Neutrophils are the main effector cells of inflammation
in the airway epithelium.(1) Neutrophil priming has been
reported to play a crucial role in the pathogenesis of
post-injury hyper-inflammation leading to transfusion-
related acute lung injury (TRALI) and multiple organ
failure (MOF) in surgery patients.(2) Although the
pathogenesis of TRALI has been linked to the presence
of antileukocyte antibodies, a significant number of
cases do not have an immunologic etiology,(3) leading
to the postulation of the two-event model for TRALI.
The two-event model suggests that TRALI is initiated
by an immune priming step (consisting of the initial
trauma), followed by exposure of neutrophils to specific
biological response modifiers via transfusion, causing
an interaction between neutrophils, platelets and lung
PRBC-derived plasma induces non-muscle myosin
type IIA-mediated neutrophil migration and
Chen Yu1, Li Xu2, Lii-fang Chen3, Ying-jie Guan3, Minsoo Kim3, Walter L. Biffl3,
and Y. Eugene Chin3
1Department of Nephrology, Tongji Hospital, Tongji Universith School of Medicine, Shanghai, China,
2Department of Immunology, School of Basic Medicine, Zhejiang Chinese Medical University Hangzhou,
Zhejiang, China, and 3Department of Surgery, Brown University School of Medicine-Rhode Island Hospital,
Providence, RI, USA
Context: Neutrophils are the primary effector cells in the pathogenesis of transfusion-related acute lung injury or
multiple organ failure after blood transfusion.
Objective: We aimed to investigate the effect of fresh (1 day preparation) and aged (42 day preparation) PRBC-derived
plasma on neutrophil morphology, migration and phagocytosis.
Materials and methods: We evaluated the production of reactive oxygen species (ROS) and the expression of non-
muscle myosin heavy chain IIA (MYH9) in neutrophils treated with PRBC-derived plasma. We used western blots and
antibody arrays to evaluate changes in signal transduction pathways in plasma-treated neutrophils.
Results: Aged PRBC-derived plasma elicited a stronger oxidative burst in neutrophils when compared with fresh PRBC-
derived plasma (p < 0.05). Antibody arrays showed increased phosphorylation of NF-ĸB proteins (p105, p50 and Ikk)
in aged PRBC-derived plasma-treated neutrophils. The expression of non-muscle myosin IIA (MYH9), a cytoskeleton
protein involved in immune cell migration and morphological change, was also significantly upregulated in
neutrophils treated with aged PRBC-derived plasma compared to fresh plasma (p < 0.05). Pretreatment of neutrophils
with blebbistatin (a specific type II myosin inhibitor), ascorbic acid (an antioxidant), or staurosporine (a protein
tyrosine kinase inhibitor), effectively abrogated the morphological changes, neutrophil migration, and phagocytosis
induced by aged PRBC-derived plasma.
Conclusion: Upregulation of MYH9 in neutrophils treated with aged PRBC-derived plasma and abrogation of neutrophil
migration in blebbistatin-treated neutrophils suggested a functional role of MYH9 in the directional migration of
immune cells. Our data help elucidate the cellular and molecular mechanisms of transfusion-related injury.
Keywords: Neutrophils, MYH9, migration, NFĸB, plasma
Address for Correspondence: Prof. Y. Eugene Chin, Department of Surgery Brown University School of Medicine Rhode Island Hospital, 593
Eddy Street, Providence, RI 02903, USA. Tel: 401–444-0172 (O). Fax: 401–444-3278 (O). E-mail: email@example.com
(Received 29 December 2011; revised 24 February 2012; accepted 14 March 2012)
Immunopharmacology and Immunotoxicology, 2013; 35(1): 71–79
© 2013 Informa Healthcare USA, Inc.
