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Blood, Vol. 94 No. 3 (August 1), 1999:
pp. 884-894
Loss of Endothelial Surface Expression of E-Selectin in a
Patient With Recurrent Infections
By
Horace M. DeLisser,
Melpo Christofidou-Solomidou,
Jing Sun,
Marian
T. Nakada, and
Kathleen E. Sullivan
From the Pulmonary and Critical Care Division, University of
Pennsylvania Medical Center, Philadelphia, PA; Centocor, Malvern, PA;
and the Division of Immunologic and Infectious Diseases, Children's
Hospital of Philadelphia, Philadelphia, PA.
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ABSTRACT |
Neutrophil accumulation at sites of inflammation is mediated by
specific groups of cell adhesion molecules including the  2 (CD18)
integrins on leukocytes and the selectins (P- and E-selectin on the endothelium and L-selectin on the leukocyte). This is supported by studies of patients with leukocyte adhesion deficiency syndromes whose leukocytes are genetically deficient in the expression of 2
integrins or selectin carbohydrate ligands (eg,
sialyl-Lewisx). However, inherited deficiency or
dysfunction of endothelial cell adhesion molecules involved in
leukocyte recruitment has not been previously described. In this report
we describe a child with recurrent infections and clinical evidence of
impaired pus formation reminiscent of a leukocyte adhesion deficiency
syndrome, but whose neutrophils were functionally normal and expressed
normal levels of CD18, L-selectin, and sialyl-Lewisx. In
contrast, immunohistochemical staining of inflamed tissue from the
patient showed the absence of E-selectin from the endothelium, although
E-selectin mRNA was present. However, E-selectin protein was expressed
as significantly elevated levels of circulating soluble E-selectin were
detected, the molecular size of which was consistent with a
proteolytically cleaved form of E-selectin. Gene sequencing failed to
show evidence of a secreted mutant variant. These data represent, to
our knowledge, the first description of a potentially inherited
dysfunction of an endothelial cell adhesion molecule involved in
leukocyte recruitment and provide additional human evidence of the
importance of endothelial selectins in the inflammatory response.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE RECRUITMENT of neutrophils out of the
circulation, and across the vascular endothelium into extravascular
sites of infection or injury, involves a cascade of events mediated by
soluble inflammatory mediators and specific groups of cell adhesion
proteins.1-5 The process begins with the leukocyte rolling along the surface of the endothelium, a step that has been classically described as mediated by the selectins (L-selectin on the neutrophil and P- and E-selectin on the endothelium) and their carbohydrate-rich counterligands. As the neutrophil rolls, L-selectin is shed and its
2 (CD18) integrins are activated by at least one of a variety of
chemokines and chemoattractants that associate with the endothelial surface.6 Leukocyte activation results in arrest of rolling and firm adhesion of the leukocyte to the endothelium, events that
result from the binding of the 2 integrins expressed on the
leukocytes to the immunoglobulin (Ig) superfamily members, ICAM-1 and
ICAM-2, expressed on the endothelium. Once the leukocyte has adhered to
the endothelium, it migrates over the luminal surface, locates an
endothelial intercellular junction and then "squeezes" between
endothelial cells to enter the extravascular space. Although not well
defined, there is evidence to suggest that the 2 integrins, ICAM-1,
and PECAM-1 participate in this final step of transendothelial migration.1
Evidence supporting this paradigm has come from investigations of
patients with leukocyte adhesion deficiency (LAD) syndromes who are
genetically deficient in the expression of 2 integrins (LAD type
1)7 or selectin carbohydrate ligands such as
sialyl-Lewisx (LAD type 2)8,9 on the surface of
their leukocytes. Affected patients suffer from severe recurrent
bacterial infections, typically without pus formation, despite markedly
elevated circulating neutrophil counts. Although these patients are
phenotypically similar, in vivo studies of chemoattractant-induced
neutrophil recruitment have shown that the neutrophils from the 2 groups are functionally different. Specifically, neutrophils from
patients suffering from LAD type 1 showed normal rolling behavior but
failed to adhere or emigrate across vascular endothelium in response to
chemotatic stimulation under flow or static conditions.7 In
contrast, neutrophils from patients with LAD type 2 rolled poorly and
failed to stick to the vasculature during flow, but were observed to adhere or emigrate under static conditions.8,9 Recently, a
novel syndrome designated LAD-1/variant (or LAD-1b) has been described
with clinical features consistent with classic LAD type 1 but with
normal 2 integrin levels. In the patient reported, leukocyte
activation did not trigger the conformational changes required for
converting 2 integrins to an activated high-avidity ligand-binding
state.10
Although deficiencies of leukocyte adhesion proteins involved leukocyte
emigration have been described as outlined above, to date inherited
deficiency or dysfunction of endothelial cell adhesion molecules
involved in leukocyte recruitment have not been previously reported. In
this report, we describe a patient with recurrent infections and
clinical evidence of impaired pus formation suggestive of a LAD
syndrome but whose neutrophils were functionally normal and expressed
normal levels of CD18, L-selectin, and sialyl-Lewisx
(sLex). However, analysis of inflamed tissue from this
patient showed markedly decreased endothelial surface expression of
E-selectin in conjunction with increased levels of circulating soluble
E-selectin. Based on these data, we speculate that in this patient
there may be upregulation of proteolytic activity at the surface of the vascular endothelium that results in markedly accelerated release of
E-selectin from the endothelial surface. This represents, to our
knowledge, the first description of a potentially inherited dysfunction
of an endothelial cell adhesion molecule involved in leukocyte
recruitment and provides further human evidence of the role of
endothelial selectins in the accumulation of leukocytes at inflammatory sites.
