1079-6061/00/4801-0039
The Journal of Trauma: Injury, Infection, and Critical Care
Copyright © 2000 by Lippincott Williams & Wilkins, Inc.
Impact of Burn Injury on Hepatic TGF-b1 Expression and
Plasma TGF-b1 Levels
Tetsuro Nishimura, MD, Teruhiro Nishiura, MD, Suzan deSerres, BA, Takao Nakagawa, MD, David A. Brenner, MD, and
Anthony A. Meyer, MD, PhD
Background: The liver plays a critical regulatory role in the
acute inflammatory response to injury, although the mecha-
nisms of this regulation are not well understood. transforming
growth factor-b1 (TGF-b1) is induced after burn injury and
may contribute to an inhibitory or fatal effect on hepatocytes.
We investigated the association over time between plasma con-
centration of TGF-b1, expression of TGF-b1 m-RNA in liver
tissue, and histologic analysis of liver apoptosis after burn in-
jury.
Methods: Male BALB/c mice were anesthetized and random-
ized to receive 0% (sham), moderate (approximately 25%) (M),
or large (approximately 50%) (L) body surface area full-thick-
ness contact burn, followed by resuscitation and analgesia. An-
imals were killed over a time course from 15 minutes to 24 hours
after burn injury, and liver tissue and peripheral blood were
collected. Plasma levels of TGF-b1 (nanograms per milliliter)
were measured by enzyme-linked immunosorbent assay.
T
ransforming growth factor b (TGF-b) is a 25-kDa
polypeptide found in normal and transformed tissues.1
TGF-b can now be considered the prototype of a mul-
tifunctional cytokine, especially after the discovery that it
could act both as an inhibitor and stimulator of cell replica-
tion. In addition to restricting cell proliferation and differen-
tiation, TGF-b can also affect the synthesis of many of the
components of extracellular matrix. Therefore, TGF-b has a
critical role in tissue repair.2 TGF-b has at least five different
isoforms, three of which have been identified in mammals,
i.e., TGF-b1, b2, and b3.
Recently TGF-b1 has been shown to inhibit hepatocyte
DNA synthesis in vitro. Furthermore, after partial hepatec-
tomy, TGF-b1 inhibits in vivo hepatocyte DNA synthesis.3,4
It has also been shown that TGF-b1 is involved in the initi-
ation of hepatocyte apoptosis, both in vitro and in vivo.
Results from Ponchel et al. suggest that TGF-b1 is a signal
factor of apoptosis in the liver.5 Apoptosis is suggested to be
involved in the control of cell number, for example, during
embryonic development, and during involution of liver
hyperplasia.6 –10 It has been reported that the concentration of
Submitted for publication May 21, 1999.
Accepted for publication September 9, 1999.
From the Departments of Surgery and Jaycee Burn Center, University of
North Carolina, Chapel Hill, North Carolina.
Poster presentation at the 58th Annual Meeting of the American Associ-
ation for the Surgery of Trauma, September 24 –27, 1998, Baltimore, Mary-
land.
Address for reprints: Anthony A. Meyer, MD, PhD, Department of
Surgery, University of North Carolina, 163 Burnett-Womack Clinical Sci-
ences Building, Chapel Hill, NC 27599-7210.
39
Vol. 48, No. 1
Printed in the U.S.A.
TGF-b1 m-RNA was extracted from liver and measured by
reverse transcription-polymerase chain reaction. Histology of
liver apoptosis was examined after fixation and staining with
TdT-mediated dUTP nick-end labeling (TUNEL) method.
Results: The plasma concentration of TGF-b1 in burn group
L was significantly increased at 4 hours after burn when com-
pared with sham and M burn groups. This rise in plasma
TGF-b1 was preceded by an increase in hepatic TGF-b1 m-RNA
expression at 30 minutes, 1, 2, and 4 hours after burn in the L
group. Histologic analysis found greater hepatocyte death in the
L group than in the M group at 8 hours after burn.
Conclusion: The levels of induced TGF-b1 and TGF-b1
m-RNA after L burn injury are higher and peak earlier than
after M burn injury. Elevated TGF-b1 may be associated with
cell death in hepatocytes. The TGF-b1 rise may be associated
with hepatocyte injury and systemic response to massive burn.
plasma TGF-b1 increases after trauma11 and TGF-b1 mRNA
is increased after partial hepatectomy.12 But the effect on
TGF-b1 by burn injury has not been well investigated.