ISSN 0892-3973 print/ISSN 1532-2513 online
72 C. Yu et al.
Immunopharmacology and Immunotoxicology
endothelium.(2,4) Neutrophil priming is characterized
by oxidative burst (release of reactive oxygen species
(ROS) via activation of NADPH oxidase), morphological
changes, delayed apoptosis and enhanced microbicidal
activity during phagocytosis.(2,5) Stored blood was shown
to cause more significant priming compared to freshly
prepared blood and there is a direct correlation between
the duration of storage time of red blood cells (RBCs) and
neutrophil priming.(6) Transfusion with stored plasma
was also shown to delay neutrophil apoptosis(7,8) and to
cause more tissue damage than fresh plasma.(2)
ROS is a term that describes free oxygen radicals such
as superoxide anion and hydroxyl radicals.(9,10) Oxidative
burst and ROS are known to regulate protein phosphory-
lation in cells stimulated with growth factors/cytokines,
leading to the activation of different signal transduction
pathways such as the TNF, NF-kB, STAT, PKC and MAP
kinase pathways,(11,12) A number of intracellular processes,
such as modulation of cell function, inflammation and
apoptosis are regulated by these signaling pathways.(9,10)
Transfused blood induces an oxidative burst and upreg-
ulation of inflammation-related cytokines like IL-6, IFN,
IL-18 and TNF in neutrophils, suggesting that transfusion
plays a role in activation of these signaling pathways.(13)
Neutrophil migration into tissues is the hallmark of
all types of inflammatory responses. Neutrophil crawl-
ing is characterized by F-actin polymerization, cytoskel-
eton contraction and adhesion to surrounding tissue.(14)
We previously showed that non-muscle myosin heavy
chain IIA (MYH9) formed the link between the integrin
LFA-1 and the cytoskeleton and regulated T lymphocyte
migration.(15) Non-muscle myosin has also been shown
to play a critical role in migration of T cells towards the
site of inflammation.(16–18) Interestingly, ROS was recently
shown to be an important regulator of neutrophil che-
motaxis.(19,20) However, the role of blood derivatives on
the morphology and migration of neutrophils remains
The present study was therefore undertaken to inves-
tigate the effect of fresh (1 day preparation) and aged (42
day preparation) PRBC-derived plasma on ROS produc-
tion, MYH9 expression and phagocytosis in neutrophils.
We also investigated changes in the morphology and
migration of neutrophils exposed to aged and fresh
PRBC-derived plasma and explored the mechanisms
underlying these changes.
Materials and methods
Blood leuko-reduction and neutrophil isolation
Ten healthy volunteers each donated 1 unit of whole
blood (WB) which was collected in citrated 500 mL
Triple Blood-Pack Unit bags (Baxter International Inc.,
Deerfield, IL) and processed per the standards of the
American Association of Blood Banks, Bethesda, MD.
One half of each packed red blood cell (PRBC) fraction
was leuko-depleted using a gravity driven leukocyte
reduction filter (Sepacell R-500 II; Baxter International
Inc.) and all the PRBC units were stored at 4°C. At
biweekly intervals, a portion of the unit was removed and
centrifuged (at 6470 rpm for 7 min, then at 10,230 rpm for
10 min) to remove the RBCs, leaving a small plasma frac-
tion that was divided into aliquots and stored at −80°C
until assayed. This was done every other week until day
42. Prior to use, the plasma aliquots were thawed to room
temperature and centrifuged at 4°C at 1000g for 15 min to
ensure complete removal of residual platelets.
Neutrophils were isolated from EDTA (0.5%)-treated
peripheral venous blood of healthy human volunteers
using a 4-step discontinuous Percoll gradient (Sigma,
St. Louis, MO). Erythrocytes were removed by hypotonic
lysis, and neutrophils were resuspended in RPMI-1640
medium (Invitrogen, Carlsbad, CA). Neutrophil purity
and viability were always higher than 99% and 96%,
respectively. Neutrophils were incubated for 1 h at 37°C
in the presence of 5% CO2, with the RBC plasmas pre-
pared as described above (20% plasma/80% RPMI 1640).
This study was approved by the Institutional Review
Board of the Lifespan Human Subject Research
Committee, Providence, RI, USA and informed consent
was obtained from all the volunteers.
Superoxide production was measured by the O2
dismutase-inhibitable reduction of cytochrome c.