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MATERIALS AND METHODS |
Patient history.
The patient presented to us at 10 years of age after a long
history of bacterial infections. She developed omphalitis with Pseudomonas species at 5 weeks of age. In the first year of
life, she additionally had mastoiditis and adenitis both requiring
surgical drainage, as well as otitis media multiple times. Between 2 and 5 years of age she was intermittently on prophylactic antibiotics. In that period she had cervical adenitis twice and an uncomplicated varicella infection. Between the ages of 5 and 10 years, she was on
constant antibiotic prophylaxis. During that time she developed septic
arthritis twice, multiple episodes of impetigo, periorbital cellulitis,
Pseudomonas cellulitis, and sepsis twice (Haemophilus influenza and Escherichia coli, respectively).
The patient's family history is remarkable only for a previous sibling
died at 32 weeks of gestation of a staphylococcal infection of the
fetus, amniotic fluid, and placenta. The patient also has 2 half-sisters who are completely well. There is no history of recurrent
infections in either parent or more distant relatives.
At 10 years of age, she developed a rapidly progressive gangrene with
Clostridium septicum involving her right lower extremity. After
surgical debridement, she was left with an infected, nonhealing ulcer
that failed to respond to prolonged intravenous antibiotics and a
muscle flap procedure. Biopsy of the ulcer showed very modest numbers
of neutrophils in small nests of cells. Eventually, a below-the-knee amputation of her right leg was performed. She has had a
mild neutropenia (absolute neutrophil count [ANC], 400 to 600 cells/µL) since birth, with appropriate increases in
response to infection. Cyclic neutropenia was not shown. Following her amputation, she was treated with granulocyte colony-stimulating factor
to maintain an ANC of approximately 7,000 cells/µL and trimethoprim-sulfamethoxazole prophylaxis. Despite these interventions, in the 2 years since her amputation she has continued to have multiple
bouts of infection including episodes of cellulitis (4 times),
pyelonephritis (3 times), and dental abscesses (2 times).
Although she is capable of generating pus and inflammatory infiltrates,
clinically the magnitude is much less than what is typically seen,
particularly in the early stages of an infection. For example, at 11 years of age she developed a Klebsiella pyelonephritis with
flank pain, fever and greater than 200,000 colonies of pure K
pneunomiae, and no white blood cells on the initial urinalysis. However, repeat urinalysis 20 hours later subsequently showed a modest
number of white blood cells with decreasing bacterial counts.
Furthermore, her skin and dental abscesses have always been associated
with fever and pain, but typically only very modest swelling and/or
fluctuance have been observed with these processes.
An immunologic evaluation11,12 was performed during a
period of good health when she was 10 years old (Table
1). Standard immunologic evaluations of
neutrophil function were normal as were standard laboratory evaluations
of antibody production and function. T-cell function was intact with
decreased proliferative responses to diphtheria and tetanus that were
not considered to be clinically significant. CD4 counts were mildly
reduced. Neutrophil chemotaxis was performed according to standard
protocols using 2.5 × 107 neutrophils per assay. A
control was run simultaneously and migration toward 10 5
mol/L f-met-leu-Phe was measured. Random migration toward buffer was
subtracted. Neutrophil killing was tested by incubating 3 × 106 neutrophils/mL with log-phase growth S
aureus. Aliquots were removed at 0, 30, 60, 90, 120 minutes and
plated onto blood agar plates according to standard protocols. Colony
counts were generated the next day. Results within 50% to 150% of the
normal control are considered normal. Proliferative responses to
phytohemagglutin, pokeweed mitogen, and Con A were measured in
triplicate cultures of 3 dilutions of stimulus obtained 72 hours after
stimulation. Responses to recall antigens (diphtheria, candida, and
tetanus) were also measured in triplicate cultures with 2 dilutions of each stimulus. These cultures were obtained at 7 days. Lymphocyte subset analyses and proliferative responses were determined in the
Clinical Immunology Laboratory at The Children's Hospital of
Philadelphia. A bone marrow biopsy performed during a period of health
showed normal cellularity for her age. The myeloid to erythroid ratio
was 2:1. All lineages were present. HIV serological evaluations were
negative. Informed consent was obtained for all studies described.
Neutrophil isolation and labeling.
Human neutrophils (PMNs) were isolated from EDTA anticoagulated blood
by density gradient centrifugation using MonoPoly Resolving Medium
(Flow/ICN; Biomedical, Aurora, OH).13 Isolated PMNs were washed once with Hanks' balanced salt solution (HBSS) without calcium
and magnesium (JRH Biosciences, Lanexa, KS) and resuspended in buffer
as indicated.
Binding of labeled antibodies.
PMNs were resuspended in HBSS with calcium to a concentration of 1 × 107 cell/mL and transferred to a 96-well plate (0.1 mL/well). PMNs were primed with buffer or 2 ng/mL tumor necrosis
factor- (TNF-; Genzyme, Cambridge, MA) for 45 minutes at 37°C
and then activated for 30 minutes at 37°C with either buffer or FMLP
(Sigma, St Louis, MO) at 10 µmol/L. PMNs were washed once with 200 µL HBSS with calcium and incubated with 50 µL of 5 µg/mL of
125I-labeled anti-L-selectin (Immunotech, Westbrook, ME),
anti-CD11b (AMAC, Westbrook, ME), anti-CD18 (clone CLB54; Centocor,
Malvern, PA) or anti-PECAM-1 (Centocor) for 30 minutes at room
temperature. PMNs were washed twice with 100 µL HBSS with calcium,
solubilized with 100 µL 1N NaOH and counted in a gamma counter.
