the inserts (3-␮m pore size) and placed in the
24-well plate. Cell migration was assessed by measuring fluorescence in the
lower compartment at 2.5-minute intervals for 45 minutes with the
HTS7000⫹ plate reader (Perkin Elmer, Norwalk, CT) at an excitation
wavelength of 485 nm and emission wavelength of 535 nm. Maximal slope
of migration was estimated over a 10-minute interval.19
Adhesion was determined in 96-well maxisorp plates (Nunc, Wiesba-
den, Germany), precoated or not with human plasma-derived fibronectin for
60 minutes at room temperature. Calcein-labeled cells (100 ␮L; 2 ⫻ 106/
mL) were pipetted in the 96-well plate, and cells were stimulated with 25
␮L solvent with PAF (final concentration, 1 ␮M), TNF (final concentration,
10 ng/mL), or PMA (final concentration, 100 ng/mL) as stimulus. Plates
were incubated for 30 minutes at 37°C. Thereafter, the plates were washed 3
BLOOD, 15 APRIL 2007 䡠 VOLUME 109, NUMBER 8
times with PBS at room temperature. Adherent cells were lysed with 125
␮L H2O, containing 0.5% (wt/vol) Triton X-100 (10 minutes, room
temperature). Fluorescence was measured with the HTS7000⫹ plate reader
at an excitation wavelength of 485 nm and emission wavelength of 535 nm.
Adhesion was determined as a percentage of total cell input (2 ⫻ 106/mL).
Analysis of neutrophil chemotaxis in the micropipette assay
Neutrophils (5 ⫻ 105) in HEPES buffer were allowed to adhere for 10
minutes to glass slides precoated with ICAM-1-Fc chimera (a generous gift
of Dr D. Staunton, ICOS, Bethell, WA). Chemotaxis toward a micropipette
tip (Eppendorf Pipetman, Hamburg, Germany) containing 10⫺9 M C5a was
then visualized using an Axiovert 200M microscope (Zeiss, Jena, Germany)
equipped with an LD Achroplan 20⫻/0.40 numerical aperture objective.
Images were taken using an Axiocam MRm with Axiovision version 4.5
software. The software used for image processing was Image-Pro Ams
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version 6 (Media Cybernetics, Silver Spring, MD).
Induction of NADPH-oxidase activity
NADPH-oxidase activity was assessed as hydrogen peroxide release
determined by an Amplex Red kit (Molecular Probes). Neutrophils
(0.25 ⫻ 106/mL) were stimulated with PAF and fMLP (both 1 ␮M, added
simultaneously), 1 mg/mL STZ, or 100 ng/mL PMA, in the presence of
Amplex Red (0.5 ␮M) and horseradish peroxidase (1 U/mL). Fluorescence was
measured at 30-second intervals for 20 minutes with the HTS7000⫹ plate reader.
Maximal slope of H2O2 release was assessed over a 2-minute interval.19,20
Using unopsonized zymosan, the same steps and procedures were
followed, but fluorescence was measured for 60 minutes at similar
30-second intervals. Maximal slope of H2O2 release was assessed over a
2-minute interval.
In order to exclude cell death during blood transportation or improper
handling, control experiments were performed for annexin-V binding,
mitochondrial staining, and morphology, exactly as described.21,22 Morphol-
ogy was determined after Giemsa staining of cytospin preparations.
Apoptotic morphology was defined as the presence of condensed nuclei and
simultaneous loss of the polysegmented nuclear appearance.
Rap activation assay
Rap1 activation was determined essentially as described.23 After warming
the cell suspensions, neutrophils (10 ⫻ 106 cells/mL), lymphocytes
(20 ⫻ 106 cells/mL), or platelets (200 ⫻ 106 cells/mL) were stimulated
with the relevant stimuli or buffer alone: that is, TRAP-6 (thrombin-receptor-
derived activation peptide-6; Bachem, Bubendorf, Switzerland) at 10 ␮M,
human SDF1␣-10a (Strathmann Biotec, Hamburg, Germany) at 12.5 nM,
and PAF and fMLP (both from Sigma) at 1 ␮M final concentration.
At various time points, samples (0.5 mL) were taken and lysed
immediately in 250 ␮L ice-cold modified RIPA (3 ⫻) buffer containing 30
mM Tris-HCl (pH 7.4), 450 mM NaCl, 3% NP-40, 1.5% deoxycholic acid,
0.3% SDS, and a mixture of protease inhibitors (Complete EDTA-free;
Roche, Basel, Switzerland) and DFP (Fluka Gmb, Seelze, Germany).
Cell lysates were put on ice for 10 minutes and clarified by centrifuga-
tion at 20 000g in an Eppendorf centrifuge for 10 minutes at 4°C. Per
sample, 15 ␮g glutathione S-transferase (GST)–RalGDS-RBD was pre-
coupled for 1 hour to 20 ␮L glutathione beads (GST-RalGDS-RBD). After
coupling, beads were washed 4 times with RIPA (1 ⫻) buffer, added to the
cell lysate, and incubated for 60 minutes at 4°C. Samples were washed
3 times in RIPA (1 ⫻) buffer, and bound proteins were eluted in 30 ␮L
Laemmli sample buffer. The samples were subjected to SDS–polyacrylam-
ide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene
difluoride membranes (PVDF; NEN, Boston, MA). Rap1 was detected with
a monoclonal antibody (Transduction Laboratories, Lexington, KY) and
horseradish peroxidase–coupled goat anti–mouse Ig (Amersham Bio-
sciences, Buckingham, United Kingdom), using enhanced chemilumines-
cence (Amersham Biosciences). Western blot of total cell lysates served as a
control for equal input into the Rap-binding domain affinity purification. In
some experiments, Rap2 antibodies (Santa Cruz Biotechnology, Santa