We hypothesized that burn injury would be followed by an
increase in plasma TGF-b1 concentration and an increase in
hepatocyte TGF-b1 mRNA expression and that this increase
in TGF-b1 would be followed by an increase in hepatocyte
apoptosis. We tested our hypothesis by examining changes in
levels of TGF-b1 plasma concentration, hepatocyte mRNA
expression, and hepatocyte apoptosis over time after burn
injury.
MATERIALS AND METHODS
Animal Protocols
Twenty- to 22-g, 6- to 8-week-old, inbred adult male BALB/c
mice (Harlan Sprague-Dawley, Inc., Indianapolis, Ind) were
used as subjects in all experiments. Animals were provided
food and water ad libitum throughout the experiment. All
protocols were approved by the Committee on Animal Re-
search, University of North Carolina at Chapel Hill, and
followed National Institute of Health (NIH) guidelines.
Experimental Design
BALB/c mice were randomized to three groups. The first
group contained sham mice. Sham animals (S) received treat-
ment identical to all other animals but did not receive the
actual burn. The second group (M) contained animals that
received a moderate, approximately 25% total body surface
area burn to the dorsum and bilateral flanks. The third group
(L) contained animals that received a large, approximately
The Journal of Trauma: Injury, Infection, and Critical Care
50% total body surface area burn to the dorsum and bilateral
flanks. The size of the area injured was confirmed by remov-
ing the entire skin from animals after injury and measuring
burn area.
Burn Injury
Burn injury was created as previously described.13 Briefly,
animals were anesthetized with inhalation of methoxyflurane
vapor (Pitman-Moore, Washington Crossing, NJ), and their
back and flank hair was clipped. A full-thickness contact burn
(moderate or large) was produced by applying a copper rod,
heated in boiling water, to the animal’s dorsum/flank for 10
seconds. Four applications of a 65 g rod (1.9 cm in diameter)
were used to produce the M burn, whereas four applications
of a 134 g rod (2.5 cm in diameter) produced the L burn.
Previous biopsy of the wound and deeper muscular tissue
demonstrated full-thickness cutaneous burn but with visible
unburned muscle beneath the skin.
Mice were resuscitated with intraperitoneal (i.p.) lactated
Ringer’s solution (0.1 mL/g body weight) and were given
subcutaneous buprenorphine (2 mg/kg body weight) for pain
control immediately after injury and as needed after burn.
Animals were returned to individual cages and allowed to
feed ad libitum. Sham controls, defined as 0% total body
surface area burn, underwent all of the above interventions,
except for the actual burn injury.
Animals were killed and terminal samples of liver tissue
and peripheral blood were collected over a time course with
intervals at 0 (sham), 15 minutes, 30 minutes, 1, 2, 4, 8, 12,
and 24 hours after burn. Four mice were killed from each
group at each time point. Total mRNA was extracted from
whole liver tissue for assay. Levels of TGF-b1 mRNA were
measured by reverse transcription coupled polymerase chain
reaction (RT-PCR) technique. Plasma levels of TGF-b1 were
measured by enzyme-linked immunosorbent assay.
Assessment for Plasma TGF-b
Immediately after collection by heart puncture, blood was trans-
ferred to ethylenediaminetetraacetic acid–coated tubes. Plasma
was separated by centrifugation at 4°C for 15 minutes at 3000 g.
Plasma was stored at 270°C. The concentration of plasma
TGF-b1 was measured by using an enzyme-linked immunosor-
bent assay (TGF-b1 KIT, GENZYME, Cambridge, Mass).
Among the isoforms, TGF-b1 has been conventionally mea-
sured to reflect plasma TGF-b concentration.14,15
RNA Isolation and RT-PCR
Total RNA from liver was isolated by ultracentrifugation
through a CsCl cushion as previously described by Chomczyn-
ski and Sacchi16 and was quantified by measurement of ultra-
violet absorption at 260 nm. RNA was diluted with diethyl
pyrocarbonate (Sigma, St Louis, Mo) –treated water containing
RNase inhibitor (Boehringer Mannheim, Indianapolis, Ind). One
microgram of total RNA was primed with oligo-d(T)16 (Perkin
Elmer, Norwalk, Conn) and reverse transcribed into first strand
cDNA (Gibco BRL, Gaithersburg, Md) by using Moloney mu-
rine leukemia virus reverse transcriptase (Gibco BRL). Upon
completion of synthesis, each cDNA reaction was diluted to
40
January 2000
final volume of 25 mL and 1 mL was removed for PCR. Glyc-
eraldehyde-3-dehydrogenase (GAPDH) mRNA levels as an
internal standard were determined by using GAPDH specific
primers (59-CATCTCTGCCCCCT CTGCTGA-39 (sense)
and 59-GGATGACCTTGCCCACAGCCT-39 (antisense);17
TGF-b1 mRNA was assessed by using TGF-b1 specific primers
(59-CGGGGCGACCTGGGCA CCATCCATGAC-39 (sense)
and 59-CTGCTCCACCTTGGGCTTGCGACCCAC-39 (anti-
sense); (STRATAGENE, La Jolla, Calif). With the PCR master
reagent mixture (Boehringer Mannheim), reaction was carried
out under the following thermocycling parameters: 94°C, 4
minutes denaturation; 50°C, 45 seconds annealing; 72°C, 1
minute extension for 37 cycles for TGF-b mRNA. The last cycle
extension was for 7 minutes (GeneAmp PCR System 9600,
Perkin Elmer). PCR products were electrophoresed on 2% aga-
rose gel in Tris/acetic acid/ethylenediaminetetraacetic acid
buffer for 3 hours at 60V, stained with ethidium bromide, and
photographed. Intensities of PCR product bands were quantified
by using NIH image software from the digital scanned gel
image.
