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TRANSFUSION MEDICINE
Soluble CD40 ligand accumulates in stored blood components, primes neutrophils
through CD40, and is a potential cofactor in the development of
transfusion-related acute lung injury
Samina Yasmin Khan, Marguerite R. Kelher, Joanna M. Heal, Neil Blumberg, Lynn K. Boshkov, Richard Phipps, Kelly F. Gettings,
Nathan J. McLaughlin, and Christopher C. Silliman
Transfusion-related acute lung injury (TRALI)
is a form of posttransfusion acute pulmonary insufficiency that has been linked to
the infusion of biologic response modifiers
(BRMs), including antileukocyte antibodies
and lipids. Soluble CD40 ligand (sCD40L) is
a platelet-derived proinflammatory mediator
that accumulates during platelet storage.
We hypothesized that human polymorphonuclear leukocytes (PMNs) express CD40,
CD40 ligation rapidly primes PMNs, and
sCD40L induces PMN-mediated cytotoxicity
of human pulmonary microvascular endothelial cells (HMVECs). Levels of sCD40L
were measured in blood components and in
platelet concentrates (PCs) implicated in
TRALI or control PCs that did not elicit a
transfusion reaction. All blood components
contained higher levels of sCD40L than fresh
plasma, with apheresis PCs evidencing the
highest concentration of sCD40L followed
by PCs from whole blood, whole blood, and
packed red blood cells (PRBCs). PCs implicated in TRALI reactions contained significantly higher sCD40L levels than control
PCs. PMNs express functional CD40 on the
plasma membrane, and recombinant
sCD40L (10 ng/mL-1 ␮g/mL) rapidly (5 min-
utes) primed the PMN oxidase. Soluble
CD40L promoted PMN-mediated cytotoxicity of HMVECs as the second event in a
2-event in vitro model of TRALI. We concluded that sCD40L, which accumulates during blood component storage, has the capacity to activate adherent PMNs, causing
endothelial damage and possibly TRALI in
predisposed patients. (Blood. 2006;108:
2455-2462)
© 2006 by The American Society of Hematology
Introduction
CD40 is a 48-kDa transmembrane glycoprotein and a member of
the tumor necrosis factor (TNF) receptor family expressed on
endothelial and epithelial cells, monocytes, and macrophages.1
CD40 ligand (CD40L [CD154]) is a primarily platelet-derived
pro-inflammatory mediator found in soluble (sCD40L) and cellassociated forms in transfused blood.2,3 Soluble CD40L activates
macrophages and elicits the production and release of multiple
proinflammatory cytokines.4 Furthermore, inhibition of the CD40CD40L system in animal models reduces acute lung injury (ALI)
caused by endotoxin (lipopolysaccharide [LPS]) or oxygen toxicity.5-7 In addition, sCD40L is present in platelet concentrates and
accumulates over routine 3- to 5-day storage times.3
Polymorphonuclear leukocytes (PMNs) are critical in host
defense against pathogens and exert their major microbicidal
function in the tissues.8,9 PMN priming is initiated by the attraction
and adhesion of PMNs to activated vascular endothelium and
continues until the pathogens are phagocytosed and destroyed.6,10-12
PMN-mediated acute lung injury (ALI) requires at least 2 separate
events: endothelial activation, which includes the synthesis and
release of chemokines and the increased surface expression of
adhesion molecules that elicit PMN adhesion, and activation of
adherent PMNs, which causes the release of their microbicidal
arsenal and results in endothelial damage, capillary leak, and
ALI.10,11,13-16 Such a 2-event model has been proposed for ALI,
especially for transfusion-related acute lung injury (TRALI).14,16,17
The 2-event model of TRALI postulates that the first event is the
clinical status of the patient leading to pulmonary sequestration of
PMNs and that the second is the infusion of biologic response
mediators (BRMs), including antibodies directed against PMN
antigens or lipids, leading to the activation of PMNs and the release
of cytotoxic substances and PMN-mediated ALI.11,14-16 We hypothesized that PMNs express CD40 and ligation causes changes in
From the Departments of Surgery and Pediatrics, University of Colorado
School of Medicine, Denver, CO; the Departments of Transfusion Medicine,
Hematology-Oncology, Environmental Medicine, Immunology, and Lung
Biology, University of Rochester, Rochester, NY; the Department of Pathology,
Oregon Health Sciences University, Portland, OR; and Bonfils Blood Center,
Denver, CO.
design, data analyses, and manuscript preparation. N.B. provided the idea for
the manuscript with J.M.H., and provided data analyses and helped to write the
manuscript. L.K.B. obtained all samples in Edmonton (AB, Canada) and helped
in manuscript preparation. R.P. provided expertise in CD40:CD40L
interactions, provided important data analyses, and helped to write the
manuscript. K.F.G. performed the modified sCD40L ELISA in Rochester. N.J.M.
completed the microscopy. C.C.S. was instrumental in experimental design,
data analyses, and the writing of the manuscript.
Submitted April 14, 2006; accepted May 13, 2006. Prepublished online as
Blood First Edition Paper, June 13, 2006; DOI 10.1182/blood-2006-04-017251.
Supported by Bonfils Blood Center, the National Heart, Lung and Blood
Institute (grants HL59355-06 and HL078603), the National Institute of General
Medical Sciences (grant P50 GM49222-12), and the United States Public
Health Service (grants ES01347 and DE0113910).