Fluorescence-activated cell sorting (FACS) analysis.
Human neutrophils were treated with mouse anti-human sLex
(clone 2H5; PharMingen, San Diego, CA) and mouse
anti-human PSGL-1 (clone 3E2.25.5; Immunotech, Westbrook, ME) for 1 hour at 4°C. The primary antibody was then removed, the cells washed
with phosphate-buffered saline (PBS) and a 1:200 dilution of
fluorescein isothiocyanate-labeled goat anti-mouse (Cappell
Laboratories, West Chester, PA) was added for 30 minutes
at 4°C. After washing in PBS, flow cytometry was performed using an
Ortho Cytofluorograph 50H cell sorter equipped with a 2150 data
handling system (Ortho Instruments, Westwood, MA).
Neutrophil adhesion to selectin IgG coated plates.
P-selectin-IgG and E-selectin-IgG fusion proteins (10 µg/mL in
HBSS), produced as previously described,14 were captured on
anti-human IgG Fc specific (Jackson Immuno Research, West Grove, PA)
coated 96-wells. Plates were blocked with 5 mg/mL human serum albumin
in HBSS before use. PMNs were fluorescently labeled with BCECF (Molecular Probes, Eugene, OH) as previously
described12 and diluted to 2 × 106 cells/mL
in HBSS with calcium. PMNs (100 µL per well) were added to the
selectin fusion protein coated plates and incubated for 30 minutes at
room temperature. Unbound cells were removed by two 150 µL washes
with HBSS. Specificity of this assay has been previously shown with
selectin-specific blocking antibodies.14
Neutrophil endothelial adhesion.
Human umbilical vein endothelial cells (HUVEC) (Cell Systems, Kirkland,
WA), passage 4 were grown to confluence in 96-well tissue culture
plates. Where indicated, cells were stimulated with 2 ng/mL TNF- in
HUVEC media. PMNs were isolated and resuspended in 5 mL HBSS without
calcium and radioactively labeled with 0.5 mCi 111Indium
(DuPont/NEN, Boston, MA) in the presence of 0.4 mmol/L tropolone
(Sigma) for 15 minutes at 37°C. PMNs were washed twice with HBSS
without calcium and resuspended to 2 × 106 cells/mL in
HBSS with calcium. PMNs (100 µL per well) were added to HUVEC plates
and phorbol myristate acetate (PMA) (Sigma) at 1 µg/mL
final or fMLP 10 µmol/L final were added if indicated. Plates were
incubated 37°C for 30 minutes. Unbound PMNs were removed with 2 × 150 µL washes with HBSS. Cells were solubilized with 100 µL 1N NaOH
and counted in a gamma counter. The number of PMNs bound was calculated
using the specific activity of the cells.
Neutrophil transendothelial migration.
HUVEC at passage 4 were seeded onto 24-well fibronectin-coated 3.0 µm
Transwell inserts (Becton Dickinson, Bedford, MA) and grown to
confluence. HUVECs were stimulated on the basolateral side with 2 ng/mL
TNF- for 4 hours. The inserts and underlying wells were rinsed to
remove TNF- and fresh media was added to the top and bottom
chambers. 111Indium-labeled PMNs (see above) were
resuspended to 1 × 107 cells/mL and 100 µL added to the
top chamber of the transwell. Plates were incubated for 90 minutes at
37°C and PMNs that had traversed into the bottom chamber were counted
in a gamma counter. The number of PMNs that had transmigrated was
calculated using the specific activity of the cells.
Neutrophil chemotaxis.
The chemotaxis assay was performed similarly to the transmigration
assay, but the HUVEC monolayer was not activated with TNF-. PMNs
were added to the top of the HUVEC transwells and fMLP (10 µmol/L)
was added to the bottom chambers. PMNs responding to the chemotactic
stimulus and migrating to the lower chamber were quantified as described.
Short-term skin organ culture.
Skin organ culture was adapted from previously described
methods.15 Patient skin acquired through punch biopsy and
neonatal foreskin from elective circumcisions were immediately placed
in RPMI 1640 medium supplemented with 10% fetal calf serum, 1%
penicillin-streptomycin and 10 mmol/L L-glutamine. Skin specimens were
cut into smaller pieces (2 × 2 mm in size), placed in 6-well
culture plates, partially submerged in 0.5 mL of media with dermal side
down and epidermis uncovered and incubated overnight for 18 to 24 hours
at 37°C in 5% CO2-humidified air. This overnight
incubation allowed for resolution of any changes in endothelial cell
adhesion molecule expression because of the physical trauma of
obtaining and preparing the skin samples. On the following day the
original media was replaced with fresh media without or with TNF-
(12,000 U/mL; Boehringer Mannheim, Indianapolis, IN) and
incubated for 8 hours at 37°C in 5% CO2-humidified air.
In separate experiments these were determined to be sufficient induce
ICAM-1 and E-selectin expression in adult and neonatal control skin.
After this incubation the skin explants were snap frozen in O.C.T.
embedding compound (Tissue-Tek/Sakura, Torrance, CA) and processed for
immunohistochemical staining.
Immunohistochemical staining.