Cruz, CA) were used instead.
BLOOD, 15 APRIL 2007 䡠 VOLUME 109, NUMBER 8
Molecular studies
Genomic DNA from peripheral mononuclear cells and fibroblasts from the
patients were extracted by standard methods. All coding exons of the CD18
gene were amplified in separate polymerase chain reactions (PCRs).1,24
Sequencing was performed with the PE dye terminator kit and products
were analyzed on an automated sequencer (ABI3100; Applied Biosystems,
Foster City, CA).
Hematologic studies
Morphologic analysis demonstrated hypercellular bone marrow (BM) in
most of the BM smears, in some patients confirmed by BM biopsies. No
excess of collagen or signs of fibrosis, disturbed bone marrow stroma
development, or disorganized hematopoiesis were observed. In some
patients, absolute numbers of progenitor B cells (CD19⫹, CD10⫹, CD24⫹),
T cells (CD2⫹, CD3⫹, CD4⫹, CD8⫹), natural killer (NK) cells (CD3⫺,
CD16⫹, CD56⫹), and myeloid cells (CD15⫹, CD14⫹, CD16⫹, CD65⫹)
were determined by standard FACScan procedures. Colony-forming units
of the erythroid and granulocyte/macrophage progenitors (BFU-Es and
CFU-GMs, respectively) were determined in a 10- to 14-day semisolid culture.
Cytogenetic analysis
Freshly obtained bone marrow samples were collected, cultured (without
stimulation) in RPMI-1640 supplemented with 15% (wt/vol) fetal calf
serum for 24 hours, and harvested according to standard protocols.
PHA-stimulated peripheral blood lymphocytes were cultured for 72 hours.
Metaphase chromosomes were analyzed with a routine Q-banding method
(QFA) and abnormal clones were defined according to the International
System for Human Cytogenetic Nomenclature 18, as described earlier.20
Statistics
Statistical analysis was performed with the SPSS package for windows,
version 10.0 (SPSS, Chicago, IL). For normally distributed data, the
Student t test was used to compare group means; otherwise the Mann-
Whitney U test was applied. A 2-sided P value of less than .05 was
considered statistically significant.
Results
Clinical features
We identified 7 families with 9 members suffering from LAD-1/
variant syndrome (Figure 1), including the original family de-
Figure 1. Pedigrees of 7 unrelated families with LAD-1/
variant syndrome patients. The diamonds in families 2, 3,
and 7 denote stillbirths after a pregnancy of a duration in
weeks as indicated beneath the genetic symbols used.
LAD-1/VARIANT SYNDROME 3531
scribed in 1997.1 All these families are known for their consanguin-
eous offspring, and all members are descendants of Turkish
ancestors from Anatolia, a region in central Turkey, although most
of the patients were born in Europe. Nonpussing infections and
severe bleedings are the apparent early and repeatedly recurring
findings in these patients (Table 1). Patients diagnosed with the
classical LAD-1 defect caused by mutations in the ITG2 gene,
show late detachment of the umbilical cord.24-26 In the LAD-1/
variant syndrome, the umbilical cord detached after 4 weeks in (at
least) 3 of the children. Omphalitis was noted in 2 patients during
the neonatal period and treated by antibiotics; in 1 of these 2
patients, information confirmed late detachment of the umbilical
cord. Cultures did not show any particular pathogen involved. The
omphalitis never resulted in necrotizing lesions or necessitated
surgical intervention.
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The infectious agents cultured were mostly Gram-negative
strains causing early sepsis (eg, Escherichia coli, Pseudomonas
spp), but staphylococcal infections were found as well. Fungal
disease occurred in 3 patients, one with superficial skin infections
and assumed mediastinal lymph node involvement (family 1), one
with pulmonary Aspergillus fumigatus infection as proven by
bronchoalveolar lavage after treatment with multiple antibiotics for
chronic bilateral pulmonary infiltrates (family 3), and another with
Fusarium oxysporum infection of the blood and the simultaneous
presence of painful subcutaneous nodules (family 4). In the first
case, voriconazole was administered for 8 weeks to treat the
mediastinal mass. The proven pulmonary aspergillosis in the
6-month-old patient of family 3 was successfully treated with
intravenous amphotericin B. In the patient of family 4 at 2 years of
age, the fusariosis was treated with lipid-formulated amphotericin
B for 12 weeks, combined with flucytosine for the first 4 weeks; the
infected Port-a-Cath was removed. Some patients had been immu-
nized very early in life by the attenuated bacillus Calmette Guérin
(BCG) vaccine strain, without any infectious consequence.
In 2 patients, CMV infection was diagnosed by CMV-specific
PCR, serology, and cultures from the urine, but in both cases the
disease was not severe. Overall, there was no evidence for
increased susceptibility to viral infections. No indications for
congenital CMV were found (negative virus cultures in the first
14 days of life; lack of clinical stigmata such as microcephaly,
vision impairment, or neuronal deafness upon follow-up).
3532 KUIJPERS et al BLOOD, 15 APRIL 2007 䡠 VOLUME 109, NUMBER 8