All authors contributed significantly to this article. S.Y.K. performed most of the
experiments at Bonfils Blood Center and wrote the article. M.R.K. performed
some experiments and helped to write the article. J.M.H. performed some of
the experiments in Rochester and provided substantive input for experimental
BLOOD, 1 OCTOBER 2006 䡠 VOLUME 108, NUMBER 7
An Inside Blood analysis of this article appears at the front of this issue.
Reprints: Christopher C. Silliman, Bonfils Blood Center, 717 Yosemite St,
Denver, CO 80230; e-mail: [email protected].
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2006 by The American Society of Hematology
2455
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2456
BLOOD, 1 OCTOBER 2006 䡠 VOLUME 108, NUMBER 7
KHAN et al
PMN function, that transfusion of cellular products containing
sCD40L rapidly primes PMNs, and that sCD40L can serve as a
second event in the 2-event model of TRALI, in which sCD40L
causes PMN-mediated cytotoxicity of pulmonary endothelium.
Patients, materials, and methods
Materials
All reagents, unless otherwise specified, were purchased from Sigma
Chemical (St Louis, MO). Solutions were made from sterile water for
injection (United States Pharmacopeia; Baxter Healthcare, Deerfield, IL).
All buffers were made from the following stock USP solutions: 10% CaCl2,
23.4% NaCl, 50% MgSO4 (American Reagent Laboratories, Shirley, NY);
sodium phosphates (278 mg/mL monobasic and 142 mg/mL dibasic), and
50% dextrose (Abbott Laboratories, North Chicago, IL). Furthermore, all
solutions were sterile-filtered with Nalgene MF75 series disposable sterilization filter units purchased from Fisher Scientific (Pittsburgh, PA). FicollPaque was purchased from Amersham Biosciences (Piscataway, NJ).
Plastic microplates, manufactured by Nunc, T-25 tissue-culture flasks,
12-well plates, and sterile pipettes were purchased from Life Sciences
Products (Frederick, CO). Human pulmonary microvascular endothelial
cells (HMVECs) and all media and tissue-culture reagents were obtained
from Cambrex (Walkersville, MD). Recombinant sCD40L and sCD40L
(human) ELISA kits were obtained from R&D Systems (Minneapolis,
MN). Murine antibodies to CD40 and goat anti–mouse F(ab⬘)2 fragments
were purchased from BioSource International (Camarillo, CA).
Patient population
All patients included within the study satisfied the definition of TRALI as
previously published.16 In addition, all TRALI reactions met the clinical
criteria for this diagnosis delineated in a National Institutes of Health (NIH)
consensus panel in June 2003 and the Canadian Consensus Conference in
April 2004.18,19 Informed consent was obtained from all study patients
through an institutional review board–approved protocol from the University of Alberta (Edmonton, Canada) prior to enrollment in this study.
Measurement of sCD40L
Soluble CD40L concentrations in human plasma or the plasma fraction of
whole blood (WB) or blood components were measured in duplicate with a
modified ELISA protocol that captured monomeric, dimeric, and multimeric forms of sCD40L. Briefly, a 96-well plate was coated with 3 ␮g/mL
mouse anti–human sCD40L antibody (a gift from Marilyn Kerry, University of Rochester, Rochester, NY) and were allowed to incubate overnight at
room temperature. A blocking buffer consisting of PBS, 1% BSA (fraction
V), and 0.1% NaN3 was added to the ELISA plate and allowed to incubate
for 1 hour. The plate was then washed 3 times with a washing buffer
consisting of PBS, 0.05% Tween 20, and 0.1% NaN3. Recombinant human
CD40L (Bender Med Systems, Vienna, Austria) was added to each plate
with 1 to 2 serial dilutions in blocking buffer to create a standard curve of
6.25 to 400 pg/mL. ELISA plates were allowed to incubate for 2 hours and
were washed 3 times with washing buffer. Mouse anti–human CD40L
biotinylated antibody (Ancell, Bayport, MN) was added to each well (2
␮g/mL in blocking buffer) and allowed to incubate 2 hours for capture.
ELISA plates were then washed 3 times with washing buffer. Streptavidin–
alkaline phosphatase (1:100; Bio-Rad Laboratories, Hercules, CA) was
added to each well and allowed to incubate for 30 minutes before it was
washed 3 times. The ELISA plates were then developed using an alkaline
phosphatase substrate kit (Bio-Rad Laboratories) according to the manufacturer’s directions. After 30 minutes of incubation, optical density was
determined at a wavelength of 405 nm with a reference of 610 nm.
Soluble CD40L concentrations were measured in fresh plasma (FP),
fresh frozen plasma (FFP), whole blood (WB) plasma, packed red blood
cells (PRBCs) leukoreduced or unmodified before storage, and platelet
concentrates (PCs) collected by apheresis techniques (A-Plts). For studies
of PRBCs, 5 healthy subjects donated 450 mL WB that was separated into
PRBCs by standard techniques according to American Association of Blood
Banks (AABB) criteria.20 Half the PRBC units (50% by weight) were
leukoreduced (LR) before storage with the use of a Hemasure r/Ls filter
(Whatman, Maidstone, United Kingdom), the standard leukoreduction filter
used at Bonfils Blood Center (Denver, CO), and the other half was
unmodified (nonleukoreduced [NLR]; PRBCs) before storage, and samples
were taken through a sterile coupler at days 1, 28, and 42 (the last day the
unit may be transfused). Healthy human volunteers donated 1 U each of
A-Plts (Cobe Trima; Gambro BCT, Lakewood, CO) after informed consent
had been obtained in accordance with the Human Subjects Committee of
the University of Colorado School of Medicine. A-Plts were stored
according to AABB standards, and samples (50-75 mL) were obtained
through sterile couplers (Baxter, Deerfield, IL) on days 1, 3, 5 (the last day
the unit could be transfused), and 7.16,20 All platelet units received standard
bacterial cultures before release for sampling, and all units or bags were
visually inspected for bacterial growth during the storage interval and
before they were discarded.20 Leukocyte counts were also performed on all
5 units, and each unit had fewer than 0.5 ⫻ 106 leukocytes. FP samples
from 5 healthy donors (3 women, 2 men) were obtained through standard
venipuncture using 18-gauge butterfly needles connected to syringes.
