Hindawi Publishing Corporation
Advances in Pharmacological Sciences
Volume 2011, Article ID 691928, 7 pages
TheImmunomodulatoryEffects ofAlbumin InVitro and InVivo
Derek S.Wheeler,1,2JohnS.GiulianoJr.,1PatrickM. Lahni,1AlvinDenenberg,1
HectorR.Wong,1,2and Basilia Zingarelli1,2
1Division of Critical Care Medicine, The Kindervelt Laboratory for Critical Care Medicine Research,
Cincinnati Children’s Research Foundation, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229-3039, USA
2Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229-3039, USA
Correspondence should be addressed to Derek S. Wheeler, firstname.lastname@example.org
Received 19 November 2010; Revised 17 February 2011; Accepted 23 February 2011
Academic Editor: William J. Wheeler
Copyright © 2011 Derek S. Wheeler et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
Albumin appears to have proinflammatory effects in vitro. We hypothesized that albumin would induce a state of tolerance to
subsequent administration of lipopolysaccharide (LPS) in vitro and in vivo. RAW264.7 and primary peritoneal macrophages were
treated with increasing doses of bovine serum albumin (BSA) and harvested for NF-κB luciferase reporter assay or TNF-α ELISA.
In separate experiments, RAW264.7 cells were preconditioned with 1mg/mL BSA for 18h prior to LPS (10μg/mL) treatment
and harvested for NF-κB luciferase reporter assay or TNF-α ELISA. Finally, C57Bl/6 mice were preconditioned with albumin
via intraperitoneal administration 18h prior to a lethal dose of LPS (60mg/kg body wt). Blood was collected at 6h after LPS
administration for TNF-α ELISA. Albumin produced a dose-dependent and TLR-4-dependent increase in NF-κB activation and
TNF-α gene expression in vitro. Albumin preconditioning abrogated the LPS-mediated increase in NF-κB activation and TNF-α
gene expression in vitro and in vivo. The clinical significance of these findings remains to be elucidated.
Latta in 1832 . Since that time, early and aggressive
intravenous fluid resuscitation has become one of the top
management priorities in critically ill children and adults
with shock. However, the optimal type of resuscitation
fluid, crystalloid or colloid, remains controversial [2–5].
For example, a systematic review of 26 randomized trials
involving over 1,500 critically ill patients suggested that the
use of albumin for fluid resuscitation increased the absolute
risk of mortality by 4% (95% confidence interval 0% to
8%) . A follow-up systematic review suggested that, while
there were no differences in mortality or length of hospital
stay between isotonic crystalloid and colloid resuscitation,
a subgroup analysis suggested that crystalloid resuscitation
in critically ill patients with trauma was associated with a
lower mortality . A large, multicenter, prospective, ran-
domized trial (SAFE Study) involving nearly 7,000 patients
showed that there were no differences in organ failures,
intensive care unit (ICU) length of stay, the number of days
on mechanical ventilatory support, or mortality between
patients assigned to 4% albumin versus normal saline for
fluid resuscitation . However, a post hoc study suggested
that fluid resuscitation with albumin was associated with
higher mortality in patients with traumatic brain injury
. Finally, albumin administration was associated with
increased mortality and length of stay in a large, European
multicenter, observational cohort study performed in over
3,000 critically ill adults . The choice of intravenous
fluid may therefore impact outcome in critically ill patients
depending upon the particular clinical context.
The effects of various intravenous fluid preparations on
the host inflammatory response are well described [9, 10].
The conflicting data on albumin administration in critically
ill patients discussed above support the observation that
intravenous fluidpreparations are not completelyinnocuous
and may, in fact, potentiate or modulate cellular injury,
again depending upon the clinical context. Several studies
suggest that albumin circulating in the blood at physi-
ologic concentrations facilitates the interactions between
endotoxin- and lipopolysaccharide-binding protein (LBP)
2Advances in Pharmacological Sciences
and CD-14, thereby assisting with pathogen recognition and
the subsequent host inflammatory response . Recent ex-
perimental data suggest that exogenous albumin treatment
produces a dose-dependent increase in proinflammatory
gene expression in vitro, primarily through a mechanism
involving activation of the transcription factor, NF-κB [12–
17]. Albumin may act via the mitogen-activated protein
kinase (MAPK)pathway,in thisregard [18,19].Wetherefore
hypothesized that albumin treatment would modulate the
proinflammatory response to LPS in vitro and in vivo.