Surgically removed tissue and explants from skin organ cultures were
snap frozen in O.C.T. compound and subjected to immunohistochemical staining with light counterstaining with hematoxylin as previously described.16 Antibodies used17 were 1)
anti-human PECAM-1 (Immunotech, Westbrook, ME); 2) anti-E-selectin
antibodies and P-selectin antibodies (Becton Dickinson Advanced
Cellular Biology, San Jose, CA); 3) anti-E-selectin
antibodies HEL 3/2 (provided by Drs Dale Cummings and Tim Ahern of
Genetics Institute, Cambridge, MA) and ES1 (provided by Rodger
McEver, University of Oklahoma, Oklahoma City, OK); and 4) anti-ICAM-1
and anti-Mac-1 (PharMingen). Antibody concentrations used for
staining ranged from 1 to 10 µg/mL.
Reverse transcription-polymerase chain reaction (RT-PCR),
single-strand conformation polymorphism (SSCP) analysis, and
gene sequencing.
Total RNA was extracted from surgical skin tissue,18
incubated at 65°C for 10 minutes and then used to synthesize cDNA. cDNA was obtained by RT using an oligo d(T)16, M-MLV
Reverse Transcriptase (Perkin-Elmer-Cetus, Norwalk, CT). To amplify the
lectin-binding and epidermal growth factor-like domains,19
reverse transcribed cDNA was subjected to PCR using a sense primer
(5'-CACAGATCTGGTCTTACAACACCTCCA-3') and an antisense primer
(5'-TTTACACGTTGGCTTCTCGTT-3'). For the SSCP analysis, cDNA from
surgical tissue was amplified using overlapping primers (available on
request) to generate PCR products of approximately 400 bp in the
presence of 35S. These products were run on a nondenaturing
polyacrylamide gel to screen for mutations.20,21 Two coding
region polymorphisms/mutations were identified using this strategy.
Based on this, the patient's E-selectin gene was fully sequenced in
both directions from PCR products specific for each exon amplified from
genomic template (available on request). Mutations were confirmed using
separate DNA preparations from the patient and her parents.
Amplification protocols for each primer pair are available on request.
Measurement of soluble E-selectin.
The concentration of soluble E-selectin present in patient and control
serum was determined by enzyme-linked immunosorbent assay (ELISA) using
a commercially available assay kit (R&D Systems, Minneapolis, MN)
according to manufacturer's instructions.
Immunoblot analysis.
The molecular species of soluble E-selectin present in patient and
control serum was determined by immunoblot analysis with the
anti-E-selectin monoclonal antibody, 1.2B6 (Immunotech) using previously described procedures.22
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RESULTS |
Expression of leukocyte cell adhesion molecules.
Because the clinical features of this patient were suggestive of a
leukocyte adhesion deficiency, the surface expression of cell adhesion
molecules on neutrophils known to be involved in leukocyte emigration
was investigated. The expression L-selectin, CD18, and PECAM-1 on the
patient's neutrophils was initially assessed and was noted to be
comparable with the expression found on neutrophils from normal control
donors (Fig 1). Stimulation with
inflammatory mediators (TNF- and fMLP) decreased the expression of
L-selectin and increased the surface level of CD18 as has been
previously reported,23 suggesting that for neutrophils,
these proteins are regulated normally in this patient.
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| Fig 1.
Neutrophil binding of 125I-labeled
antibodies. Neutrophils (1 × 106) from the patient and
2 normal donors, unactivated (control,  ) or activated with the
combination of TNF- and fMLP (TNF/fMLP,  ), were incubated with
125I-labeled antibodies against L-selectin, CD18, and
PECAM-1 in a 96-well plate. The expression of these cell adhesion
molecules was assessed by the binding of labeled antibody as determined
by the total counts per well of cells. The expression of L-selectin,
CD18, and PECAM-1 on the patient's neutrophils were comparable with
the expression found on neutrophils from normal control donors and
stimulation with TNF- and fMLP decreased the expression of
L-selectin and increased the surface level of CD18 as has been
previously reported. These data are representative of 3 experiments
done in triplicate in which the patient was compared with 2 normal
donors and in which different normal donors were used in each
experiment.
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Normal levels of sLex, the carbohydrate
binding motif present on selectin ligands, and P-selectin glycoprotein
ligand 1 (PSGL-1) were detected on neutrophils from the
patient (Fig 2). To evaluate further the neutrophil ligands for endothelial selectins, neutrophils from the patient and normal donors were allowed to adhere to
immobilized P- and E-selectin IgG chimeric proteins. No significant
differences in neutrophil adhesion to immobilized endothelial selectin
was noted between the patient and normal donors (Fig
3), suggesting that the neutrophil ligands
for P- and E-selectin are present and functional in the patient.
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| Fig 2.
Neutrophil expression of sLex and PSGL-1. The
surface expression of sLex (A and C) and PSGL-1 (B and D)
on neutrophils from a normal donor (A and B) and the patient (C and D)
was determined by FACS analysis. Filled and unfilled tracings
represent, respectively, the background staining and staining for the
targeted protein. For the normal donor and the patient comparable
levels of expression of sLex and PSGL-1 were noted.
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| Fig 3.
Neutrophil binding to immobilized selectin-IgG chimeric
proteins. 111Indium-labeled neutrophils from the patient
and 2 normal donors were added to uncoated wells () or wells coated
with P- () or E-selectin IgG ( ) chimeric proteins incubated at
37°C for 30 minutes and the number of adherent cells per well after
washing was determined following incubation. No significant differences
in neutrophil adhesion to immobilized endothelial selectin was noted
between the patient and normal donors. These data are representative of
2 experiments done in triplicate in which the patient was compared with
2 normal donors and in which different normal donors were used in each
experiment.
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Neutrophil endothelial adhesion and transendothelial migration.