Citrated whole blood was then centrifuged at 5000g for 7 minutes to remove
cellular debris; this was followed by a second centrifugation step at 12 500g
for 5 minutes to remove acellular debris.21 Plasma was removed, aliquoted,
and stored at ⫺70°C. In addition, plasma samples from FFP, NLR-PRBCs,
LR-PRBCs, WB-Plts, and A-Plts were obtained through sterile couplers and
underwent separation identical to that for FP, and concentrations of sCD40L
were measured in these samples using FP and FFP as controls on each
ELISA plate.
Soluble CD40L concentrations were also quantified in PCs implicated
in TRALI reactions, derived from WB-Plts or A-Plts, compared with
control WB-Plt concentrates stored for an identical length of time that did
not cause transfusion reactions when infused into hospital patients16 or
compared with 5 U A-Plts stored for an identical length of time. Samples
from TRALI patients and the implicated platelet units, either WB-Plts or
A-Plts, were drawn immediately when the reaction was recognized and the
transfusions were stopped. Patient samples and remaining implicated
platelet units were transported to the Blood Bank (University of Alberta
Hospital) and were received within 60 minutes of the TRALI reaction (these
reactions were part of an IRB-approved study under the direction of Dr
Lynn Boshkov, University of Alberta Hospital). Transportation of all
samples occurred within the hospital without notable temperature variation,
and the plasma was separated, aliquoted, and frozen at ⫺70°C within 2
hours of identification of a possible TRALI reaction. Informed consent was
obtained for all control and TRALI patients included in this study. Samples
from control patients who received platelet transfusions and who did not
have transfusion reactions were treated in an identical manner. Before
measurement of the sCD40L concentration, the samples were thawed and
centrifuged at 12 500g for 5 minutes.
PMN isolation
Heparinized whole blood was drawn from healthy human donors, after
informed consent, with the use of a protocol approved by the Colorado
Multiple Institutional Review Board. PMNs were isolated by standard
techniques, including dextran sedimentation, Ficoll-Hypaque gradient
centrifugation, and hypotonic lysis of contaminating red blood cells.21
Whole cell lysates were made by sonication and centrifugation (700g) to
obtain the postnuclear supernatant and then were boiled in Laemmli–SDS
lysis buffer, as previously reported.22,23 We also obtained whole cell lysates
as previously described.22
CD40 expression in PMNs and activation by antibody
cross-linking
The presence of CD40 on PMNs was initially determined by Western blot
analysis of whole cell lysates. Cross-linking of CD40 on PMNs was
accomplished by incubation of PMNs with a primary mouse monoclonal
antibody to CD40 followed by incubation with goat F(ab⬘)2 antimouse
fragments for 5 minutes at 37°C, pH 7.35.24 PMNs were then activated by
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BLOOD, 1 OCTOBER 2006 䡠 VOLUME 108, NUMBER 7
1 ␮M formyl-Met-Leu-Phe (fMLP), and the maximal rate of superoxide
anion release was measured as the reduction of cytochrome c, as previously
published.21 Priming activity was determined as the augmentation of the
fMLP-activated respiratory burst in control cells (eg, PMNs treated with
isotypic antibody controls).7,21
Digital fluorescence microscopy
PMNs (5 ⫻ 105) were warmed to 37°C and prepared as previously
described.12 Images were acquired with a Zeiss Axiovert microscope (Carl
Zeiss, Oberkochen, Germany) fitted with a 63 ⫻/1.4 NA oil-immersion
objective lens (Carl Zeiss) and a Cooke CCD SensiCam (Applied Scientific
Instrumentation, Eugene, OR) using a Chroma Sedat Multiple Bandpass
filter wheel (Chroma Technology, Brattleboro, VT) and a Sutter filter
control (BD Biosciences Bioimaging, Rockville, MD). Images were
acquired with Slidebook 4.1 software (Intelligent Imaging Innovations,
Denver, CO). All images compared within a single figure were acquired as
Z-stacks in 0.2-␮m intervals. All Z-stack images were deconvolved by the
application of constrained iterative deconvolution and Gaussian noise
smoothing from system-specific point spread functions.25 After deconvolution, images were cropped to represent the middle-most planes (center ⫾ 10
planes), and the proteins in question were masked to represent zero
fluorescence in IgG-negative controls, prepared as previously described.12
In short, slides were incubated with a mouse IgG isotype, with the host
species as the primary antibody against CD40. Positive binding of the CD40
antibody was determined by setting the lowest fluorescence threshold to a
value identical to the greatest fluorescence threshold seen from the negative
controls. Fluorescence of CD40 (red) was detected with the use of a donkey
antimouse secondary antibody conjugated with Cy3, which binds directly to
the monoclonal antibody to CD40. The membrane was identified by the
association of the lectin wheat germ agglutinin (WGA) to salicylic acid
residues linked to the outer surfaces of membrane proteins.26 WGA was
conjugated with AlexaFluor 488 (Ex-495 Em-519; Invitrogen, Carlsbad,
CA) and was acquired on the FITC channel (green). The nucleus was
identified by the DNA-specific stain bis-benzimide, which was fluorescent
(blue) and acquired on the Cy5 channel.26,27 Overlay images were displayed
in pseudocolor with the Slidebook (Intelligent Imaging Innovations).