Table 1. Patient characteristics

Family 1 Family 2 Family 3 Family 4 Family 5 Family 6 Family 7

No. 1 No. 1 No. 1 No. 1 No. 2 No. 1 No. 1 No. 2 No. 1

Sex (y of birth) Male (1992) Male (2002) Male (1998) Male (1992) Male (1994) Male (2002) Female Female Male (2005)
(1993)† (1993)†
First symptoms Skin infection Skin infection, CNS bleeding Omphalitis Nose bleeding Omphalitis CNS TF-dep Anorectal Sepsis
petechiae bleeds and anemia fistula mucosal and
anemia skin bleeding
Average no. 2, perianal 7-8 (1st y), perianal 6 4 2 3-4, perianal 1 2 4
infections/y abscesses abscesses abscesses
Bacterial Y Y Y Y Y Y Y Y Y
Viral N N N N NA (CMV) N N (CMV)
Fungal Presumed Candida albicans Aspergillus Fusarium sp NA N N N N

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fumigatus
Necrotic skin lesions Y N N Y N N Y Y N
Late detachment of Y Unknown Unknown Y Unknown Y N N Y
umbilical cord
Wound healing Poor Normal Poor Poor Poor Normal Poor Poor Normal
Bleeding tendency Severe Severe Severe Severe Moderate Severe Moderate Moderate Moderate
Average no. platelet ⬎ 50 ⬎ 50 ⬎ 50 10-20 ⬍ 10 ⬎ 50 ⬍ 10 ⬍ 10 5
transfusions/y
Average no. Ery ⬎ 20 ⬎ 20 ⬎ 20 5-10 ⬍5 ⬎ 20 5-10 5-10 ⬎ 10
transfusions/y
Birth (weight, g) AT (3870) AT (3650) 35⫹4 (2040) AT (3560) AT (unknown) AT (3500) 35⫹6 35⫹6 3000
(1935) (1945)
Growth* p 10 p 3-10 p3 p 40 p 25 p 3-10 p⬍3 p⬍3 p 3-10
Dysmorphic features Strabism Hypospadia None ASD (ll) None Coarse face, Anus Anus None
large tongue, praeter praeter
psychomotor
retardation
Physical examination Splenomegaly Normal Hepatospleno- Transient Transient Hepatospleno- Transient Transient Hepatospleno-
megaly hepatospleno- hepatospleno- megaly hepato- hepato- megaly
megaly megaly spleno- spleno-
megaly megaly
Major surgery Y Orchidectomy, N Y N IV cath placement Y Y N
perianal abscesses
Surgical complication Hemothorax — — Skin graft — — Fistula Bleeds —
Hb level, g/dL, 12-14 10-12 8-10 6-9 6-10 6-11 8-10 10-14 6-10
normal range
Plt count, ⫻ 109/L, 120-180 21-90 41-250 130-210 130-240 40-320 150-250 114-230 50-280
normal range
WBC count, ⫻ 109/L, 12-35 20-45 20-70 20-59 20-65 22-54 20-50 18-49 30-60
normal range
% neutrophils, range 60-80 70-90 60-90 60-90 55-80 30-70 60-90 60-90 30-50
% lymphocytes, 20-30 10-20 10-15 10-35 20-40 15-60 10-40 10-40 30-50
range
BM ery/gran 1:4.0 1: 1.0 1:2.5 1:12 1:8.0 1:8.0 1:1.6 1:5.0 1:4.5
Cytogenetics Normal Normal (4% blasts) Normal Normal Normal Normal Normal Normal Normal
(⬍ 5% blasts)
Immunization Normal Normal (VZV) — Normal Normal — Normal Normal HBV only
BCG status BCG⫺ BCG⫺ BCG⫹ BCG⫺ BCG⫺ BCG⫺ BCG⫹ BCG⫹ BCG⫺
Alive Y; BMT 2005 Y; BMT 2006 N; BMT 1999 N N Y Y Y Y
Mortality — — BMT (VOD) Pseudomonas Nose bleed — — — —
sepsis and
soft tissue
infections

