© 2001 Schattauer GmbH, Stuttgart
Hepatic Thrombopoietin mRNA Is Increased in
Acute Inflammation
Eva-Maria Wolber 1, Joachim Fandrey 2, Urszula Frackowski 1, Wolfgang Jelkmann 1
1 Institute of Physiology, Medical University of Luebeck, Luebeck, Germany
2 Institute of Physiology, University of Essen, Essen, Germany
Keywords
Thrombopoietin, inflammation, reactive thrombocytosis, acute
phase reaction, LPS
Summary
The plasma concentration of thrombopoietin (TPO) in general is
inversely related to the mass of platelets and megakaryocytes. How-
ever, reactive thrombocytosis of inflammatory disease is accompanied
by elevated TPO levels. To investigate whether the rate of TPO mRNA
expression is altered during acute inflammation, rats were injected with
bacterial lipopolysaccharide (LPS). After 6 h, total RNA from liver and
kidney was reverse transcribed and analyzed by competitive PCR
for TPO and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
LPS-treated rats showed a significant increase in hepatic TPO mRNA
concentration. The ratio of TPO to GAPDH mRNA was 3.5 ± 0.6% in
the livers of control rats and 8.3 ± 2.0% in the livers of LPS-treated rats
(mean ± SD). Thus, reactive thrombocytosis of inflammatory disease
might result from an increase in hepatic TPO production. Since plate-
lets are involved in the immune reaction, reactive thrombocytosis may
be a mechanism of host defense.
Introduction
Thrombopoietin (TPO), the primary regulator of thrombopoiesis and
megakaryopoiesis (1), is mainly produced by the hepatocytes in the
liver (2). TPO mRNA has also been detected in kidney, bone marrow,
lung, brain, intestine, and other organs (2, 3). The rate of the hepatic
formation of TPO mRNA is independent of the concentration of circu-
lating platelets (4). The inverse relationship between TPO and platelet
concentration in blood is probably achieved by binding, internalization,
and degradation of TPO via the TPO receptor on platelets, mega-
karyocytes and their progenitors (4-6).
The reactive thrombocytosis in patients with inflammatory or
malignant diseases typically associates with a relatively high level of
circulating TPO (7-9). This might be due to decreased TPO clearance or
increased TPO production. Recent studies have shown that in vitro
TPO production of the human hepatic cell line HepG2 can be increased
by interleukin-6 (IL-6) treatment while several other cytokines were
without effect (10, 11). IL-6 is produced during systemic inflammation
and plays an important role in the induction of the acute phase re-
Correspondence to: Dr. E.-M. Wolber, Institute of Physiology, Medical
University of Luebeck, Ratzeburger Allee 160, D-23538 Luebeck, Germany –
Tel.: +49 451 500 4167; Fax: +49 451 500 4171; E-mail: wolber@physio.
mu-luebeck.de
Thromb Haemost 2001; 86: 1421–4
sponse, stimulating hepatocytes to secrete acute phase proteins such as
C-reactive protein or fibrinogen (12). A widely used experimental
model to study inflammatory reactions and IL-6 activation in vivo is the
injection of bacterial lipopolysaccharide (LPS) (12).
Here, we investigated whether acute inflammation leads to increased
hepatic and renal TPO mRNA levels. To this aim, rats were treated with
Downloaded by: Georgetown University Medical Center. Copyrighted material.
LPS, and TPO mRNA expression in the liver and the kidney was
measured by reverse transcription and competitive polymerase chain
reaction (PCR).
Materials and Methods
Animal experiments. All animal experiments were approved by the govern-
mental committee and performed according to the law for the protection of
animals. Rats were injected intraperitoneously with either LPS (0.1 mg/kg
body weight; Sigma, Munich, Germany) dissolved in sterile NaCl solution
(0.9% w/v) or equal volumes of sterile NaCl solution (13). After 6 h, the
abdomen was opened under Nembutal anesthesia, and animals were bled to
death via aortic puncture. Liver and kidneys were rapidly removed and snap-
frozen in liquid nitrogen.