PMN priming
PMN priming assays were completed as follows: PMNs (3.75 ⫻ 105) were
incubated with buffer (Krebs-Ringers phosphate with 2% dextrose [KRPD])
controls, sCD40L (1 ng/mL, 10 ng/mL, 100 ng/mL, and 1 ␮g/mL), or 2 ␮M
platelet-activating factor (PAF) for 3, 5, or 15 minutes at 37°C, followed by
activation with 1 ␮M fMLP, and the maximal rate of superoxide production
was measured.28 Priming activity was calculated as the augmentation of the
maximal rate of O2⫺ in response to fMLP.
sCD40L ACCUMULATES IN STORED BLOOD: ROLE IN TRALI
2457
Two-event in vitro model of PMN-mediated pulmonary
endothelial damage
A 2-event in vitro model of PMN-mediated pulmonary endothelial damage
was performed as previously described.17 Briefly, HMVECs were grown to
80% to 90% confluence on 12-well plates and incubated with 2 ␮g/mL
endotoxin from Salmonella enteritides LPS or buffer for 6 hours at 37°C in
5% CO2. PMNs (1 ⫻ 106) were added at a 10:1 effector-cell–to–target-cell
ratio and were allowed to settle for 30 minutes. After settling, the PMNs
were exposed to buffer or sCD40L (10 ng/mL-1 ␮g/mL) for 30 minutes.
Supernatants were removed by quickly inverting and forcefully decanting
the plates from a height of 50 cm onto absorbent towels.17 After the addition
of warm KRPD, the number of viable HMVECs, adherent and trypan blue
negative, were counted over a 4-mm2 surface area by 3 separate observers
to exclude bias. Controls consisted of HMVECs alone without PMNs or
incubated with all reagents used in these experiments, alone or in
combination, and viability was assessed.
Statistical analysis
Statistical analysis was completed with the use of repeated-measures
analysis of variance (ANOVA) followed by post hoc Bonferroni test for
multiple comparisons. Significance was determined at a P value below .05.
Results
Soluble CD40L accumulation in blood components
Because sCD40L accumulation has only been evaluated at the
outdate (the last day the unit may be transfused) of platelet
concentrates from A-Plts (day 5), we measured sCD40L concentration serially in the plasma from A-Plts with storage time to day 7
and in other blood components including FP and FFP and the
plasma fractions from WB, WB-Plts, and PRBCs (both LR and
NLR).3,26,29 Compared with FP from healthy volunteers (3 women,
2 men), the amount of sCD40L from the plasma fraction of A-Plts
was increased on all days of storage (days 1-7); moreover, sCD40L
concentrations in A-Plts increased significantly on days 3, 5, and 7
of storage compared with day 1 (day 1, 5.0 ⫾ 0.8 ng/mL; day 3,
8.8 ⫾ 1.2 ng/mL; day 5, 11.8 ⫾ 0.8 ng/mL; day 7, 14.0 ⫾ 1.5
ng/mL; P ⬍ .05) (Figure 1A). However, the concentration of
sCD40L on day 7 was not significantly greater than the sCD40L
accumulation measured on day 5 (Figure 1A). Soluble CD40L was
also measured in the plasma fraction of LR- and NLR-stored
Figure 1. Accumulation of sCD40L in stored A-Plts and LR-PRBCs and NLR-PRBCs. (A) A-Plts were serially stored according to AABB standards, plasma samples were
obtained and separated from the cells by centrifugation, sCD40L concentration (ng/mL) was measured by commercial ELISA, and fresh, heparinized plasma taken from
healthy donors was used as a control for sCD40L concentration. The sCD40L concentration in A-Plts was significantly greater at days 1, 3, 5, and 7 of storage in the A-Plts than
in FP (P ⬍ .05; n ⫽ 5). Moreover, the levels continued to significantly increase on days 3, 5, and 7 compared with day 1. However, the concentrations of sCD40L on day 5
compared with day 7 were not significantly different from one another (P ⬍ .05). *Statistical significance from FP (P ⬍ .05); ¥,§Significance from day 1 (#) (P ⬍ .05). (B)
Accumulation of sCD40L in PRBCs in which 50% of the unit (by weight) was leukoreduced (LR) and 50% was unmodified (NLR) before storage. sCD40L concentrations
(ng/mL) were measured serially in LR-PRBCs and NLR-PRBCs on days 1, 28, and 42. Day 28 and day 42 NLR (hatched bars) were statistically significant (P ⬍ .05; n ⫽ 5)
compared with day 1 NLR. Days 28 and 42 NLR were also significant from days 1, 28, and 42 LR (solid bars). *Statistical significance (P ⬍ .05) compared with day 1 of storage.