All families were consanguineous and of Turkish descent. For % lympho-subsets/proliferative capacity, all families were normal. Relative CD3⫹CD4⫹, CD3⫹CD8⫹, CD19⫹,
CD16/56⫹ lymphocyte cell fractions and CD45RA/CD45RO distribution in the T-cell subsets tested. Lymphocyte proliferation upon activation by mitogens and CD3/CD28
MoAb combinations were tested as previously described.1 For “Birth (weight, g)” numbers outside parentheses indicate pregnancy duration in weeks (plus days in superscript).
To convert hemoglobin level from grams per deciliter to grams per liter, multiply grams per deciliter by 10.
Ery indicates erythrocyte; WBC, white blood cell; NA, no information available; CMV, cytomegalovirus; AT, at term (about 40 wk); —, none; VZV, varicella-zoster virus; HBV,
hepatitis B virus; BMT, bone marrow transplantation; VOD, veno-occlusive disease; N, no; Y, yes.
*Percentiles are given according to local growth charts.
†Homozygous twins.
BLOOD, 15 APRIL 2007 䡠 VOLUME 109, NUMBER 8 LAD-1/VARIANT SYNDROME 3533

Splenomegaly or hepatosplenomegaly was observed in almost

all patients, in some only later during infancy, which may result
from extramedullary hematopoiesis.
Three children with the syndrome were born with mulberry
spots and in one child extramedullary hematopoiesis was proven by
skin biopsy (data not shown).
Patients from families 4, 5, and 6 were suspected of presumed
juvenile myelomonocytic leukemia (JMML). All patients were
tested in one way or another for the differential diagnosis of
JMML. Granulocyte-macrophage colony-stimulating factor (GM-
CSF) hypersensitivity of bone marrow (BM) or blood cells, as the
hallmark for JMML,27 was negative. Moreover, the thrombasthenia
was present in all patients, being another feature to distinguish
JMML from LAD-1/variant syndrome. Hematologic malignancy