The finding of normal neutrophil expression of CD18 and selectin
ligands did not exclude the possibility of functional disturbances in
these molecules that may interfere with the ability of the patient's
neutrophils to interact with the endothelium. We therefore studied the
adhesion of unactivated neutrophils to cytokine-stimulated endothelium,
or activated neutrophils to unstimulated endothelium. In these studies,
normal endothelial cells (HUVEC) were used. For the patient, adhesion
of unactivated neutrophils to 4- and 24-hour TNF--stimulated
endothelium, as well as adhesion of fMLP- or PMA-treated neutrophils to
quiescent endothelium was intact (Fig 4).
Adhesion stimulated by fMLP and PMA was predominately mediated by CD18
binding, as evidenced by the effectiveness of a blocking anti-CD18
antibody. Adhesion to TNF--stimulated endothelial cells was only
partially blocked by anti-CD18, because E-selectin also mediates
adhesion to these cells.
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| Fig 4.
Neutrophil adhesion to endothelial cells. The adhesion of
111Indium-labeled neutrophils from the patient and 2 normal
donors, without () or with () anti-CD18 antibody, to monolayers
of normal endothelial cells (HUVEC) was studied in 2 ways. Neutrophils
were activated with fMLP (106 mol/L) or PMA (2 µg/mL)
and added to unstimulated endothelium or unstimulated neutrophils were
added to endothelium stimulated with TNF- (2 ng/mL) for 4 hours or
24 hours. Neutrophils bound to the endothelium per well was determined.
Control indicates the adhesion of unactivated neutrophils to
unstimulated endothelial cells. In these multiple conditions, adhesion
of the patient's neutrophils to endothelium did not differ
significantly from that of the normal donors. These data are
representative of 3 experiments done in duplicate or triplicate in
which the patient was compared with 2 normal donors and in which
different normal donors were used in each experiment.
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Neutrophil transendothelial migration was also investigated in a
migration assay involving HUVEC grown on transwell inserts. In these in
vitro studies, it was observed that neutrophil migration across
TNF--stimulated HUVEC was preserved in this patient (Fig 5). Neutrophil chemotaxis through HUVEC in
response to fMLP was also intact (data not shown). Taken together,
these studies suggest that the patient's neutrophils are able to
appropriately engage normal endothelium.
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| Fig 5.
Neutrophil transendothelial migration. The ability of
111Indium-labeled neutrophils from the patient and 2 normal
donors to migrate across unstimulated endothelial cells (), or
endothelial monolayers stimulated previously for 4 hours with TNF-
(2 ng/mL) (), was studied. Normal endothelial cells (HUVEC) were
used. After 90 minutes of incubation at 37°C, transendothelial
migration of the patient's neutrophils was equivalent to or exceeded
that of normal donors. These data are representative of 3 experiments
done in duplicate or triplicate in which the patient was compared with
2 normal donors and in which different normal donors were used in each
experiment.
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Expression of endothelial cell adhesion molecules from inflamed
tissues.
After the patient's amputation (see Patient history), tissue from the
necrotic muscle flap, the nonhealing ulcer and the surgical margin were
obtained and processed for immunohistochemical staining. The
availability of this tissue provided the opportunity to evaluate the
expression of cell adhesion molecules on the endothelium of her
vasculature known to be involved in leukocyte recruitment. Immunohistochemical staining of multiple (>6) tissue sections from
three distinct sites were performed. The margins of the ulcer showed
that although PECAM-1, ICAM-1, and P-selectin were expressed on the
vasculature, no expression of E-selectin was detectable on the vessels
(large or small) from inflamed tissue (Figs 6 and 7). Staining
with 3 different anti-E-selectin antibodies gave similar results.
There was also patchy staining for VCAM-1 on her endothelium (data not
shown). Figure 8 shows serial sections of
tissue from the junction between necrotic and viable tissue. In this
obviously inflamed tissue, as evidenced by the presence of Mac-1
positive leukocytes (neutrophils and monocytes) and intense neovascularization, no expression of E-selectin was noted on the vasculature. In contrast, staining of tissue from the margins of
chronically infected nonhealing wounds/ulcers located on the lower
extremities of 2 diabetics who were at risk for amputation showed the
expression of E-selectin on vessels in these tissues (Fig
9). Of note, although our patient appeared
clinically to manifest less pus formation, the presence of
extravascular leukocytes as indicated in Fig 7, suggests that her
leukocytes still had some capacity to migrate into sites of infection
or injury.
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| Fig 6.
Expression of endothelial cell adhesion molecules in
inflamed tissues. Serial tissue sections from visually and
histologically inflamed ulcerated tissue removed at the time of the
patient's below-the-knee amputation were stained immunohistochemically
with antibodies against PECAM-1 (A), ICAM-1 (B), P-selectin (C), and
E-selectin (D). Two vessels (long and short arrows) are identified by
staining with antibody against PECAM-1. Although ICAM-1 expression (B,
long arrow) and P-selectin expression (C, short arrow) were detected on
the vasculature, no expression of E-selectin was detected on these or
other vessels in the surrounding tissue. Staining with 3 anti-E-selectin antibodies gave similar results.
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| Fig 7.
Expression of P-selectin and E-selectin in inflamed
tissue. Immunohistochemical staining of serial tissue sections from
another sample of visually and histologically inflamed ulcerated tissue
different from that presented in Fig 5. Shown are sections stained with
antibodies against PECAM-1 (A) to identify the vessels, P-selectin (B),
and E-selectin (C). Although a subpopulation of the vasculature
expressed P-selectin (V and arrows), no significant expression of
E-selectin was detected on any of the vessels in this tissue.
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| Fig 8.