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2458
BLOOD, 1 OCTOBER 2006 䡠 VOLUME 108, NUMBER 7
KHAN et al
PRBCs on days 1, 28, and 42 (Figure 1B). Compared with the FP
from healthy donors, a mild but significant elevation in sCD40L
was observed in the plasma fraction of LR-PRBCs and NLRPRBCs; moreover, the levels of sCD40L increased in the plasma
fraction of NLR-PRBCs throughout storage and were significantly
higher than concentrations from LR-PRBCs at days 28 and 42
(*P ⬍ .05) (day 28: LR 0.62 ⫾ 0.17 ng/mL vs NLR 5.6 ⫾ 0.9*;
day 42: LR 0.45 ⫾ 0.1 vs NLR 7.4 ⫾ 1.5*) (Figure 1B). These data
demonstrate that sCD40L levels were elevated in NLR-PRBCs
compared with LR-PRBCs. Because LR-PRBCs had much lower
levels of sCD40L than NLR-PRBCs, we investigated whether the
Hemasure r/LS leukoreduction filters removed platelets as well as
leukocytes. In a study of 11 whole blood donations, 50% of the
PRBCs were filtered and the other 50% were left as an unmodified
control (NLR-PRBCs). The Hemasure rL/S filters significantly
removed platelets from LR-PRBCs by almost 2 logs compared
with NLR-PRBCs (3.36 ⫾ 0.69 Plt ⫻ 103/␮L [LR-PRBCs] vs
285.55 ⫾ 27.31 Plt ⫻ 103/␮L [NLR-PRBCs]; P ⬍ .01). Lastly,
sCD40L concentrations were also measured in 5 U WB at
component outdate and in 5 U FFP, and these concentrations were
significantly elevated compared with FP levels from 5 healthy
donors (15.0 ⫾ 2.0 ng/mL [WB] and 1.3 ⫾ 0.1 ng/mL [FFP] vs
0.01 ⫾ 0.003 ng/mL [FP]; *P ⬍ .05).
Plasma sCD40L concentrations in implicated platelet
concentrates and in TRALI patients before
and after transfusion
Soluble CD40L concentrations were measured in WB-Plts (n ⫽ 62)
implicated in TRALI reactions and were compared with WB-Plts
infused into hospital patients (n ⫽ 59). Platelet concentrates were
stored for identical amounts of time and did not cause transfusion
reactions (Table 1). In addition, sCD40L concentrations were
measured in the plasma of A-Plts implicated in TRALI (n ⫽ 29)
and were compared with 5 control A-Plts units, stored for identical
amounts of time, and were not infused (Table 1). In both cases, the
platelet concentrates implicated in TRALI reactions, whether WB
derived or collected by apheresis, had significantly greater concentrations of sCD40L in the plasma fraction than did control platelet
concentrates stored for an identical period of time (P ⬍ .05) (Table
1). These data represent the results from individual A-Plt and
WB-Plt units and from pooled WB-Plts, depending on the products
transfused; however, all units infused into TRALI patients or
control subjects were tested without exception. Moreover, if one
takes into account the total amount of sCD40L infused into the
TRALI patients from the implicated WB-Plt concentrate units
compared with the control WB-Plt concentrate units, the TRALI
Table 1. sCD40L concentrations in blood components
patients were exposed to 4.4 ⫾ 0.4 ␮g sCD40L compared with
2.5 ⫾ 0.3 ␮g in control patients, which demonstrates that TRALI
patients received significantly larger amounts of sCD40L than
control subjects (P ⬍ .05).
Soluble CD40L concentrations were also measured in 12
TRALI patients’ plasma samples before and after transfusion, and
the circulating levels of sCD40L increased in 8 of 12 patients at the
time TRALI was clinically recognized after transfusion (3.3 ng/mL)
compared with the levels in the pretransfusion plasma level (3.1 ng/mL)
(Figure 2). The increased level of sCD40L was not statistically
significant from sCD40L concentrations in the pretransfusion
plasma samples.
PMNs express CD40 on the plasma membrane
Because the presence of CD40 has not been investigated in PMNs,
CD40 expression was initially determined by immunoblotting
proteins from whole cell lysates, which demonstrated a band of
immunoreactivity at 48 kDa, the reported molecular weight of
CD401 (Figure 3A). Fixed PMNs were then smeared onto slides
and incubated with human CD40 antibody, and images were
prepared as previously reported.12,29 Compared with the negative
controls consisting of fluorescently labeled isotypic immunoglobulins, CD40 immunoreactivity was present at the periphery of the
cell (Figure 3B). Colocalization of CD40 at the plasma membrane
is displayed in pseudocolor; red represents high amounts of
colocalization, and blue represents low colocalization between
CD40 and the plasma membrane (Figure 3B).
PMNs express an active CD40 receptor
sCD40L, ng/mL
Blood components
Nonimplicated TRALI, U
Implicated TRALI, U
FP from healthy donors
0.01 ⫾ 0.003
N/A
FFP
1.3 ⫾ 0.13*
N/A
WB-Plts
7.9 ⫾ 1.1*
14.0 ⫾ 3.0*†
11.8 ⫾ 0.82*
44.7 ⫾ 4.5*†
A-Plts
Figure 2. Soluble CD40L concentration in the plasma of TRALI patients before
and after transfusion. Each line reflects the sCD40L in a pretransfusion typing
plasma (left) compared with the sCD40L concentration in a plasma sample drawn
when TRALI was clinically recognized (right).16 In 8 of 12 TRALI patients, levels of
sCD40L concentrations increased at the time TRALI was diagnosed.