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or solid tumors have not been noted in any of the patients thus far.
Our index patient from family 1 showed the unexpected
potential to form thrombosis. He was admitted because of clinical
vena cava superior syndrome owing to occlusion of the subclavian
veins and the right-sided vena jugularis communis by phlebogra-
phy, most likely as a result of the repeated platelet infusions in the
presence of an indwelling Port-a-Cath (which he had for more than
8 years without any problem). A well-prepared mediastinal biopsy
of an enlarged lymph node resulted in a severe bleeding (despite
optimizing platelet function by prior transfusions and a thoraco-
scopic approach); the procedure ended in an iatrogenic hematotho-
rax. The patient was in the intensive care unit for respiratory failure
and bilateral pneumonia and septicemia by Pseudomonas aerugi-
nosa, for which he was treated with antibiotics. In addition, this
patient received 4 granulocyte transfusions. The boy survived and
subsequently, at the age of 13 years, successfully received a bone
marrow transplant from a matched unrelated donor. Although we
have not formally proven that the donor-derived neutrophils have
tilted the balance into a favorable condition of pathogen clearance
and initiation of wound healing, we strongly believe that the patient
was saved by these transfusions.
In family 4, the eldest son (born in 1992) died from P
aeruginosa septicemia following surgical débridement of a deep
and necrotic wound on his thigh after initial resolution with
granulocyte transfusions at the age of 13 years. These transfusions
have not been administered during or around the surgical approach
chosen. The youngest son in this family died from a severe nose
bleed after a trauma at the age of 6 years.
The 13-year-old female twin sisters of family 6 are relatively
healthy. One has started menstruation. Contraceptives are being
used to regulate the frequency of bleeding, which until now could
be controlled with single platelet transfusions.
Neutrophil chemotaxis and adhesion defects; normal
Rap 1 activation
Adhesion was defined in uncoated wells or in wells coated with
fibronectin. Under all circumstances, the stimuli tested most often
induced weak adhesion, in contrast to purified neutrophils from
healthy controls (Figure 2A). Neutrophil chemotaxis was measured
and found defective in all patients tested (n ⫽ 7; Figure 2B).
The severely reduced ability to invoke some directed motility
seemed due to the lack of polarization and attachment sites
generated by the neutrophils, as shown in an assay of directed
migration toward a gradient of chemoattractant from a micropipette
(Figure 3 and Videos S1-S2, which are available on the Blood website;
see the Supplemental Videos link at the top of the online article).
Figure 2. Neutrophil adhesion and chemotaxis in LAD-1/variant syndrome.
(A) Neutrophil adhesion to fibronectin upon activation by various stimuli (n ⫽ 7
patients). Neutrophils were isolated from blood of (local) healthy individuals shipped
in the same manner and are included as controls. (B) Neutrophil chemotaxis to
various chemotactic factors. Data of 7 patients have been combined. Controls as in
panel A. Data represents mean ⫾ SD.
These findings on adhesion and chemotaxis are surprising. We
had previously found F-actin polymerization in neutrophils from
the patient in family 1 to be completely normal upon cellular
activation by various chemoattractants.1 Therefore, the inside-out
signaling capacity through activation of Rap1 was tested in
neutrophils, as well as in lymphocytes and platelets. As shown in
Figure 4, no abnormalities were observed in the activation of Rap1.
There was active Rap present in the LAD-1/variant neutrophils
(and platelets), also prior to stimulation, at equal levels as in control
neutrophils, as demonstrated by blotting the same pull-down
experiments with Rap1- or Rap2-specific antibodies (data not
shown). In contrast to the time-dependent activation and deactiva-
tion of Rap1, Rap2 activity was stable and not subject to strong
alterations in activity as in the case of the Rap1 activity shown in
Figure 4. The data on Rap1 activation shown are representative for
the results obtained with cells from 5 patients.
Moreover, in the purified granulocyte fractions, the major GEFs
for Rap1 (ie, C3G and smg-GDS) were normally present on
Western blots, with a similar molecular weight after gel electrophore-
sis when compared with the proteins blotted in lysates of control
neutrophils. Epac (or cyclic AMP [cAMP]–GEF) was absent from
both controls’and patients’neutrophils. Similar results were obtained for
GAPs for Rap1, rap1GAPII, and SPA-1 (data not shown).
Together, these findings of normal Rap1 activation and identical
amounts of the proteins involved in the GDP/GTP exchange of
Rap1 in neutrophils from patients and controls render a defective
Rap1 activity in these patients unlikely.
3534 KUIJPERS et al
Zymosan activity
As previously described for the first patient,1 we have subsequently
confirmed defective up-regulation of the activation epitope on
CD11b/CD18 (CR3) as recognized by the MoAb CBRM1/5 in 3