Morphologic Evaluation of Apoptosis
A portion of liver tissue from each animal was fixed over-
night with neutral buffered 10% formaldehyde solution.
Specimens were embedded in paraffin, sectioned at 5 mm,
and attached to microscope slides. After washing and perme-
abilization, specimens were processed by using the TdT-
mediated dUTP nick-end labeling (TUNEL) procedure with
fluorescein-12-dUTP for 1 hour at 37°C according to the
protocol provided with the Apoptosis Detection System Flu-
orescein (Promega, Madison, Wis). Slides were washed and
counterstained with 1 mg/mL propidium iodide for 15 min-
utes. Slides were mounted and photographed with BHT Sys-
tem microscope (OLYMPUS, Tokyo, Japan). Micrographs
were recorded at 1323 magnification by using fluorescein
isothiocyanate and Texas red dual-band filter set.
Statistical Analysis
The difference between samples was compared by analysis of
variance. p values of less than 0.05 were assigned statistical
significance. The graphic depiction of data in the figures
facilitates the depiction of group differences. Error bars
shown in these figures represent standard error of the mean
(SEM).
RESULTS
Time Course of TGF-b Concentration in Plasma
TGF-b1 was detectable in the plasma of both burn injured and
control mice. As shown in Figure 1, at 4 hours after L burn
injury, circulating TGF-b1 levels were significantly elevated
compared with control ( p , 0.05). TGF-b1 levels returned to
control values by 8 hours after burn injury. There was no
significant difference between control and M burn values
( p , 0.05).
Expression of TGF-b in the Liver
To investigate the mRNA expression of TGF-b1, we isolated
RNA from whole liver at different intervals after burn injury
Vol. 48, No. 1
FIG 1. Plasma concentration of TGF-b1 after burn injury. Asterisk, p less than 0.05
versus sham; dagger, p less than 0.05 versus M at same time after burn.
and performed RT-PCR. For each time point, four liver spec-
imens excised from four mice were used for RNA isolation.
The photographs shown (Figs. 2 and 3) are representative of
four different experiments. TGF-b1 mRNA was slightly ex-
pressed in sham liver. Upon injury, a strikingly increased
expression of TGF-b1 was observed in L burn. Expression of
TGF-b1 mRNA in L burn mice increased within 1 hour after
burn injury but decreased by 8 hours, as shown in Figure 2.
Expression of TGF-b1 mRNA in M burn mice increased
within 2 hours and subsided by 12 hours, as shown in Figure
3. However, TGF-b1 mRNA level was low during the entire
period compared with that of L burn mice. The levels were
essentially unchanged in control animals at various times
points within 24 hours (data not shown).
Quantitative Analysis of TGF-b Expression
We scanned the densities of both TGF-b1 mRNA as target
and GAPDH mRNA expression as an internal standard to
assess the ratio of RT-PCR product for TGF-b to that for
GAPDH, as previously described.18 The TGF-b1/GAPDH
ratios were normalized by comparing with the value obtained
from sham mice. As shown in Figure 4, the intensity of
FIG 2. TGF-b1 mRNA expression in L burn mouse liver. Data shown are repre-
sentative of four different experiments.
FIG 3. TGF-b1 mRNA expression in M burn mouse liver. Data shown are repre-
sentative of four different experiments.
41
Impact of Burn Injury on Hepatic TGF-b1
TGF-b1 mRNA expression in L burn mice increased at 30
minutes after burn and remained elevated through 4 hours.