Neutrophils (3.75 × 105/well) were incubated for 3 min
nuclear factor-kappa B
the Signal Transducers and Activators of
Nonmuscle myosin IIA
transfusion-related acute lung injury
multiple organ failure
red blood cells
reactive oxygen species
mitogen-activated protein kinase
tumor necrosis factor
reduced form of nicotinamide-adenine
NMMHCA non-muscle myosin heavy chain-A
AA ascorbic acid
STS tyrosine kinase inhibitor staurausporin
Plasma induces neutrophil activation 73
© 2013 Informa Healthcare USA, Inc.
with the different plasma preparations and immedi-
ately placed in a microplate reader (THERMOmax with
Softmax software, Molecular Devices, Menlo Park, CA)
for kinetic measurement of O2
Leu-Phe (fMLP; 1 mM/L) obtained from Sigma was used
as positive control. Absorbance at 550–450 nm was mea-
sured every 20 s for 5 min. The maximal rate of O2
duction (Vmax) was determined by the absorbance curve
over 5 points. An extinction coefficient of 8.4 × 10−3 L/mol/
min was used as determined for the 150 µL reaction vol-
ume and the 550-nm filter in the microplate reader. Data
were recorded as Vmax, nmol O2
Antibody array analysis and western blot analysis
Four hundred commercial antibodies (20–40 ng in a
volume of 0.4 mL or less) were spotted on a 5 × 4 cm
nitrocellulose membrane (Bio-Rad) as previously
described.(21) The spots were less than 500 µm in diameter
and all antibodies were obtained from Santa Cruz Biotech
Inc., Santa Cruz, CA. Freshly prepared antibody arrays
were immersed in PBS containing 3% bovine serum
albumin for 2 h prior to use. Neutrophils were subjected
to different PRBC-derived plasma treatments and whole
cell extracts were prepared with RIPA lysis buffer (50 mM
Tris [pH 8.0], 5 mM EDTA, 150 mM NaCl, 0.5% NP-40,
0.1% sodium dodecyl sulfate [SDS], 1 mM dithiothreitol,
1 mM Na3VO4, 0.5 mM phenylmethylsulfonyl fluoride,
1 mM NaF) containing a protease inhibitor cocktail
(Roche, Indianapolis, IN). Whole cell extracts (1.5 mg)
were incubated with the antibody arrays for 3 h at room
temperature. The arrays were extensively washed with
TBST and incubated with a 1:1000 dilution of HRP-
conjugated phospho-tyrosine antibody pY20 (Santa Cruz
Biotech Inc.) for 2 h at 37°C. Signals were detected using
enhanced chemi-luminescence (Amersham Biosciences,
Piscataway Township, NJ).
For western blots, whole cell extracts prepared from neu-
trophils were separated on 10% SDS-PAGE (100 µg/lane).
All antibody incubations were performed as previously
described(22) and immune-blotting analysis was performed
as described elsewhere.(7) Whole cell extracts were also
prepared from Jurkat and Daudio cells (ATCC, Manassas,
VA) which were cultured in in RPMI media supplemented
with 10% FBS (1 × 105–106 cells/mL). HeLa and HEK 293T
cells (ATCC) were grown in DMEM media supplemented
with 10% FBS (both from Invitrogen). Rabbit polyclonal
antibodies against Myo IIA heavy chain were purchased
from Sigma. Antibodies against actin were purchased from
Santa Cruz Biotechnology. Neutrophils were pretreated
with NADPH oxidase inhibitor, diphenylamine iodonium
(DPI; Sigma) at a concentration of 3 µM/L for 30 min prior
to incubation with PRBC-derived plasma.
− production. Formyl-Met-
−/3 × 106 neutrophils/min.
Neutrophil migration, morphological changes and
Glass chamber slides (Nalge Nunc International) were
coated for 1 h at 37°C, with ICAM-1-Fc (R&D Systems,
Minneapolis, MN) at a concentration of 80 nM. Time-lapse
video-microscopy was used to measure real time migra-
tion of human neutrophils on coated chamber slides.
Time-lapse movies were taken every 5 s. Cells were
maintained at 37°C in an FCS2 live cell imaging chamber
(Bioptechs) in glucose medium containing IL-15 (2 mg/
mL; Sigma) Zymosan-induced luminol-enhanced che-
miluminescence (CL; Invivogen, 300 µg/30 µL PBS) was
used to measure the phagocytic response of neutrophils.
The CL signal produced by phagocytosing leukocytes was
measured in a luminometer (Model Luminoscan-RT;
Labsystems). Thirty cycles of measurements using a 5 s
counting time and a 70 s interval time were performed
for each sample. Neutrophil counts were determined in
all the samples using a hemocytometer.(8) Blebbistatin
(Calbiochem), a specific type II myosin inhibitor, was used
to inhibit myosin activity. Ascorbic acid (AA) and stau-
rosporine (STS) were obtained from Sigma. Neutrophils
(2 × 106/mL) were treated for 1 h at 37°C with 100 µM
blebbistatin, 1 µM STS or 100 µM AA prepared in DMSO
Analysis of variance (ANOVA) followed by a multiple
comparison test or Student’s t-test was performed to
determine statistical significance. Values were taken to
be statistically significant at p < 0.05.