additional patients. This effect was always corrected for the
simultaneous up-regulation of CD11b, per se, as assessed by a
normal nonconformational-constrained CD11b MoAb.
To avoid the intrinsic variability in the determination of
activation epitopes, we have generated another test system that has
been found to be reproducible and more robust. Unopsonized
zymosan is used in the assay to induce uptake and NADPH oxidase
activity in purified neutrophils. This test is based on the require-
ment for CR3 to change into its active conformation before
unopsonized zymosan is taken up (Figure 5A) and the respiratory
burst is initiated (Figure 5B).28 In control neutrophils, there is a lag
time of about 10 to 12 minutes until the respiratory burst is initiated
by unopsonized zymosan, probably reflecting the time needed for
the neutrophils to become primed by the zymosan particles and
CR3 to adopt its active high-affinity conformation for binding and
uptake of zymosan. This response was absent in the neutrophils
obtained from the LAD-1/variant patients (Figure 5C). Indeed, the
lag time of normal neutrophils was very much shorter (⬍ 3-5
minutes) when a high concentration of Mg2⫹ (10 mM) was added
simultaneously with the zymosan, because this induces the active
conformation of CR3 instantly (data not shown). In contrast to the
STZ response, which we have never found to be defective in
neutrophils from any of the patients tested, uptake and concomitant
NADPH oxidase activity induced by bare, unopsonized zymosan
was absent, even when measured over 60 to 90 minutes of
incubation. Activation of CR3 by high Mg2⫹ concentrations
resulted in uptake of zymosan, even in LAD-1/variant neutrophils,
proving that CR3, once in its active conformation, is functional in
Figure 4. Rap-1 activation in leukocytes and platelets of patients suffering from
LAD-1/variant syndrome. TRAP indicates thrombin receptor-derived activation
peptide. Representative for 5 patients.
BLOOD, 15 APRIL 2007 䡠 VOLUME 109, NUMBER 8
Figure 3. Lack of polarization and chemotaxis of purified neutrophils
toward a micropipette from which an optimal dose of fMLP is diffusing in a
prewarmed chamber containing healthy control cells (top panel) or
patients’ cells (bottom panel). Representative for 4 patients.
these patients (Figure 5C). Altogether, the pattern of respiratory
burst activity seen in the “zymosan test” makes it easy to discern
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the defective response with LAD-1/variant patients’ neutrophils
over a period of about 30 minutes in total.
In 4 families, we have shown this defective induction of
NADPH oxidase activity by unopsonized zymosan in purified
neutrophils from patients. In neutrophils from unaffected siblings,
healthy parents, and controls, from all of whom blood was
simultaneously drawn, shipped, and handled in the same way as the
patients’ blood samples, normal levels of neutrophil NADPH
oxidase activity (as well as adhesion and chemotaxis) were found.
The dependence on the presence of functional CD11b/CD18
was further proven by the lack of NADPH oxidase activity as
verified in 3 classical LAD-1 patients with known CD18 mutations
and no CD11b/CD18 expression at all. Also, the addition of MoAb
44a, a blocking CD11b MoAb (10-minute preincubation at 10
␮g/mL), completely prevented uptake and induction of NADPH
oxidase activity by unopsonized zymosan particles, supporting the
major role of CD11b/CD18 in this screening test (data not shown).
Mutation analysis of the CD18 gene was performed in all
patients, with negative results. Other candidates contributing to
inside-out signaling are cytohesins,29 L-plastin,30 talin,31 and the
recently described RAPL.32 At the protein level, these molecules in
the neutrophils from our patients were normally present, and their
respective genes were without mutations (data not shown).
Discussion
The data from 9 patients in 7 unrelated families indicate that the
LAD-1/variant syndrome can be recognized early in life by
necrotic nonpussing lesions, poor wound healing, and a severe
life-threatening bleeding tendency. Infections range from bacterial
pneumonia and early septicemia to fungal disease. Severe intracra-
nial hemorrhages may affect neonates, followed by a period of
persistent mucosal bleeding and fragile gums due to chronic
periodontitis during childhood. Three patients died: 1 patient died
after BMT and graft failure and 2 died in the same family, 1 from
pseudomonal septicemia during wound débridement and the other
from a severe nose bleed after an unlucky fall from a staircase. Of
the 2 patients who underwent transplantation, the case with the
longest follow-up from his birth in 19921 has recently undergone
transplantation successfully, demonstrating that BMT is curative.
There may be phenocopies of the syndrome with a different
genetic background. Following our first case,1 a number of similar
patients have been reported with defective LAD–Glanzmann-like
syndrome.2-4 All these patients suffered from the typical complex
of clinical features that were highly similar to the original patient
we had described before (Table 1).
BLOOD, 15 APRIL 2007 䡠 VOLUME 109, NUMBER 8 LAD-1/VARIANT SYNDROME 3535