There are significant differences between these values and
sham. At their peak level, the L burn group showed approx-
imately a threefold rise compared with the sham group. As
shown in Figure 5, expression in M burn mice tends to
increase gradually and peaked at 2 hour after burn, then
approached normal at 12 to 24 hours after burn. But there was
no significant difference against sham value.
Analysis of TUNEL Staining of Liver Tissue
Figure 6 (A and B) shows the result of TUNEL staining of
liver tissue harvested. Apoptotic cells have incorporated flu-
orescein-12-dUTP (yellow-green fluorescence), indicating
the presence of DNA fragments. Propidium iodide (red) was
used as a counterstain for all cells. The induction of apoptosis
by L burn mice was only exhibited at 8 hours after burn
injury. We could not detect any evidence of apoptosis in M
burn and control mice.
DISCUSSION
Our laboratory has been studying the effect of burn injury for
several years. Previously, we identified the early expression
of c-Jun and NFkB in the mouse liver after burn injury. The
expression of c-Jun is required for liver development.19
NFkB is a ubiquitous transcription factor that is activated by
a variety of cytokines and mitogens.20 The effect of these
factors are most often investigated in a damaged liver model,
such as partial hepatectomy.21 The kinetics of the increase in
TGF-b1 mRNA in liver have been reported after partial
hepatectomy.22 Furthermore, after traumatic injury, the in-
crease of plasma TGF-b1 concentration has been reported.11
In the present study, we compared the plasma concentrations
of TGF-b1 over the 24-hour time frame after burn injury.
TGF-b1 was detectable in all groups. By 4 hours after burn
injury, plasma TGF-b1 concentration was significantly ele-
vated in massively burned animals, compared with control
FIG 4. L burn TGF-b1/GAPDH activity. Asterisk, p less than 0.05 versus sham;
dagger, p less than 0.05 versus M at same time after burn.
FIG 5. M burn TGF-b1/GAPDH activity. Dagger, p less than 0.05 versus L at same
time after burn.
The Journal of Trauma: Injury, Infection, and Critical Care
FIG 6. (A) TUNEL stain for detection of apoptosis in liver after L burn (original magnification, 1323). Labeled apoptotic nuclei were observed in the liver tissue at 8 hours
after L burn injury. (B) TUNEL stain for detection of apoptosis in liver after M burn (original magnification, 1323) Labeled apoptotic nuclei were not observed in the
liver tissue even at 8 hours after M burn injury.
groups in our experimental model. The concentration of
TGF-b1 at this point was approximately threefold above that
of control groups. Recently, Frank et al. have shown elevated
expression of TGF-b1 in burn wounded skin within 13 days
after burn injury.23 Although TGF-b1 must be an essential
and beneficial cytokine in burn wound healing, circulating
plasma TGF-b1 seems to be taken up by the liver in a
concentration-dependent manner.24
In our study, we demonstrated the increase of expression of
TGF-b1 mRNA in L burn mice and in M burn mice. The
RT-PCR method was used in a quantitative manner to study
the expression of TGF-b1 mRNA relative to the expression of
GAPDH gene product, which is widely used as an internal
standard for gene expression studies.25 To control for errors
that could arise because of differences in the quality of RNA
samples or variations in the PCR reaction efficiency, we have
used an endogenously expressed internal standard that is
expressed equally in the tissues.
It has been reported that TGF-b plays a part in distinct
types of liver injury, partial hepatectomy, hepatic fibrosis,
and inflammation. After partial hepatectomy, loss of liver cell
mass occurs. To compensate for this loss, hepatocyte prolif-
eration is stimulated. To down-regulate this hepatocyte pro-
liferation, TGF-b is increased.3,26,27 In the case of liver in-
flammation such as hepatitis, TGF-b stimulates production of
extracellular matrix.28 –31 TGF-b correlates closely with fi-
brogenesis in animal models and patients with cirrhosis.32–34
When comparing burn injury with partial hepatectomy, we
may expect similar effects on the liver. In 70% partial hep-
atectomy, the mRNA of TGF-b1 is not changed during the
first 1 to 4 hours.12 Czaja et al. demonstrated that, after oral
administration of hepatotoxic carbon tetrachloride, TGF-b1
mRNA began to increase after 24 hours of one-time
administration.35 In our study, TGF-b1 mRNA expression is
different. It occurs just after the burn injury. After a rapid
increase in TGF-b1 mRNA, the expression level returned to
normal range in both M and L groups. Consistent with the
percentage of burn wound, the peak expression of TGF-b1
mRNA in L burn group was approximately 1.5 times that of
42
January 2000
the peak expression in the M burn group. The impact of fluid
resuscitation on the different size burns is another potential
variable in these changes. In a preliminary experiment, we
examined different levels of resuscitation in mice with M and
L burn. We found no significant differences in liver enzyme
concentration and TUNEL result in the different resuscitation
groups. We also demonstrated that plasma TGF-b1 concen-
tration in the L burn group was increased, whereas in the M
burn group it was not. After these results, it could be assumed
that the plasma elevation of TGF-b1 occurred after expres-
sion of its mRNA in liver tissue after burn injury. Of course,
there is a possibility of TGF-b secretion by other cells in the
liver. Study of individual liver cell populations after partial
hepatectomy reveals that endothelial and Kupffer cells ex-
press TGF-b1 at relatively high levels. Moreover, they pro-
duce an active form of the factor.12 This finding may account
for the delay between the plasma and mRNA expression in
our study.