Differential oxidative burst and protein
phosphorylation patterns in neutrophils treated with
different PRBC-derived plasma preparations
We evaluated the effect of plasma on oxidative burst by
comparing oxygen consumption in neutrophils incu-
bated with PRBC-derived plasmas prepared under dif-
ferent conditions. There was an increase in superoxide
production when neutrophils were incubated with the
different PRBC-derived plasma preparations, suggesting
that PRBC-derived plasma induced an oxidative burst
in human neutrophils. fMLP was used as a positive con-
trol and untreated neutrophils were used as a control.
Aged PRBC-derived plasma (42-day storage; NLR-42D)
induced a significantly higher magnitude of oxidative
burst when compared with fresh PRBC-derived plasma (1
day storage; NLR-1D) (p < 0.05; Figure 1A). Preincubation
of neutrophils with the NADPH oxidase inhibitor, DPI,
resulted in a significant abrogation of superoxide pro-
duction evoked by aged PRBC-derived plasmas (p <
0.05), suggesting the involvement of the NADPH oxidase
machinery in aged PRBC-derived plasma-evoked super-
Since oxidative burst triggered by UV or cytokines is
known to induce protein tyrosine phosphorylation, we
evaluated the effect of plasma on protein tyrosine phos-
phorylation in neutrophils. We incubated whole cell neu-
trophil extracts with different plasma preparations for 1 h
and immunoblotted with anti-pY20 antibody to show that
74 C. Yu et al.
Immunopharmacology and Immunotoxicology
Figure 1. Fresh and aged plasmas trigger oxidative burst and protein phosphorylation in neutrophils. (A) Representative results of
ROS generation in untreated normal human neutrophils (Control), and neutrophils treated for 1 h with fresh (1 day preparation) non-
leukocyte-reduced plasma (NLR-1D), aged (42 day preparation) non-leukocyte-reduced plasma (NLR-42D), aged leukocyte-reduced
plasma (LR-42D), and NLR-42D plus NADPH oxidase inhibitor DPI (NLR-42D + DPI). The results are expressed as means ± SD from 3
experiments. fMLP: formyl-Met-Leu-Phe (as a positive control). (*p < 0.05, compared with NLR-42D). (B) Western blot analysis of protein
tyrosine phosphorylation in response to different preparations of plasma. Normal human neutrophils were incubated with the different
plasma preparations for 1 h. Whole cell lysates were blotted with anti-pY20 antibody. (C) Normal human neutrophils were incubated with
the different plasma preparations for 1 hr. Whole cell lysates prepared from these neutrophils were subjected to antibody array analysis
with immunoblotted with anti-pY20-HRP. (D-E) Western blot analysis demonstrated tyrosine phosphorylation of IKK, p105 and p50 in
response to plasma treatment (*p < 0.05, compared with NLR-42D). 1D represents the ratio of p-IKK value over IKK. 1E represents the ratio
of p105 value over p50.
Plasma induces neutrophil activation 75
© 2013 Informa Healthcare USA, Inc.
aged PRBC-derived plasma induced higher levels of pro-
tein phosphorylation when compared with fresh PRBC-
derived plasma (p < 0.05; Figure 1B). We performed an
antibody array analysis in order to identify the proteins
that were tyrosine phosphorylated. Whole cell extracts
were prepared from neutrophils which were incubated
with different plasma preparations. The extracts were
incubated with our antibody arrays immobilized with
400 different antibodies as previously described.(21–24) The
arrays immunoblotted with anti-pY20 antibody showed
that freshly prepared and aged stored PRBC-derived
plasmas induced differential protein tyrosine phosphory-
lation (Figure 1C). The criterion for differential protein
tyrosine phosphorylation was a 3-fold difference in signal
intensity between the spots in the NLR-42D group and
the corresponding spots in the control group. Neutrophils
incubated with aged PRBC-derived plasma preparations
showed higher levels of phosphorylation of c-Abl (I11),
c-Rel (C2), Hdac6 (E1), IKK (E16), Lyn (N6), p16 (G17),
and RIP (I20, I21) when compared with neutrophils
incubated with fresh PRBC-derived plasma (Figure 1C).