N/A indicates not applicable.
*P ⬍ .05 from FP obtained through venipuncture from 5 healthy subjects.
†P ⬍ .05 from WB-Plt concentrates that did not cause a transfusion reaction or
from 5 A-Plt concentrates from healthy donors, respectively. Soluble CD40L concentrations were measured in duplicate by commercial human sCD40 ELISA. Numbers
of units tested in all categories were as follows: 5 U FP, 10 U FFP, 57 samples from
WB-Plt concentrates that did not cause transfusion reactions, 62 samples from
WB-Plt concentrates implicated in TRALI, 5 U A-Plts from healthy donors, and 29
samples from A-Plt concentrates implicated in TRALI.
To determine whether the CD40 receptor was functional, isolated
PMNs were incubated with a monoclonal antibody to CD40,
isotypic antibody controls, or buffer, followed by the addition of
goat anti–mouse F(ab⬘)2 fragments to cross-link the anti–CD40
antibody bound to CD40. Admixtures were then activated by 1 ␮M
fMLP, and the maximal rate of superoxide anion was measured.
These data demonstrated that cross-linking of antibodies specific
for CD40 caused significant priming (P ⬍ .05) of the fMLPactivated PMN oxidase compared with the isotypic controls alone,
CD40 antibodies alone, or PMNs incubated with buffer plus goat
anti–mouse F(ab⬘)2 or PMNs incubated with isotype controls plus
goat anti–mouse F(ab⬘) (Figure 4).
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BLOOD, 1 OCTOBER 2006 䡠 VOLUME 108, NUMBER 7
sCD40L ACCUMULATES IN STORED BLOOD: ROLE IN TRALI
2459
Figure 3. Expression of CD40 in human PMNs by Western blot analysis and localization of CD40 by digital microscopy. (A) Experiments were performed on 3 donors.
Proteins from PMN whole cell lysates (1.25 ⫻ 106) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and were immunoblotted with a
monoclonal antibody to human CD40. The CD40 glycoprotein band is present at 48 kDa. (B) Fixed PMNs were incubated with a human CD40 monoclonal antibody, and then
the images were prepared for digital microscopy as previously described.12 (i) CD40 immunoreactivity is red because of the secondary donkey antimouse antibody conjugated
to Cy3. (ii)The membrane is green because of the association of the wheat germ agglutinin (WGA)–AlexaFluor 488 with salicylic acids, and the nucleus is stained blue with the
fluorescent bis-benzimide nuclear dye. (iii) Colocalization of CD40 and the plasma membrane is displayed in pseudocolor. Arrows point to the red area, where a high amount of
colocalization is present. Blue represents low colocalization between CD40 and the plasma membrane. These images are representative of 3 different experiments on the
PMNs of 3 donors.
Recombinant soluble CD40L primes the PMN oxidase
PMN-mediated HMVEC damage
Given that PMNs express functional CD40, we investigated the
ability of recombinant sCD40L to prime the fMLP-activated
oxidase in human PMNs. The sCD40L priming activity was
measured over a range of incubation times (3-15 minutes) and
concentrations (1 ng/mL-5 ␮g/mL). Significant priming activity of
sCD40L compared with buffer-treated controls was present at 3 and
5 minutes, with priming of the fMLP-activated respiratory burst
reaching a relative maximum at 5 minutes (P ⬍ .05) (Figure 5). At
longer incubation times (10-15 minutes), the priming activity
decayed and was not significantly increased at 15 minutes compared with identical buffer-treated controls (results not shown).
With respect to concentration, sCD40L significantly primed PMNs
at a range of concentrations from 10 ng/mL to 1 ␮g/mL compared
with buffer-treated controls (P ⬍ .05) (Figure 5). In addition,
preincubation of PMNs with antibodies specific for CD40 inhibited
the priming activity of sCD40L by 50% ⫾ 7%.
Because PMN-mediated injury of pulmonary endothelium is
thought to require at least 2 separate cellular events, we developed
an in vitro model of PMN-mediated pulmonary HMVEC damage
that required HMVEC activation leading to PMN adherence and
activation of these adherent PMNs resulting in the activation and
release of cytotoxic agents from the microbicidal arsenal and the
killing of HMVECs.17 HMVECs were incubated for 6 hours with
buffer or 2 ␮g/mL LPS, which resulted in proinflammatory
activation of these endothelial cells as documented by the synthesis
and release of chemokines, specifically IL-8, GRO-␣, and ENA-78,
and increased ICAM-1 surface expression compared with the
buffer-treated PMNs.17 LPS-induced adherence was significantly
greater (P ⬍ .05; n ⫽ 6) than in buffer-treated controls
(18.4% ⫾ 3.5% PMNs [LPS] vs 1.5% ⫾ 0.6% PMNs [buffer
controls]). This LPS-induced adhesion could be abrogated by
preincubation of the HMVECs with antibodies to adhesion molecules or immunoglobulin neutralization of the released chemokines (18.4% ⫾ 3.5% [LPS] vs 1.5% ⫾ 0.9% [anti-CD18] and vs
1.8% ⫾ 0.5% PMNs [chemokine neutralization]) and was not
different from PMN adherence to buffer-treated HMVECs
Figure 4. Cross-linking CD40 primes the PMN oxidase. PMNs were incubated
with a mouse monoclonal antibody to human CD40, isotype controls, or buffer for
60 minutes at 4°C, followed by the addition of a buffer control or a goat anti–mouse
F(ab⬘)2 (GAM) to cross-link the CD40 antibody. Activation of the PMN oxidase was
accomplished with the addition of 1 ␮M fMLP, and priming of the fMLP-activated
oxidase was calculated as the augmentation of the maximal rate of O2⫺ in response
to fMLP in buffer-treated controls. *Significant increase in oxidase activity compared
with the buffer-treated controls (P ⬍ .05). The only group to significantly prime the
fMLP-activated respiratory burst was CD40 ⫹ GAM ⫹ fMLP, indicating that crosslinking CD40 directly causes priming of the NADPH oxidase. Importantly, incubation
of PMNs treated with isotype controls with the F(ab⬘)2 cross-linkers did not cause
priming of the fMLP-activated oxidase (results not shown). This figure is representative of 7 separate experiments using disparate blood donors as the source of the
PMNs. Data are presented as mean ⫾ standard error of the mean.