These findings seem to correlate with the hepatocyte apopto-
sis shown in the morphologic evaluation. Morphologically, pro-
grammed cell death is characterized by apoptosis, including
nuclear pyknosis and formation of membrane-bound apoptotic
bodies, chromatin margination, membrane blebbing, cell con-
densation with preservation of organelles, and detachment from
adjacent cells.36,37 However, it is not easy to identify apoptotic
cells in routinely stained tissue sections. In our study, we used
the TUNEL method for detecting the apoptotic cells. Apoptotic
cells have incorporated fluorescein-12-dUTP (yellow-green), in-
dicating the presence of DNA fragments. Propidium iodine (red)
was used as an intercalating DNA dye and counterstain for all
cells. Although some controversy still exists as to TUNEL
method specificity of apoptotic cells, the many reports already
existing generally argue the validity of the method. The present
study revealed the occurrence of apoptosis in liver tissue after
burn injury. We demonstrated the occurrence of apoptosis at 8
hour after L burn injury, but we were unable to detect it under
other conditions. The hepatocyte apoptosis may be partially
caused by TGF-b1. It has been reported that TGF-b is a potent
inhibitor of hepatocyte proliferation.38 – 40 Furthermore, it has
Vol. 48, No. 1
been shown that TGF-b1 induces apoptosis in hepatocytes in
vitro and in vivo conditions.41,42 Oberhammer et al. also re-
ported that in vivo apoptotic hepatocytes in normal and preneo-
plastic liver exhibited immunostaining for TGF-b1. From tissues
of fulminant hepatitis and bile retention, Takiya et al. suggested
that TGF-b1 may play a role in these diseases and account for
inadequate hepatic regeneration and induced apoptosis.43 In an
in vitro study, adding exogenous TGF-b caused apoptosis of
hepatocytes after 16 to 20 hours.41,44 In our in vivo study,
apoptosis occurs within 8 hours after burn injury. This result is
consistent with previous in vivo results that exogenous TGF-b1
injection induces apoptosis in hepatocytes 5 hours after
injection.42 The differences between these may depend on the
TGF-b1 dose, exposure time and possibly other cytokines.
Hepatocytes might be under different influences of paracrine
and circulating TGF-b in in vivo studies compared with in vitro
studies. Furthermore, it has been demonstrated that administra-
tion of TGF-b alone in vivo induces a small amount of apoptosis
in the normal liver but that administration of TGF-b with a liver
mitogen induces a large amount of apoptosis.42 Our in vivo burn
study may be affected by these factors.
Additionally, some authors demonstrate the release of liver
enzymes into blood during cell death or apoptotic processes.42,44
Previously, we reported the release of liver enzymes in the burn
model. Indeed, it is the general concept that the apoptotic cell
undergoes cell death without release of intracellular ma-
terial,45– 48 yet it is assumed that a certain amount of enzymes are
released after the induction of apoptosis during fragmentation of
the cell.42 Given this concept, the elevation of liver enzyme in
our model supports the occurrence of apoptosis.49 We are cur-
rently undertaking studies that use anti–TGF-b1 antibody ad-
ministration. Preliminary results suggest that anti–TGF-b1 anti-
body can decrease the plasma concentration of GOT and GPT
(unpublished data).
We investigated the expression of TGF-b1 after burn injury
and its change over time. In severe burn injury, high expres-
sion of TGF-b1 mRNA may contribute to increased serum
TGF-b1 levels and to the induction of apoptotic cells in liver
tissue. These results may provide insight into the mechanism
of liver dysfunction after burns. Further study of other mech-
anisms of TGF-b expression after burn injury, specifically
looking at other organs will continue.
Acknowledgments
The authors thank Virginia Godfrey, DVM, PhD, and Nariaki Mat-
suura MD, PhD, for performing the histologic analysis of samples.
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