We used western blots to confirm the phosphorylation of
proteins involved in NF-kB pathway such as p105 and Ikk
and p50 (Figure 1D–1E).
Plasmas induce type II nonmuscle myosin expression
Since NF-kB activation is thought to play a role in
cell migration via activation of the myosin-actin
cytoskeletonsome,(22) we evaluated the expression of
MYH9 in plasma-treated neutrophils. Interestingly,
although MYH9 was expressed in T cells, B cells, and
macrophages, we were unable to detect MYH9 expression
in untreated neutrophils (Figure 2A). However, we
showed induction of MYH9 in neutrophils treated with
different plasma preparations. Aged PRBC-derived
plasma induced higher levels of MYH9 expression when
compared with fresh PRBC-derived plasma (p < 0.05;
Figure 2B). Additionally, the NLR-42D plasma group
had significantly higher levels of MYH9 compared to the
LR-42D group (p < 0.05). Induction of MYH9 by plasma
was abolished by NADPH oxidase inhibitor DPI (p < 0.05;
Figure 2C), suggesting that superoxide played a role in
the regulation of MYH9 expression.
Plasmas induce morphological change, migration and
phagocytosis in neutrophils
We investigated the effect of plasma on neutrophil
migration. We showed that neutrophils incubated with
different PRBC-derived plasma preparations, became
polarized and migrated on ICAM-1 coated surfaces
with a steady-state migration velocity of approximately
10µm/min. Analysis of 100 cells showed that the migrat-
ing neutrophils exhibited rapid changes in shape, formed
constriction rings, and showed concomitant cytoplasmic
streaming (Figure 3A).
Based on the hypothesis that oxidative burst and
tyrosine phosphorylation may play a critical role in
MYH9 accumulation in plasma-treated neutrophils, we
investigated the effect of anti-oxidant AA and tyrosine
kinase inhibitor staurosporin (STS) on neutrophil migra-
tion and morphological changes. We also investigated if
blebbistatin, a specific inhibitor of myosin type II, could
inhibit neutrophil migration in vitro. Neutrophils were
pre-treated with AA (an anti-oxidant), STS (a tyrosine
kinase inhibitor) or blebbistatin for 1 h before incubat-
ing with different plasma preparations. We showed that
incubation with aged PRBC-derived plasma induced
more significant morphological changes when com-
pared with fresh PRBC-derived plasma (p < 0.05; Figure
3C). We also showed that pretreatment of neutrophils
with AA, STS or blebbistatin abolished the morphologi-
cal changes and cell migration induced by PRBC-derived
plasma (p < 0.05; Figure 3B and 3D). DMSO was used as
a vehicle control.
We investigated if plasma induced neutrophil phago-
cytosis. Our results showed that neutrophils exhibited
more efficient phagocytosis of yeast in the presence of
aged PRBC-derived plasma compared to fresh PRBC-
derived plasma (p < 0.05; Figure 4A–4B). Pre-treatment
of neutrophils with blebbistatin, AA or STS blocked this
process efficiently (p < 0.05; Figure 4C).
Figure 2. Fresh and aged plasmas induce nonmuscle type IIA
myosin (MYH9) expression in neutrophils. (A) Of the immune
cells tested, myosin type II was detected in T cells, B cells and
macrophages, but not in neutrophils (B) The NLR-42D plasma
group had significantly higher levels of MYH9 compared to the
LR-42D plasma group (*p < 0.05). (C) NADPH oxidase inhibitor
DPI abolished the plasma-induced expression of MYH9 in the
NLR-42D group (*p < 0.05).
76 C. Yu et al.
Immunopharmacology and Immunotoxicology
In this study, we showed that aged PRBC-derived plasma
was more potent than fresh PRBC-derived plasma at
inducing superoxide production in neutrophils. Antibody
array analysis showed that this oxidative burst was
accompanied by increased protein tyrosine phosphory-
lation. Some of the key proteins phosphorylated were
members of the NF-kB family (p105, IKK and p50). We
showed upregulation of non-muscle myosin IIA, MYH9,
in plasma-treated neutrophils. MYH9 upregulation was
reversed by NADPH oxidase inhibitor, DPI. We also
showed that aged plasma promoted neutrophil migra-
tion and induced morphological changes and phago-
cytosis. These processes were reversed by pretreating
neutrophils with blebbistatin, a myosin type II inhibitor.