Figure 5. Soluble CD40L primes the PMN oxidase. PMN priming assays were
completed with incubation of PMNs with buffer, sCD40L (1 ng/mL-1 ␮g/mL), or PAF
(2 ␮M) (positive control). The ability of sCD40L to prime the fMLP-activated oxidase in
human PMNs was measured over a range of concentrations for 5 minutes. Significant
priming of the PMN oxidase occurred with the addition of 10 ng/mL, 100 ng/mL, and
1 ␮g/mL sCD40L compared with buffer-treated controls (#P ⬍ .05; *P ⬍ .05; n ⫽ 7);
significant differences exist between groups with different symbols. Data are
presented as mean ⫾ standard error of the mean.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2460
KHAN et al
Figure 6. A 2-event in vitro model of PMN-mediated pulmonary endothelial
damage. HMVECs were grown to 80% to 90% confluence on 12-well plates and were
incubated with buffer or LPS for 6 hours, followed by the addition of buffer or freshly
isolated PMNs that were allowed to settle for 30 minutes at a target-effector ratio of
10:1. After the addition of PMNs or buffer, a range of human recombinant CD40L
concentrations or buffer controls was added to the HMVECs. (dark gray bars) Control
cells; buffer-treated HMVECs with no PMNs. (light gray bars) HMVECs pretreated
with 2 ␮g/mL LPS for 6 hours at 37°C, followed by treatment with buffer or a range of
sCD40L concentrations (as delineated on the x-axis). (black bars) HMVECs incubated with LPS for 6 hours, followed by the addition of PMNs and sCD40L. Incubation
of the adherent PMNs with concentrations of sCD40L at 100 ng/mL, 10 ng/mL, and 1
␮g/mL resulted in a significant decrease in the number of viable HMVECs. *P ⬍ .05
(n ⫽ 3) compared with buffer. These data demonstrate that sCD40L may cause
concentration-dependent PMN-mediated damage of LPS-activated HMVECs. Importantly, HMVECs stimulated with LPS and exposed to PMNs without sCD40L did not
demonstrate PMN-mediated damage but did evidence widespread PMN adherence.
Data are presented as mean ⫾ standard error of the mean.
(1.5% ⫾ 0.6%), identical to previous results.17,24 The addition of
sCD40L (10 ng/mL-1 ␮g/mL) to LPS-activated HMVECs with or
without PMNs resulted in significant HMVEC cytotoxicity compared with buffer-treated HMVEC controls with or without PMNs
activated with sCD40L or buffer, LPS-activated HMVECs with or
without PMNs activated with buffer, and buffer or LPS-activated
HMVECs with sCD40L (Figure 6). Furthermore, the concentrations of sCD40L that caused significant neutrophil oxidase priming
caused extensive HMVEC killing. Lastly, HMVEC viability was
not affected by any reagents, alone or in combination, without
PMNs (Figure 6 and results not shown; data available on request).
Discussion
The data presented demonstrate that sCD40L accumulated in the
plasma fraction of most cellular blood components (WB, PCs,
and PRBCs) and that sCD40L concentrations accumulated in
A-Plts to higher concentrations than any other blood product. In
addition, prestorage leukoreduction significantly decreased
sCD40L levels, which may be explained by an almost 2-log
reduction of contaminating platelets by the Hemasure r/LS
filters used in these experiments. Moreover, sCD40L levels were
increased in PCs implicated in TRALI reactions compared with
the levels in control PCs that did not cause transfusion reactions
and were stored for an identical length of time. Furthermore,
increased posttransfusion levels of sCD40L were found in the
plasma from 8 of 12 patients with documented TRALI. Because
PMNs are the effector cells in TRALI15,16 and CD40 expression
had not been demonstrated in PMNs, we determined that human
PMNs express an active CD40 receptor and that receptor
ligation, especially with sCD40L, can activate this receptor,
which caused priming of the fMLP-activated oxidative burst. In
BLOOD, 1 OCTOBER 2006 䡠 VOLUME 108, NUMBER 7
addition, recombinant sCD40L could also serve as the second
event in a well-characterized 2-event in vitro model of PMNmediated endothelial damage.17 Importantly, our previous data
demonstrated that recombinant, monomeric sCD40L, identical
to that used in the priming and PMN-mediated HMVEC damage
experiments, has decreased biologic activity compared with
native (trimeric or dimeric) CD40L such that the supernatant of
stored platelets stimulate lung fibroblast release of IL-6 and IL-8
partially inhibited by anti-CD154, an activity displayed to a
lesser degree by recombinant sCD40L.3,30,31 The data presented
demonstrate that, at physiologic levels, native sCD40L or even
monomeric sCD40L could cause priming of the PMN oxidase
and could serve as the second event in an in vitro model of
PMN-mediated pulmonary endothelial damage. This in vitro
model of PMN-mediated pulmonary endothelial damage required a first event to activate the pulmonary endothelium,
resulting in PMN adherence (priming) similar to sequestration
in the lung in vivo.15,17 In this model, LPS was used as the first
event to mimic sepsis, a clinical condition that predisposes
patients to TRALI and to other forms of ALI.14,17,31 Soluble
CD40L, over a range of concentrations, could serve as the
second event and could cause activation of the microbicidal
arsenal of the adherent primed PMNs, resulting in HMVEC
damage. Thus, the infusion of platelet concentrates or other
blood components with significant amounts of sCD40L may
have the capacity to cause TRALI in a susceptible host.