(American Type Culture Collection, Bethesda, Md) were
maintainedin Dulbecco’s Modified
(DMEM, Gibco BRL, Grand Island, NY) containing 10%
fetal bovine serum (FBS), 100U/mL penicillin, 0.1mg/mL
streptomycin, 20mM HEPES buffer, and 2.2g/L sodium
bicarbonate (Sigma, St. Louis, Mo) at 37◦C in a room
air/5% CO2 tissue culture incubator. The human acute
monocytic leukemia cell line, THP-1, was maintained in
RPMI 1640 medium containing 10% FBS, kanamycin,
2-β-mercaptoethanol, and 2% glutamine (pH 7.35) at 37◦C
in a room air/5% CO2tissue culture incubator. Cells were
used between passages 3–5.
2.2. Isolation of Murine Peritoneal Macrophages. In separate
experiments, primary peritoneal macrophages were isolated
from C3H/HeJ and C3H/HeOuJ mice (Jackson Labora-
tory, Bar Harbor, Me) via peritoneal lavage. C3H/HeJ and
C3H/HeOuJ mice were housed in a laminar hood in a virus-
free animal facility prior to isolation of macrophages. All
Institutes of Health Guidelines for the Use of Laboratory
Animals (National Institutes of Health Publication 85-23,
revised 1996) and with approval of the Institutional Animal
Care and Use Committee, Cincinnati Children’s Research
Foundation. Animals were acclimatized for 7 days prior to
surgical manipulation and maintained on 12-h light/dark
cycles with access to food and water ad libitum. Briefly, mice
were anesthetized with isoflurane, and the peritoneal fascia
was exposed by dissection. Three mL of sterile PBS was
injected through the fascia into the peritoneal cavity using
a sterile 22-G needle. Peritoneal fluid was then withdrawn
through the fascia with an 18-G needle. The recovered peri-
toneal fluid was centrifuged at 1500rpm×10min, and the
pellet was isolated, resuspended in DMEM containing 10%
FBS, and plated on a 96 well plate. Peritoneal macrophages
were allowed to adhere for 1 hour at 37◦C. Cytospin of the
sample was performed to confirm the percentage of macro-
phages present, which was consistently greater than 90% of
the cells in the sample.
2.3. Transient Transfection and Luciferase Reporter Assay.
RAW264.7 cells were transiently transfected with a 3xNF-κB
luciferase reporter plasmid in duplicate, in six-well plates,
at a density of 200,000 cells per well by incubation with
FuGENE 6 (Roche Molecular Biochemicals, Indianapolis,
Ind) and serum-free DMEM overnight. After transfection,
cells were washed once with PBS and treated with increasing
concentrations of bovine serum albumin (BSA) (Sigma-
Aldrich, St. Louis, Mo). In separate experiments, cells were
preconditioned with BSA×18h at 37◦C prior to subsequent
treatment with LPS, 10μg/mL×6h at 37◦C.
In separate experiments, THP-1 cells were transfected
using DEAE-dextran. Briefly, 1×106THP-1cells/mL were
seeded into tissue culture flasks the day before transfection.
On the next day, 6mL of cell suspension were washed twice
with STBS (25mM Tris·Cl, pH 7.4, 137mM NaCl, 5mM
KCl, 0.6mM Na2HPO4, 0.7mM CaCl2, and 0.5mM MgCl2)
and pelleted. One μg/mL of NF-κB reporter plasmid was
mixed with DEAE-dextran (400μg/mL) in 140μL of STBS
buffer and immediately added to the pelleted THP-1 cells.
The cells were incubated at 37◦C for 20min, washed twice
with STBS, resuspended, and cultured in complete RPMI
medium. The transfected cell lines were cultivated for 48h
Cellular proteins were extracted and analyzed for lucif-
erase activity according to the manufacturer’s instructions
Luciferase activity wascorrected fortotal cellularproteinand
reported as fold induction over control cells (cells that were
transfected and treated with medium alone).