Figure 5. Unopsonized zymosan uptake and activation of human neutrophils. (A) Neutrophil phagocytosis of zymosan is shown for neutrophils purified from a healthy
donor (left panel) and a patient (right panel); in case of patient samples, particles were easily washed off during the preparation of cytospins because of the defective
phagocytosis (n ⫽ 4 patients). (B) Neutrophils from a healthy control were stimulated with serum-treated zymosan (STZ) or unopsonized zymosan as described in “Patients,
materials, and methods” (n ⬎ 100 controls). (C) Identical experiments with neutrophils from LAD-1/variant patients demonstrate the lack of NADPH oxidase activity in the
absence of phagocytosis of the unopsonized zymosan particles (left bars; n ⫽ 12 controls and n ⫽ 5 patients [following similar procedures and timing], mean ⫾ SD, at 10

Downloaded from https://ashpublications.org/blood/article-pdf/109/8/3529/1290827/zh800807003529.pdf by guest on 08 May 2020

minutes after addition of zymosan). The effect of simultaneous addition of Mg2⫹ and the zymosan particles indicates the potential to react in a CR3-dependent manner (right
bars).

To date, several issues remain to be clarified. In vitro studies in
the 3 reports on LAD–Glanzmann-like patients showed the involve-
ment of the ␤1 integrin in 2 of the patients, as was defined by
several test systems (Table 2). This may hold true for all cases of
LAD-1/variant syndrome but only to variable degrees. The possibil-
ity of a ␤1 integrin dysfunction cannot be excluded, but, if present,
it may be assumed to be subtle for multiple reasons. First, it seems
not to be an apparent finding in the whole group of clinically
identical patients, but this may depend on the tests performed1
(T.W.K., unpublished data, May 2004, October 2005, and January
2006). Second, ␤1 integrin involvement is expected to result in a
more outspoken or lethal syndrome when these ␤1 integrin–
dependent adhesive processes would also fail in the nonhematopoi-
etic cells.33,34 The fact that the ␤3 integrins in platelets are involved
without injury to the vasculature due to ␣v␤3 dysfunction makes us
believe that the defect is largely restricted to the hematopoietic cell
lineage. The successful BMT in one of the patients reinforces this
notion. Finally, in the absence of ␤1 integrin function, a relatively
normal hematopoiesis may be present,35 but lymphocyte trafficking
would be severely affected when both the LFA-1 (CD11a/CD18)
and VLA-4 (CD49d/CD29) integrins would be functionally im-
paired.36 Such a scenario, in which the integrin activation step to
both ␤1 and ␤2 integrin function is strongly impaired, can be
expected to result in a more severe combined immunodeficiency
with recurrent or fatal viral and Pneumocystis jiroveci infections.
CMV has been noted by us and by Harris et al2 during early
infancy, but this did not result in severe disease or death. Severe
viral disease was absent in our series of patients. The normal BCG
vaccination responses also excluded serious cellular immune
defects in the cytokine-activation loop involving IL-12 (and IL-23)
and interferon-gamma (IFN-␥).37
Of interest, Alon and colleagues have suggested that the
syndrome (called LAD-III by Kinashi et al5) is caused by an
aberrant signaling of Rap1. This hypothesis would fit with the data
on Rap1 activity in adhesion.6 In 1989, Kitayama38 was the first to
describe Rap1 by its inhibition of the transforming activity of
K-Ras. In 2001, Bos et al showed for the first time that Rap1
overcomes the active K-Ras effect by integrin function regulating
adhesion and spreading, which was suggested to prevent transfor-
mation.6 Rap1 activation occurs in response to a variety of stimuli,
including CD31 stimulation,39 T-cell receptor engagement,40 and
chemokines.41
Rap1 has 2 isoforms, Rap1a and Rap1b, which differ in only a
few amino acids. Rap1a/b are encoded by 2 Rap1 genes with more
than 95% homology.6 The 2 Rap2 proteins are 65% homologous
with the Rap1 proteins. While Rap1a proteins are ubiquitously
expressed in tissues, Rap1b is the predominant Rap1 isoform and
most abundant Ras family member in platelets.42 Recent gene

Table 2. Neutrophil characteristics and discordant features with similar patient reports
LAD-1 variant LAD-1 classical LAD-2 Harris et al2 McDowall et al3 Alon et al (LAD-3)4

WBC count 10-30 50-120 ND ND ND ND

% Neutrophils 60-90 60-90 ND ND ND ND
CD11/CD18 expression N Absent N N N N
CD18 DNA sequence N Mutated N N ND ND
GPCR-induced integrin activation Absent Absent N Absent Absent Absent
Chemotaxis Absent Absent Reduced? Absent ND N*
PMA-induced adhesion Reduced Absent N Reduced Reduced N
STZ-induced oxidase N 50% ND ND ND ND
Zym-induced oxidase Absent Absent ND ND ND ND
Spreading in cell track assay (ICAM) N Absent ND ND ND ND
Random migration in cell track Absent Absent ND ND Normal (T cells) ND
Consanguinity (region) Yes (Anatolia) Yes (worldwide) Yes (Arab/Brazil) No (United States) No (Malta) Yes (Arab)
Discordant features ND No bleeding No bleeding; PCP infection ND ND
retardation
Special remarks ND ND Fucosylation defect ␤1 defect† ␤1 defect† ␤1 defect†

ND indicates not described.