Soluble CD40L concentrations in all cellular blood components were significantly greater on day 1 than FP from healthy
volunteers. This increase appeared to be caused by the isolation
of the component itself from the activation of contaminating
platelets, either by the tubing or the isolation technique, because
the Cobe Spectra (Gambro BCT) machines are reported to cause
activation of a small amount of platelets during collection.32
When platelet contamination is decreased by prestorage leukoreduction with a filter that removes platelets, the generation of
sCD40L is decreased. These results are congruous with estimates that 95% of all human sCD40L is platelet derived,33 both
in apheresis procedures and in the collection of whole blood for
separation into components. Although these concentrations are
not large, a significant amount may be infused into patients
requiring massive blood support.34,35
Although sCD40L has the capacity to prime the PMN
oxidase and to activate the microbicidal arsenal of PMNs
adherent to HMVECs (ie, primed PMNs), other proinflammatory compounds also increase during the storage time of PCs,
especially lipids,15 which have the capability to cause TRALI in
susceptible hosts.11,36,37 Furthermore, sCD40L actually decreased or was unchanged in 4 of 12 patients with TRALI. Thus,
determining the biologic significance of sCD40L in patients’
plasma requires further study. Importantly, sCD40L may be
rapidly internalized or it may become ligated to CD40, which
resides on a number of cells in the human vasculature.38-40
Soluble CD40L could also serve as a cofactor in TRALI because
of its ability to activate circulating monocytes or the vascular
endothelium causing increases in the levels of pro-inflammatory
chemokines.17 Moreover, though A-Plts are thought to be a purer
platelet product than WB-Plts with respect to the increased rate
of adverse events, sCD40L was increased in these components
compared with WB-Plts and thus may have unexpected effects
in the transfused host.41,42 A recent report revealed that sCD40L
is associated with adverse events in patients requiring platelet
transfusion (in press).43 Furthermore, sCD40L may have direct
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
BLOOD, 1 OCTOBER 2006 䡠 VOLUME 108, NUMBER 7
sCD40L ACCUMULATES IN STORED BLOOD: ROLE IN TRALI
effects on the innate and adaptive immune system and may
contribute to or cause TRALI and other adverse events in human
patients, especially those requiring aggressive blood support for
organ transplantation, a group with an unexplained increase in
TRALI reactions in one series.3,44-48
In summary, sCD40L is a proinflammatory protein that accumulates during the storage of A-Plts and other cellular blood components. In PRBCs, it appears that leukocytes directly influence its
accumulation in the plasma; however, in PCs, even those with
minimal leukocyte contamination, sCD40L is released into the
plasma in high concentrations. A-Plts have been implicated in
TRALI,3,36,37,49 and injured patients with thrombocytopenia requiring platelet support are at risk for postinjury multiple organ failure,
which invariably includes acute lung injury.50-52 Little research has
focused on the mediators contained within platelet membranes or
in storage granules—␣-granules, dense bodies, or lysosomes—
agents that may be responsible for proinflammatory and immunomodulatory effects in the transfused host.53 To date, the only
2461
effective method of removing proteins or polypeptides from the
plasma fraction of cellular blood components is washing, which
can be expensive and time consuming and is not suitable for all
patient groups.29 Further work is required to optimize platelet
support for patients at risk for transfusion-related inflammation or
immunomodulation and to determine the biologic affects of native
sCD40L with respect to its role in transfusion reactions, especially
TRALI and its inherent ability to directly cause activation of the
PMN microbicidal arsenal in vivo.
Acknowledgments
We thank Daniel Ambruso (Bonfils Blood Center and the Department of Pediatrics, University of Colorado School of Medicine),
Larry Dumont (Gambro BCT), and Deborah Dumont (Whatman)
for providing data regarding the removal of platelets by the
Hemasure r/LS leukoreduction filter.
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2006 108: 2455-2462
doi:10.1182/blood-2006-04-017251 originally published
online June 13, 2006
Soluble CD40 ligand accumulates in stored blood components, primes
neutrophils through CD40, and is a potential cofactor in the
development of transfusion-related acute lung injury
Samina Yasmin Khan, Marguerite R. Kelher, Joanna M. Heal, Neil Blumberg, Lynn K. Boshkov,
Richard Phipps, Kelly F. Gettings, Nathan J. McLaughlin and Christopher C. Silliman
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