Expression of E-selectin in inflamed tissue.
Immunohistochemical staining of serial sections at the junction between
viable and necrotic tissue indicated by the dotted line. Sections were
stained with antibodies against PECAM-1 (A), Mac-1 (B), and
E-selectin (C). There is intense neovascularization at the
interface of viable and necrotic tissue (A) and infiltration of
Mac-1-positive leukocytes (neutrophils and monocytes) in the necrotic
tissue (B). Despite this indication of active inflammation, no
significant expression of E-selectin was detected on any of the vessels
in this tissue (arrows).
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| Fig 9.
Expression of E-selectin in a chronic ulcer.
Immunohistochemical staining of serial tissue sections of a sample of
visually and histologically inflamed tissue from the margin a diabetic
chronic nonhealing wound/ulcer. Sections stained with antibodies
against PECAM-1 (A) to identify the vessels, ICAM-1 (B), and
E-selectin (C). In this inflamed tissue, E-selectin was detected on a
subpopulation of the vasculature (arrows).
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TNF--stimulation of organ skin cultures.
To evaluate further the observations noted above and to investigate the
ability of her endothelium to upregulate E-selectin, short-term organ
cultures of control and normal skin from the patient were incubated for
8 hours with TNF- (12,000 U/mL). In unstimulated control and patient
skin (data shown for control), 10% to 20% of the vessels expressed
ICAM-1, although there was very little expression of E-selectin (Fig
10). In control skin, but not the
patient's skin, TNF--stimulation increased the expression of
E-selectin on the dermal vessels. This difference was not because of an
inability of the patient's skin to respond to TNF-, because like
control skin, cytokine-induced upregulation of ICAM-1 was observed in
dermis and on the vasculature of the patient's skin as previously
described. These data, along with that from the in situ tissue
staining, suggest that despite appropriate inflammatory stimuli there
is an absence of endothelial surface expression of E-selectin on the
endothelium of this patient.
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| Fig 10.
Expression of E-selectin in TNF--stimulated skin
organ cultures. Immunohistochemical staining of skin organ cultures
stimulated for 8 hours with TNF- (12,000 U/mL). Serial sections of
unstimulated normal skin (A through C), stimulated normal skin (D
through F) and stimulated skin from the patient (G through I) were
stained with antibody against PECAM-1 (A, D, and G) to identify the
vessels (arrows and arrowheads), ICAM-1 (B, E, and F) and E-selectin
(C, F, and I). The epidermis (Epi) is indicated for the stimulated
normal skin. In unstimulated normal skin there was some expression of
ICAM-1 (B, arrowheads), although most of the vessels did not express it
(B, double arrows), and negligible expression of E-selectin (C). A
similar pattern was seen for unstimulated skin from the patient (data
not shown). In stimulated normal skin, there was marked upregulation in
the number of vessels expressing ICAM-1 (E) and a smaller but clearly
significant increase in the number of vessels expressing E-selectin
(F). In contrast to normal skin, stimulated skin from the patient
showed upregulation of ICAM-1 on the vasculature (H) but no increased
E-selectin expression (I). The asterisk indicates dermal interstitium
in which there is also upregulate expression of ICAM-1 on the dermal
stroma particularly evident in the sample of stimulated normal skin
(E).
|
|
Expression of E-selectin message.
One possible explanation for the absence E-selectin on the endothelium,
despite appropriate inflammatory stimuli, was an absence of E-selectin
RNA transcripts. We therefore looked for the presence of E-selectin
message in inflamed and normal tissues obtained at the time of her
amputation. Total cellular RNA was isolated from ulcerated inflamed
tissue and the surgical margin and subjected to RT-PCR using primers
designed to amplify the lectin-binding and epidermal growth factor
domains of the molecule. The products generated by RT-PCR are shown in
Fig 11. PCR with these primers of
E-selectin cDNA, (lane 2) yielded the expected product of 870 bp. An
identical product was also identified in tissues samples from the
abnormal, inflamed tissue (lanes 4 and 5), confirming that message for
E-selectin was present in this patient.
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| Fig 11.
RT-PCR of surgical tissue from the patient. RT-PCR using
primers designed to amplify the lectin and epidermal growth-like
domains was conducted on total cellular RNA isolated from surgical
tissue from the patient that included normal tissue from the margins of
the surgical amputation (lane 3) and ulcerated nonhealing inflamed
tissue from two distinct sites (lanes 4 and 5). PCR with these primers
of E-selectin cDNA (lane 2) yielded the expected product of 870 bp. An
identical product was also identified in tissue samples from the
abnormal, inflamed tissue (lanes 4 and 5) but was not readily detected
in normal tissue at the surgical margin (lane 3).
|
|
Expression of E-selectin protein.
The observation that E-selectin was not detected on inflamed
endothelium, despite the presence of E-selectin message, suggested for
this patient that E-selectin was secreted or rapidly shed from the
endothelial surface. To investigate this further, we measured by ELISA
the level of soluble (s) E-selectin in sera from the patient along with
age-matched controls. Two samples of sera from the patient were
obtained when she was clinically stable without evidence of infection.
As shown in Table 2, more sE-selectin was
detected in sera from the patient compared with controls (186 ± 38
ng/mL v 90 ± 13 ng/mL). Levels of soluble ICAM-1 (sICAM-1)
and tumor necrosis factor receptor-1 (sTNF-R1) were noted to be
slightly elevated (sICAM-1, 312 ng/mL, normal range, 115 to 306 ng/mL;
sTNF-R1, 2,245 pg/mL, normal range, 749 to 1,966 pg/mL).