2.4.Enzyme-Linked Immunosorbent Assay (ELISA).RAW264.7
cells or primary peritoneal macrophages were precondi-
tioned with BSA, 1mg/mL×18h at 37◦C prior to subse-
quent treatment with LPS, 10μg/mL×6h at 37◦C. In sepa-
rate experiments, male C57Bl/6mice were preconditioned in
vivo with BSA, 10μg/kg body wt×18h. Primary peritoneal
macrophages were then isolated, allowed to adhere, and
treated ex vivo with LPS, 10μg/mL×6h at 37◦C. Cell super-
natants were collected and clarified (5,000rpm for 10min
at 4◦C) prior to being analyzed for tumor necrosis factor-
(TNF-) α via ELISA (BioSource International, Camarillo,
2.5. Nuclear Protein Extraction and EMSA. Nuclear proteins
were isolated from treated cells as previously described
 and stored at −70◦C until further analysis. EMSA was
performed using an oligonucleotide probe for the NF-κB
consensus site as previously described .
2.6. Murine Model of Endotoxin Shock. C57Bl/6mice (Jack-
son Laboratory, Bar Harbor, Me), 20–25g body weight,
were acclimatized for 7 days prior to surgical manipulation
and maintained on 12-h light/dark cycles with access to
food and water ad libitum. Briefly, mice were precon-
ditioned with bovine serum albumin (Sigma-Aldrich, St.
Louis, Mo), 10mg/kg body wt or vehicle, administered via
intraperitoneal (i.p.) injection and were returned to their
(LPS) (Sigma-Aldrich, St. Louis, Mo) was administered
via i.p. injection. Mice were anesthetized 6 hours later
with isoflurane, and blood was obtained via direct cardiac
Advances in Pharmacological Sciences3
puncture and centrifuged at 5,000g×10 minutes in EDTA-
at −80◦C until further analysis. Plasma concentration of
tumor necrosis factor- (TNF-) α was measured via com-
mercially available enzyme-linked immunosorbent assay kits
(Biosource International, Camarillo, Calif) using the proto-
col recommended by the manufacturer.
2.7. Statistical Analysis. All continuous data are reported as
mean±SEM and were compared using one-way analysis of
variance and Student Newman-Keuls test (Stata 11.1, Stata-
Corp, College Station, Tex). A P value <.05 was considered
3.1. Albumin Dose-Dependently Induces NF-κB-Dependent,
TLR-4-Dependent TNF-α Gene Expression in Murine Peri-
toneal Macrophages. Prior studies have shown that exoge-
in proinflammatory gene expression in vitro, primarily
through a mechanism involving activation of the transcrip-
tion factor, NF-κB [12–16]. In order to confirm these results,
we transfected muirine peritoneal macrophages(RAW264.7)
with a 3x-NF-κB luciferase reporter plasmid prior to treat-
ment with increasing doses (0.5–30mg/mL) of Fraction V
BSA (Sigma-Aldrich, St. Louis, Mo). BSA treatment resulted
in a dose-dependent increase in NF-κB promoter activity.
Similarly, BSA treatment resulted in a dose-dependent
increasein theNF-κB-dependentproinflammatory cytokine,
TNF-α, as measured by ELISA (Figure 1).
In order to confirm that these results were not due
to potential endotoxin contamination of the commer-
cial albumin preparation, we repeated the experimental
conditions in the presence of the endotoxin inhibitor,
polymyxin B (100μg/mL added 1h before albumin or
LPS treatment, resp.). While polymyxin B reduced BSA-
induced TNF-α expression, the difference was not statis-
tically significant (BSA 30mg/mL: 1995±75pg/mL versus
BSA 30mg/mL+polyB: 1100±50pg/mL, P = NS). In stark
contrast, polymyxin B cotreatment significantly inhibited
LPS-mediated TNF-α expression back to baseline (LPS
10μg/mL: 7,000±200pg/mL versus LPS 10μg/mL+polyB:
50±0pg/mL, P < .05). In addition, we repeated the ELISA
experiments using a low-endotoxin albumin preparation
(Sigma-Aldrich, St. Louis, Mo). Both albumin preparations
significantly increased TNF-α expression compared to con-
trol, and there were no significant differences between Frac-
tion V BSA versus low-endotoxin albumin (data not shown).