*T cells; PMNs also normal (N) for C5a when tested in Amsterdam.
†␤1 integrin defect according to the authors (Harris et al2; McDowall et al3; and Alon et al4).
3536 KUIJPERS et al BLOOD, 15 APRIL 2007 䡠 VOLUME 109, NUMBER 8

deletion experiments have indicated that at least 40% of rap1b⫺/⫺
murine embryos die due to bleedings in utero or perinatally,43
similar to the mouse integrin ␤3 subunit knock outs.44 The
surviving rap1b-null mice were smaller but appeared otherwise
normal, without overt hematologic defect or malignancy and with a
normal lifespan. Bleeding time and platelet aggregation responses
to various stimuli were abnormal. The reduced aggregation re-
sponse was overcome at higher doses of collagen, which differs
from the responses observed in the patients described in this study1
(T.W.K., unpublished observations, October 2005 and January
2006). Rap1a activity is undetectable in platelets, and rap1a-null
platelets show normal aggregation.43 Finally, Rap2 is present in
human neutrophils.12 Its activity and function are not exactly
known,41,45,46 but seem unaffected by chemokines or other forms of
activation (N.K., D.R., T.W.K., unpublished observations, March
sensitivity and reproducibility is a large diagnostic improvement.
In the presence of normal ␤2 integrin expression levels on all
leukocytes and normal up-regulation of these integrins upon
activation on neutrophils, but abnormal neutrophil chemotaxis, the
zymosan test applied is robust and can easily discriminate the
functional defect of such patients (Table 2).
The test is based on the lack of activation of CR3 in LAD-1/
variant neutrophils in response to an external stimulus (ie, unopso-
nized bare zymosan). While in neutrophils from healthy controls,
CR3 is activated over a period of 10 to 12 minutes in response to
the presence of zymosan, leading to uptake of the particle and
NADPH oxidase activation; this activation is not seen in LAD-1/
variant neutrophils. The nature of the signal that leads to CR3
activation is not clear. Vetvicka et al52 described the involvement of
the lectin site in CD11b in priming of the receptor, while also TLR2

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and May 2004, February and October 2005).
In conclusion, apart from the findings in a single Epstein-Barr
virus (EBV)–transformed B-cell line from the patient described by
the group of Alon (Kinashi et al5), there is little evidence for
defective Rap1 activation in any of the patients described thus far
with the LAD-1/variant syndrome.
The molecular defect in LAD-1/variant syndrome is as yet
unknown. Many molecules involved in signal transduction have
been identified in regulating integrin activity, but only limited
evidence exists on the signaling pathways that control the inside-
out signaling. In this respect, the Rho family GEF Vav1 is of
interest.47,48 There are 3 homologues that become activated via
membrane recruitment and phosphorylation by Src or Syk tyrosine
kinases. However, Vav was normally expressed in our patients on
Western blotting, as also reported by McDowall et al,3 and showed
normal phosphorylation upon neutrophil activation (data not shown).
Also, the upstream kinase Syk is unlikely because of the normal
neutrophil migration in vitro and in vivo in murine syk⫺/⫺ bone
marrow chimeras,49 together with our findings on normal Vav and
phospho-Vav in patients’ neutrophils. Candidate actin-binding
integrin partners, such as talin, filamin, and calreticulin, associate
with all the activation-impaired leukocyte and platelet inte-
grins.31,50,51 Although these key integrin adaptors are not restricted
to cells of the hematopoietic lineage, it remains possible that
homologous cell-specific adaptors may be defective in LAD-1/
variant syndrome.
The LAD-1/variant syndrome can be easily defined by a simple
screening test as described in this paper, instead of using the
detection of activation epitopes as experimental evidence. The
latter test has been found not sufficiently sensitive and highly
variable, and hence, it cannot be used as the gold standard. The
important advantage of the present screening test with respect to
and Dectin-1 can respond to zymosan and are present on neutro-
phils. TLRs have been described before to be able to activate CR3
in response to pathogens,53 and are therefore good candidates to
take part in the activation of CR3 in the zymosan test. Dectin-1 is
also a very interesting molecule in this respect, as it has been shown
to function as a phagocytic receptor for zymosan in mice,
independent of integrins.54 However, from our results and previous
reports,55 it is apparent that CR3 is indispensable for the uptake of
zymosan by human neutrophils.
In conclusion, the study of all the molecules that may be
involved in integrin activation was based on an educated guess
about the causative gene defect. Family studies will be required for
genetic linkage analysis. The availability of a good screening test
makes the diagnosis of patients suffering from an identical
syndrome easier and may help to pin down the defect.
Acknowledgments
This work was financially supported by the Landsteiner Foundation
for Blood Transfusion Research.
We are grateful to Drs A. Metin, S. Berrak, and E. Ozek for their
help and collaboration in our studies. We thank Drs K. Reedquist
and J. Bos for their helpful suggestions.
Authorship
Conflict-of-interest disclosure: The authors declare no competing
financial interests.
Correspondence: T. W. Kuijpers, Emma Children’s Hospital,
Academic Medical Center (Rm G8-205), Meibergdreef 9, 1105 AZ
Amsterdam, the Netherlands; e-mail: t.w.kuijpers@amc.uva.nl.

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