To determine the molecular form(s) of the sE-selectin present in her
circulation, samples of the patient's serum along with controls were
transferred to nitrocellulose and immunoblotted with an
anti-E-selectin polyclonal antibody (Fig
12). A single molecular species was
detected in the patient's serum of ~100 to 110 kD comparable in size
with the form noted in control sera and appropriately smaller than the
full-length species found in extracts from endothelial cells stimulated
with TNF-. This suggests that the molecular form of sE-selectin in
this patient's circulation results principally from the proteolytic
cleavage of surface E-selectin.
View larger version (22K):
[in this window]
[in a new window]
| Fig 12.
Western blot analysis of sera for E-selectin.
TNF--stimulated HUVEC extracts (lane 1) and sera from the patient
(lane 2) and from 2 normal donor controls (lanes 3 and 4) were
transferred to nitrocellulose and immunoblotted with an
anti-E-selectin antibody. A single molecular species was detected in
the patient's serum of approximately 100 to 110 kD, comparable in size
with the form noted in control sera and appropriately smaller than the
full-length species found in extracts from endothelial cells stimulated
with TNF-.
|
|
Sequencing of E-selectin gene.
SSCP analysis was initially used to screen for coding region mutations.
PCR products containing the CR5 and cytoplasmic 2 domains both showed
conformational changes. These PCR products were directly sequenced and
2 heterozygous mutations were identified. Based on these findings, the
patient's E-selectin gene was fully sequenced using exon-specific PCR
products in both directions. Each PCR product included at least 50 bp
from each flanking intron. The patient was found to be heterozygous for
4277C-T histidine to tyrosine missense mutation in the CR5 exon. She
was also heterozygous for a 5822T-C mutation which does not result in
any amino acid change in the cytoplasmic 2 exon. One further intronic
mutation was identified in intron 10 just after the transmembrane exon at 5334T-C. 4277T and 5822C were previously identified in cDNA sequencing.19 These may represent common polymorphisms,
although we were unable to identify either mutation in 30 controls. The intronic mutation is thus of uncertain significance. It does not lie
within any known motif involved in splice signaling, although it could
potentially affect the ability to form membrane bound E-selectin if it
did alter splicing of the transmembrane domain exon.
| |
DISCUSSION |
At sites of acute injury and infection in the systemic circulation, the
initial interaction of the intravascular leukocyte with the endothelium
consists of the tethering and rolling of leukocytes along postcapillary
venular endothelium.1-5 These early events are believed to
be largely mediated by the interactions of selectins with their
carbohydrate-rich protein ligands.24 Evidence for the
importance of these endothelial selectins comes from patients with an
inherited defect in fucose metabolism that results in an inability to
synthesize the fucosylated tetrasaccharides (eg,
sialyl-Lewisx [sLex]) that decorate selectin
ligands and mediate ligand binding.8,9 As originally
described, patients with this so-called leukocyte adhesion deficiency
type 2 suffer from recurrent bacterial infections, typically without
pus formation, despite markedly elevated circulating neutrophil counts.
In vitro, the neutrophils of these patients are unable to adhere or
roll on E-selectin, P-selectin, or cytokine or mediator-stimulated
endothelial cells. Interestingly, for one of these patients, infections
requiring hospitalization or intravenous antibiotics have not occurred
over last 5 years, although periodontitis has been a chronic problem.
This has occurred despite the fact that his neutrophils are still
deficient in SLex and are functionally
abnormal.25
Although our patient did experience recurrent infections with clinical
evidence of reduced pus formation, the patients neutrophils were not
deficient in sLex and adhered to cytokine-stimulated
endothelial cells and recombinant P- and E-selectin proteins (Figs 2
and 3). In addition, she did not show evidence for a deficiency or
dysfunction of her -2 integrins (Figs 1, 4 and 5). Our data,
however, do not exclude the possibility of more subtle defects that
might interfere with neutrophil rolling. Interestingly, unlike
previously reported patients with LAD where neutrophilia is present,
this patient showed a mild neutropenia with an ability to appropriately
respond to infection. Increasing her neutrophil counts to normal values
with G-CSF did not appear to decrease the occurrences of bacterial
infections. This latter observation, coupled with the mild degree of
neutropenia and her demonstrated ability to mount neutrophilia in
response to infection, make it unlikely that the neutropenia alone is
responsible for her severe, recurrent infections. The mechanism(s)
responsible for this neutropenia are not clear, but we speculate that
the processes which reduce the surface expression of endothelial
E-selectin may also alter the expression of other adhesion receptors on
leukocytes and the endothelium that are involved in release of
leukocytes from the bone marrow.
Although our patient did not have evidence of a leukocyte adhesion
deficiency, she did show a striking absence of E-selectin from the
surface of the endothelium despite inflammatory stimuli (Figs 6 through
8). These observations were supported by the finding that in skin organ
cultures, TNF- increased the vascular expression of E-selectin in
normal, but not patient, skin (Fig 10). E-selectin message was found in
inflamed tissues (Fig 11), and although E-selectin was not present on
the endothelium, its message was translated as evidenced by the high
levels of circulating sE-selectin (Table 2). As significant mutations
in the E-selectin gene were not detected, the absence of endothelial
E-selectin does not appear to be the result of a secreted alternatively
spliced or mutational variant. This is supported by the finding that
the molecular species of sE-selectin detected in the patient and
control sera were of the same molecular weight (Fig 12). Of note, her
serum did not inhibit the TNF-induced expression of E-selectin on HUVEC
(data not shown), suggesting that proteolytic activity in her serum was
not responsible for the loss of surface endothelial E-selectin. We
therefore propose that there may be upregulation of proteolytic activity on the surface of the vascular endothelium of the tissues we
studied that results in markedly accelerated release of E-selectin from
the endothelial surface. As the entire vasculature was not surveyed, our data do not exclude the possibility that in specific organs, such as the lung and the gastrointestinal tract, the
vasculature may be normal.