Finally, as an additional control, we independently verified
the level of endotoxin contamination in our albumin prepa-
ration via Limulusamoebocytelysate (LAL)assay performed
at Charles River Laboratories (Charleston, SC). The level
of endotoxin contamination (0.8828EU/mL) at the highest
albumin dose studied (30mg/mL) was equivalent to a LPS
dose of 12pg/mL. We treated RAW264.7 transfected with
3x NF-κB luciferase reporter plasmid with 12pg/mL LPS,
which was not significantly different from control cells (data
(fold induction over control)
Figure 1: Treatment with fraction V bovine serum albumin (BSA)
(Sigma-Aldrich, St. Louis, Mo) dose-dependently increases TNF-
α gene expression in murine peritoneal macrophages in an NF-
κB-dependent manner.(a)RAW264.7peritoneal macrophageswere
transiently transfected with a 3x NF-κB/luc reporter plasmid prior
to treatment with increasingdoses of BSAfor 24h (dosesin mg/mL
shown on the x-axis). BSA treatment resulted in increased NF-
κB activation in a dose-dependent manner. As a comparison, LPS
treatment (10μg/mL) resulted in a 3.9-fold increase in relative
in triplicate with 3 wells per condition (∗P < .05 compared
to control). (b) RAW264.7 peritoneal macrophages were treated
with increasing doses of BSA for 24h (doses in mg/mL shown
on the x-axis). BSA treatment resulted in a significant increase in
TNF-α, as measured by ELISA. As a comparison, LPS treatment
(10μg/mL) resulted in a much greater increase in TNF-α compared
to BSA (TNF-α 7000pg/mL) (data not shown). All experiments
were performed in triplicate with 3 wells per condition (∗P < .05
compared to control).
exogenous BSA induces proinflammatory gene expression
independent of any potential endotoxin contamination.
As an additional control to show that these effects
were not due to species-dependent differences (i.e., treating
mouse peritoneal macrophages with a bovine preparation
of albumin), we treated THP-1 cells transfected with the
3x NF -κB luciferase reporter plasmid with human recom-
binant albumin (Sigma-Aldrich, St. Louis, Mo). Human
albumin dose-dependentlyincreased NF-κBactivationinthe
human THP-1 cell line (Alb 1mg/mL: 1.5-fold induction;
4Advances in Pharmacological Sciences
Alb 3mg/mL: 5.0-fold induction; Alb 10mg/mL: 6.5 fold
induction; Alb 30mg/mL: 6.0-fold induction; P < .05).
We wanted to confirm that these findings were not
just a nonspecific effect of a high concentration of a high-
molecular-weight protein. We next treated RAW264.7 cells
transfected with the 3x NF-κB luciferase reporter plasmid
with increasing doses of the high-molecular-weight protein,
Dextran (Sigma-Aldrich, St. Louis, Mo). In contrast to the
results observed with BSA above, Dextran treatment did not
increase NF-κB activation, even at the highest dose studied
(30mg/mL) (data not shown).
Finally, previous studies suggest that exogenous albu-
min does not induce proinflammatory gene expression in
macrophages . We therefore wished to confirm our
results in primary murine macrophages. We also wanted to
albumin administration were dependent upon the Toll-like
receptor- (TLR-) 4 pathway [20, 21]. We therefore treated
peritoneal macrophages obtained from both LPS-resistant
LPS-sensitive C3H/HeOuJ mice. BSA treatment resulted in a
Collectively, these data extend the findings from previous
studies [12–16] and further demonstrate that exogenous
albumin treatment produces a dose-dependent increase in
proinflammatory gene expression in a NF-κB-dependent
manner. Moreover, exogenous albumin produced a dose-
dependent increase in TNF-α in peritoneal macrophages
obtained from LPS-sensitive C3H/HeOuJ mice, but not in
LPS-resistant C3H/HeJ mice, suggesting that these effectsare
dependent upon the Toll-like receptor- (TLR-) 4 pathway.
3.2. Albumin Preconditioning Abrogates LPS-Mediated TNF-α
GeneExpression in RAW264.7Macrophages. Giventheeffects
of albumin on TNF-α expression in vitro and the potential
role of TLR-4 in this process, we hypothesized that albumin
preconditioning would attenuate subsequent LPS-mediated
TNF-α expression in RAW264.7 macrophages, similar to the
phenomenon of endotoxin tolerance [20, 21]. We therefore
preconditioned RAW264.7 macrophages with a low dose
(i.e., a dose that did not significantly activate NF-κB activity
or TNF-α expression) of BSA, 1mg/mL ×18h at 37◦C, prior
to a subsequent treatment with LPS, 10μg/mL. As shown in
Figure 3, albumin preconditioning abrogated LPS-mediated
TNF-α expression. We next transfected RAW264.7 cells
with the 3x-NF-κB luciferase reporter plasmid and repeated
these experiments. As expected, LPS treatment resulted in
a significant increase in NF-κB promoter activation. Albu-
min preconditioning significantly inhibited subsequent LPS-
mediated NF-κB promoter activation (Figure 4(a)). These
results were further confirmed by EMSA (Figure 4(b)).