TRALI is initiated by an immune priming step, followed
by exposure to specific biological response modifiers via
transfusion, causing an interaction between neutrophils,
platelets and lung endothelium.(4) Early research indi-
cated the presence of a priming agent present in PRBCs,
WB and platelet concentrates (PCs), which activated this
enzyme system.(25) This priming activity was observed
in aged blood, but not in fresh blood. Further analysis
showed the presence of lysophosphatidylcholines as well
as a lipid priming activity in WB, PRBCs and PCs.(2) Since
fresh frozen plasma did not contain this lipid priming
activity, such bioactive lipids which appeared after a few
days of storage, were thought to be generated via cellular
Our results agreed with these studies and showed
higher levels of superoxide production in neutrophils
incubated with aged PRBC-derived plasma when com-
pared with fresh PRBC-derived plasma. Since phagocyte
Figure 4. Blebbistatin, ascorbic acid, and STS suppress
phagocytosis in plasma-treated neutrophils. Neutrophils were
incubated with NLR-1D or NLR-42D from 4 different donors in
triplicate (*p < 0.05, compared with NLR-42D). (B) Blebbistatin,
ascorbic acid, and STS pretreatment significantly blocked plasma-
induced phagocytosis in 6 different donors in triplicate (*p < 0.05,
compared with NLR-42D).
Figure 3. Blebbastatin, ascorbic acid, and STS disrupt migration-
related morphological change in neutrophils. (A) Fresh and aged
plasma induced neutrophil morphological changes (n = 100
cells). (B) Blebbistatin, ascorbic acid, and STS abrogated plasma-
induced morphological changes in neutrophils (n = 7 blood
samples from 7 different volunteers; in triplicate). DMSO was used
as a vehicle control. (C) Aged plasma induced a greater number of
morphological changes than fresh plasma (*p < 0.05, compared
with NLR-42D) (n = 5 blood samples from 5 different volunteers;
in triplicate) (D) Blebbistatin, ascorbic acid, and STS significantly
abrogated plasma-induced neutrophil morphological change,
(*p < 0.05, compared with NLR-42D).
Plasma induces neutrophil activation 77
© 2013 Informa Healthcare USA, Inc.
activation is characterized by oxidative burst (increased
production of superoxide anions), these data suggested
that aged plasma would be more efficient than fresh
plasma at inducing phagocytosis. We used a yeast phago-
cytosis assay and showed higher levels of phagocytic
activity in neutrophils exposed to aged plasma when
compared to fresh plasma. Interestingly, Lyn, a member
of the Src family tyrosine kinases, was previously shown
to play an important role in phagocytosis by alveolar
macrophages via regulation of respiratory burst.(26) It will
be interesting to more fully characterize the molecular
mechanisms underlying increased phagocytosis in neu-
trophils treated with aged PRBC-derived plasma.
Migration of cells is a complex process, which results
in the generation of motor forces through cytoskeleton
reorganization. We previously showed that TNF induced
migration of T cells by regulation of the myosin-actin
cytoskeletonsome.(22) There are conflicting reports on
the effect of transfusion on cell migration and chemo-
taxis. Supernatant from standard RBC preparations
was recently shown to prime polymorphonuclear cells
(PMNs) and induce chemotaxis.(27) These effects were
directly correlated with storage time. Other reports indi-
cated that supernatants from stored RBCs could inhibit
fMLP-induced neutrophil chemotaxis as well as induce
neutrophil migration.(28) RBC transfusions were also
recently shown to inhibit neutrophil chemotaxis.(29)
Our data showed that aged PRBC-derived plasma
was more efficient than fresh PRBC-derived plasma
at inducing protein phosphorylation in neutrophils.