An important question is how might the loss of the endothelial surface
expression of E-selectin with an accompanying increase in circulating
sE-selectin increase the susceptibility to infection in this patient?
There are a number of possibilities. First, given the role of selectins
in the initial rolling phenomena, the loss of endothelial surface
expression of E-selectin may compromise the ability of leukocytes to
roll along the endothelium and therefore impair leukocyte emigration
into sites of inflammation. However, mutant mice deficient in
E-selectin do not show an increased incidence of spontaneous
infections, and neutrophil recruitment in response chemical-induced
peritonitis is impaired only if P-selectin is also simultaneously
blocked.26 These and other studies with selectin-deficient
mice27,28 suggest that with respect to the process of
leukocyte recruitment, E- and P-selectin may serve redundant functions,
and/or that in the setting of chronic loss each may be able to
compensate for the persistent absence of the other. Consistent with
this proposal is the demonstration in our patient of P-selectin
endothelial expression and some extravascular accumulation of
Mac-1-positive leukocytes in chronically inflamed tissues (Fig 8).
While data from mutant mice are very instructive, its is possible (and
probably likely) that the specific or relative functions of P- and
E-selectin in humans differ from that of mice, and therefore care
should be exercised in extrapolating from the murine studies.
Second, there is evidence that E-selectin may mediate other processes
that are crucial to host defenses. This is suggested by studies of
E-selectin-deficient mice subjected to systemic pneumococcal
infection.29 After intraperitoneal inoculation with S
pneumonie, mice deficient in E-selectin showed more prominent morbidity, substantially increased 10-day mortality, persistent bacteremia, and a higher bacterial load compared with wild-type mice.
These derangements occurred despite the fact that leukocyte emigration
into the peritoneal space was preserved in these mice. Leukocyte
activation may be one of these alternative processes, as interaction of
unstimulated neutrophils with E-selectin upregulates the adhesive
activity of Mac-1 (M2),30,31 and neutrophils from
transgenic mice constitutively expressing E-selectin show increased
oxidative activity.32 Therefore, in this patient, the
diminished surface expression of endothelial E-selectin may result in
the loss of important signals required for the activation of
neutrophils and their subsequent ability to kill bacteria.
The presence of markedly elevated levels of sE-selectin33
represents yet a third potential mechanism that may contribute to this
patient's increased susceptibility to infection. Cultured endothelial
cells have been shown to release E-selectin following activation
presumably as a result of proteolytic cleavage of surface E-selectin.34-36 Elevated levels of sE-selectin have been
detected in patients with diabetes, inflammatory skin disorders,
vasculitis, and scleroderma, although these levels have correlated only
weakly or not all with disease activity.37,38 High levels
of sE-selectin have also been observed in sepsis with higher
and/or persistent elevations correlating with greater
mortality and severity of disease.33,35 In our
patient, sE-selectin was determined on 2 occasions, 5 months apart,
when she was well without clinical evidence of infection and the levels
were noted to be 2 to 3 times that of age-matched
controls.39,40 Although its precise in vivo function is
unknown, sE-selectin in the blood is biologically active in that it is
able to bind to and mediate adhesion and activation of
neutrophils.30-32 Therefore the high circulating levels of
sE-selectin observed in this patient may result in inappropriate intravascular leukocyte activation or may compromise adhesive interactions with activated endothelium.
In conclusion, we have identified a patient with recurrent severe
bacterial infections and clinically impaired pus formation associated
with markedly reduced surface expression of E-selectin on inflamed
endothelium, although E-selectin message was present in this tissue and
significantly elevated levels of sE-selectin were detected in the
patient's serum. As gene sequencing failed to show any significant
mutations, these data suggest that in this patient, E-selectin is
rapidly shed from the endothelium of her vasculature resulting in
markedly decreased endothelial surface expression of E-selectin and
increased circulating sE-selectin. We speculate that this pattern of
expression may be part of a wider process in which there is increased
proteolytic activity on the endothelium. Regardless of the mechanism,
these data provide additional human evidence for the importance of
endothelial selectins in leukocyte recruitment and the ability to fight infections.
| |
ACKNOWLEDGMENT |
The authors are extremely grateful to Steven M. Albelda for his advice
and support of the work in this article. We also thank Mildred Daise,
Eugenia Argyris, Kate Veksler, Patricia Sassoli, Ann Leone, and Miae Oh
for their expert technical assistance. We are grateful to Enyi Okereke,
MD, for providing us with tissue from chronic diabetic lower extremity ulcers.
| |
FOOTNOTES |
Submitted July 22, 1998; accepted March 26, 1999.
Supported by grants from the Robert Wood Johnson Foundation Minority
Faculty Development Program (H.M.D.), National Institutes of Health
Grant No. K14 HL-03382 (H.M.D.), The Wallace Chair (K.E.S.), and the
Lupus Foundation (K.E.S.).
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.
Address reprint requests to Horace M. DeLisser, MD, 894 Maloney Bldg,
University of Pennsylvania Medical Center, 3600 Spruce St,
Philadelphia, PA 19104-4283; e-mail: delisser{at}mail.med.upenn.edu.
| |
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TOP
ABSTRACT
INTRODUCTION