We wanted to confirm that these findings were not
just a nonspecific effect of a high concentration of a high-
molecular-weight protein. We therefore preconditioned
RAW264.7 cells transfected with the 3x NF -κB luciferase
reporterplasmid with increasing dosesofthehigh-molecular
weight protein Dextran (1–30mg/mL) prior to subsequent
C3H/HeJ (TLR-4 mutant)
P < .05
P < .05
P < .05
Figure 2: BSA increases TNF-α gene expression in murine peri-
toneal macrophages in a TLR-4-dependent manner. Primary peri-
toneal macrophages were isolated from C3H/HeJ (TLR-4 mutant)
and C3H/HeOuJ (wild-type) and were treated with BSA for 24h.
As expected, LPS treatment (10μg/mL) resulted in a significant
difference in TNF-α induction between macrophages isolated from
C3H/HeJ mice and their wild-type counterparts. BSA treatment
resulted in a dose-dependent increase in TNF-α in macrophages
isolated from C3H/HeOuJ mice, but not in macrophages isolated
from C3H/HeJ mice. All experiments were performed in triplicate
with 3 wells per condition.
Alb + LPS
Figure 3: Albumin preconditioning abrogates LPS-mediated TNF-
macrophages were preconditioned with BSA (1mg/mL) for 18h
prior to a subsequent treatment with LPS (10μg/mL). LPS treat-
ment resulted in a significant increase in TNF-α expression, as
measured by ELISA. Albumin preconditioning, however, signifi-
cantly abrogated LPS-mediated TNF-α expression. All experiments
were performed in triplicate with 3 wells per condition (∗P < .05
compared to control;#P < .05 compared to LPS alone).
Advances in Pharmacological Sciences5
Alb + LPS
(fold induction over control)
Figure 4: Albumin preconditioning attenuates LPS-mediated NF-κB activation. (a) NF-κB luciferase experiments. RAW264.7 peritoneal
macrophages were transiently transfected with a 3x NF -κB/luc reporter plasmid. Cells were allowed to recover overnight and were then
preconditioned with BSA (1mg/mL) for 18h prior to a subsequent treatment with LPS (10μg/mL). LPS treatment resulted in a significant
increase in NF-κB promoter activation, which was significantly inhibited by albumin preconditioning. All experiments were performed in
triplicate with 3 wells per condition (∗P < .05 compared to control;#P < .05 compared to LPS alone). (b) EMSA. RAW264.7 peritoneal
macrophages were preconditioned with BSA (1mg/mL) for 18h prior to a subsequent treatment with LPS (10μg/mL). Nuclear protein
was harvested at 30min after LPS and EMSA were performed. LPS treatment resulted in a significant increase in NF-κB binding (Lane 2)
compared to either control (Lane 1) or albumin preconditioning alone (Lane 3). Albumin preconditioning abrogated NF-κB binding (Lane
4) compared to LPS alone. Lane 5 (LPS treatment, cold competitor), Lane 6 (LPS treatment, p65 supershift), Lane 7 (LPS treatment, p50
as additional controls to demonstrate specificity and the nature of the NF-κB. The EMSA shown is representative of 3 separate experiments,
all with similar results.
LPS treatment. Again, in contrast to the results observed
with BSA preconditioning, Dextran preconditioning did not
inhibit LPS-mediated NF-κB activation (LPS treatment: 3.9-
foldinductionversusDextran+LPS: 4.1-foldinduction,P =
NS). Collectively, these data confirm that albumin precondi-
tioningattenuatesLPS-mediated TNF-αgeneexpression and
NF-κB activation in vitro.
In Vivo. Albumin is one of the most abundant serum
proteins in vivo. Given the immunomodulatory effects of
physiologic levels of albumin , it is difficult to discern
whether the immunomodulatory effects observed in the
in vitro experiments above would be observed following
the administration of exogenous albumin in the clinical
albumin treatment could abrogate LPS-mediated TNF-α
gene expression in vivo. Our group has previously shown
that intraperitoneal administration of LPS, 60mg/kg results
in significant induction of TNF-α at 6h after injection .