Phosphorylation and activation of mitogen-activated
protein kinase, p38, was previously shown to be required
for priming of respiratory burst in neutrophils.(30)
Early studies also showed enhanced respiratory burst
in neutrophils stimulated with granulocyte-colony
stimulating factor (G-CSF), which resulted in activation of
transcription factor p80c-rel.(31) In this study, we showed
that among the proteins that were phosphorylated, were
members of the NF-kB family (p105, p50 and IKK). Since
NF-kB activation has been long suspected to play a role in
cell migration, presumably by transcriptional regulation
of genes involved in migration,(32) our data suggest a
regulatory role for NF-kB in plasma-induced neutrophil
Non-muscle myosin II has previously been implicated
in the migration of tumor cells and leukocytes(18,33) and
has been shown to participate in the redistribution of
adhesion receptors in the immunological synapse.(34,35)
Myosin IIA-deficient T-cells exhibit decreased interstitial
migration.(36) Furthermore, the H chain of myosin II is
known to play a role in regulation of pseudopod forma-
tion and chemotaxis in Dictyostelium discoideum.(37)
Interestingly, MYH9, an isoform of the non-muscle type
II myosin H chain, has been shown to be important for
spontaneous (matrix-induced) as well as ligand-induced
migration of leukocytes. In this study, we showed upreg-
ulation of MYH9 expression in neutrophils incubated
with aged PRBC-derived plasma. These data are consis-
tent with the increased migration observed in these cells.
We used blebbistatin, a specific myosin type II inhibi-
tor, to look at the role of MYH9 in neutrophil migration.
Blebbistatin has previously been shown to inhibit cell
migration and cell morphological changes during cytoki-
nesis.(16,38) We showed a significant abrogation of plasma-
induced cell migration and morphological changes in
neutrophils which were pretreated with blebbistatin.
Taken together, our data showed that neutrophils treated
with PRBC-derived plasma expressed significant amounts
of MYH9 protein, while resting neutrophils did not. It will be
interesting to investigate the kinetics of MYH9 upregulation
and cell migration in response to PRBC-derived plasma. A
number of studies have investigated possible mediators
of neutrophil activation and transfusion-related immu-
nomodulation. H2O2 was shown to amplify the innate
immune response in certain inflammatory disorders, by
stimulating NOX2-mediated superoxide production in
neutrophils via Ca(2+)/c-Abl signaling pathway.(39) Based
on our results, we suggest that NLR-42D PRBCs induced
superoxide production in neutrophils, leading to tyrosine
phosphorylation and activation of transcription factors
such as NF-kB, which may regulate MYH9 transcription.
MYH9 and ICAM-1 were previously suggested to play a
role in T cell migration.(18) Our study, showing inhibition of
neutrophil migration in response to blebbistatin, a myosin
type II inhibitor is further validation that MYH9 plays a role
in neutrophil migration. Furthermore, inhibition of MYH9
expression in response to DPI, an NADPH inhibitor, sug-
gested that superoxide production plays a role in NLR-42D
PRBC-induced MYH9 expression. The use of a no-plasma
control group as well as the LR-42D negative control group
in every experiment served to ensure that the difference
between the fresh and aged plasma observed in our study
were indeed authentic and not due to artifactual priming.
One limitation of our present study was that we did not
use pooled donated blood samples. Individual differences
may therefore play a role in the interpretation of the results.
We would like to validate our results using larger sample
sizes and pooled samples. We also did not explore if expo-
sure of neutrophils to activators of oxidative burst could
upregulate MYH9 expression. In our future studies, we
would like to perform a finer dissection of the molecular
mechanisms dictating the pathogenesis of transfusion-
driven acute lung injury in vitro and in vivo.
To the best of our knowledge, we are the first to
demonstrate a functional role of MYH9 in the directional
migration of immune cells. Our findings indicate that
transfusion with aged PBRC-derived plasmas resulted
in a number of biologic effects such as (i) increased
protein tyrosine phosphorylation, capable of triggering
different signal transduction pathways in neutrophils,
(ii) increased MYH9 expression in neutrophils, and
(iii) modulation of phagocytosis, cell migration and
morphological changes in neutrophils. Plasma-induced
changes in migration and morphology were likely
78 C. Yu et al.
Immunopharmacology and Immunotoxicology
mediated via MYH9 and were abrogated by pretreatment
of neutrophils with blebbistatin, a specific inhibitor
of type II myosin. Our data provide an insight into the
cellular and molecular mechanisms of transfusion-
related injury and suggest that blebbistatin is a potential
therapeutic agent for blood transfusion related disorders.
Declaration of interest
This research was sponsored by the National Natural
Science Funds 30871181 and 81070547 (to Chen Yu). The
authors declare no conflict of interest.
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