Six-week-old C57Bl/6 mice (20–25g) were preconditioned
with either saline vehicle or BSA (10mg/kg i.p.) 18h prior
to a subsequent challenge with LPS, 60mg/kg. Consistent
with our previous study , LPS injection produced a
significant increase in plasma TNF-α,which wassignificantly
abrogated by albumin preconditioning (Figure 5). Collec-
tively, these data demonstrate that albumin preconditioning
modulates the proinflammatory response to LPS in vivo,
suggesting that administration of exogenous albumin may
have immunomodulatory effects in the clinical setting.
Alb + LPS
Figure 5: Albumin preconditioning abrogates LPS-mediated TNF-
α expression in vivo. C57Bl/6 mice, 20–25g body wt, were
preconditioned with BSA (10mg/kg body wt, i.p.) or vehicle
18h prior to subsequent treatment with a lethal dose of LPS
(60mg/kg body wt, i.p.). Plasma was harvested at 6h, and TNF-
α was measured via ELISA. Albumin preconditioning significantly
inhibited LPS-mediated plasma TNF-α expression. Experiments
were performed in triplicate with 5mice per experimental group
(∗P < .05 compared to control;#P < .05 compared to LPS alone).
Herein, we confirm previous in vitro studies which demon-
strate that albumin increases proinflammatory gene expres-
sion in an NF-κB-dependent manner [12–14, 16]. We
extend these findings to show that albumin exerts these
6Advances in Pharmacological Sciences
proinflammatory effects through Toll-like receptor- (TLR-)
4. Finally, we demonstrate that albumin pretreatment, or
preconditioning, significantly attenuates subsequent LPS-
mediated TNF-α gene expression in vitro and in vivo.
Albumin preconditioning therefore elicits a cellular response
similar to classic endotoxin tolerance [21, 23]. For example,
while LPS induces a dramatic increase in TNF-α gene
expression in human peripheral blood monocytes (PBMCs),
a second exposure produces a markedly attenuated response
with decreased TNF-α gene expression . The common
assertion that endotoxin tolerance represents a global down-
regulation of proinflammatory gene expression, however, is
proinflammatory cytokines such as IL-1β and IL-6 may be
increased, decreased, or unchanged . In addition, early
studies suggested that endotoxin tolerance resulted in both
(i) a diminished proinflammatory response to a subsequent
dose of LPS in vitro and in vivo and (ii) increased phagocyto-
sis in vitro . As such, the clinical relevance of endotoxin
tolerance remains unknown.
effects that likely impact the host inflammatory response
in critical illness. The impact of these immunomodulatory
effects may, in turn, depend upon the clinical context
(i.e., sepsis, hemorrhagic shock, or trauma). For example,
the immunomodulatory effects of albumin in critically ill
patients with a predominantly proinflammatory phenotype
may improve outcome, whereas these same effects may
worsen outcome in critically ill patients with a predomi-
nantly anti-inflammatory phenotype. Consistent with this
notion, albumin fluid resuscitation prevents experimental
lung injury following hemorrhagic shock [25–27], but not
after endotoxic shock . These questions remain an active
focus of investigation in many laboratories, including our
Several endogenous molecules, including several mem-
bers of the heat shock protein (Hsp) family of proteins (e.g.
, and HMGB-1 [33, 34], have recently been shown to
modulate the host inflammatory response in vitro. Recent
studies have questioned some of these findings due to the
possibility of bacterial contamination of the recombinant
proteins used in the aforementioned studies [35, 36]. With
this in mind, we performed several additional controls
in order to assure that endotoxin contamination of our
protein preparation was not responsible for these effects.
First, we independently verified the level of endotoxin con-
tamination in our protein preparation and showed that
this concentration of endotoxin did not induce TNF-α
gene expression. Next, we treated cells with a commercially
available “endotoxin-free” albumin preparation and noted
similar results. Finally, we conducted our experiments in the
presence of polymyxin B. While polymyxin B reduced the
proinflammatory effects of albumin, the difference was not
statistically significant. Collectively, these results suggest that
albumin has immunomodulatory properties that are distinct
from any bacterial contamination oftheprotein preparation,
consistent with previous observations [12, 16].
In conclusion, we show that albumin has potent immun-
omodulatory effects in vitro and in vivo. Albumin induces
TNF-α gene expression in RAW264.7 peritoneal macropha-
ges in a TLR4- and NF-κB-dependent manner. These effects
are not due to endotoxin contamination of the recombinant
protein. The clinical significance of these effects remains to
be elucidated and warrants further investigation.
D. S. Wheeler was supported by the National Institutes of
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