RNA extraction, reverse transcription and qualitative PCR. Total RNA was
extracted by the acid guanidinium thiocyanate/phenol-chloroform method (14)
and reverse transcribed into cDNA with murine Moloney leukemia virus rever-
se transcriptase (Promega, Madison, WI) and oligo-dT primers (15). Prior to the
quantitation by competitive PCR, qualitative PCRs for glyceraldehyde 3-phos-
phate dehydrogenase (GAPDH) and TPO cDNAs were performed under pre-
viously published conditions (15, 16). Primers were: 5’GAPDH, 5’-ATC ATC
CCT GCC TCT ACT GG-3’; 3’GAPDH, 5’-TGG GTG TCG CTG TTG AAG
TC-3’; 5’rTPO, 5’-TTT CTG GTC AGG TTC GCC TC-3’; 3’rTPO, 5’-GTT
CGT GTG TCC CGT TCA GG-3’. For GAPDH cDNA 27 PCR cycles with
55° C annealing temperature, and for TPO cDNA 32 PCR cycles with 60° C
annealing temperature were used. The expected PCR product lengths were
257 bp for GAPDH cDNA and 420 bp for TPO cDNA. Comparison of the rat
TPO cDNA sequence (17) (GenBank accession No. D32207) with the human
TPO gene (3) (GenBank accession No. S76771) showed that the primers bind
to regions corresponding to exons 5 and 6 of the human TPO gene.
Competitive PCR. The TPO competitor DNA was constructed by PCR with
the 5’rTPO-composite primer, 5’-TTT CTG GTC AGG TTC GCC TCC CCT
CTG TGT CAG ACG GAC C-3’, and the 3’rTPO primer. The GAPDH
competitor DNA was constructed by restriction digestion of the GAPDH PCR
product, ligation, and subsequent PCR amplification. The correct fragment
lengths of 213 bp for GAPDH and 272 bp for TPO competitors were confirmed
by agarose gel electrophoresis. The competitor fragments were purified with
the Wizard PCR Preps DNA Purification System (Promega), and the concen-
tration was determined by absorbance at 260 nm. The competitive PCR and the
subsequent calculations were performed as described for human TPO cDNA
(15), using the appropriate primers and competitors. For further analysis, the
ratio of TPO mRNA to GAPDH mRNA was determined.
Statistical analysis. Results are given as mean and standard deviation (SD).
Statistical significance was assigned if the two-tailed P value calculated by
Student’s t-test was < 0.05.
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Thromb Haemost 2001; 86: 1421–4
Results
GAPDH and TPO mRNAs were detectable in the livers and kidneys
of all rats. There was no difference in the GAPDH PCR signal between
both groups. The 5’rTPO and 3’rTPO primers bind to different putative
exons of the rat TPO gene. Thus PCR products of different isoforms are
distinguishable by their size. The main PCR product in rat liver with a
size of 420 bp corresponds to human TPO-1.
Quantitation of the TPO and GAPDH gene expression was per-
formed by competitive PCR (Fig. 1). The results revealed an about
twofold increase in TPO mRNA concentration related to the GAPDH
mRNA concentration 6 h after LPS treatment (Fig 2). The ratio of TPO
mRNA per 100 molecules of GAPDH mRNA was 3.5 ± 0.6 in the livers
of control rats and 8.3 ± 2.0 in the livers of LPS-treated rats. This
Fig. 2 Increased TPO mRNA expression after treatment with LPS. Rats were
treated 6 h with 0.1 mg/kg LPS, and TPO and GAPDH mRNA in liver (hatched
bars) and kidney (open bars) were measured by reverse transcription and com-
petitive PCR. TPO expression is given as molecules TPO mRNA per 100 mole-
cules of GAPDH mRNA. Data are mean ± SD, n = 4, * P < 0.05 (Student’s
t-test versus control)
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Fig. 1 TPO and GAPDH mRNA expression in rat
liver and kidney. Quantitative PCR results with cDNA
from liver and kidney of two exemplary rats. Treat-
ment of the rats is indicated above the lanes. Top, TPO
competitive PCR; middle, GAPDH competitive PCR;
bottom, relative TPO mRNA expression. Arrows
indicate concentration equivalence of cDNA and
competitor
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increase was statistically significant (P = 0.004). The kidneys of
LPS-treated rats also showed higher TPO mRNA expression (9.3 ± 4.5
versus 5.4 ± 2.2 in control rats), but this difference was not significant
(P = 0.2).
Discussion
Hepatic TPO mRNA formation has been considered a constitutive
process, as the TPO mRNA content in the liver is unaffected by plate-
let counts (2, 4). However, recent reports indicate that the level of cir-
culating TPO can be altered under pathophysiologic conditions. First,
thrombocytopenic patients with liver cirrhosis show decreased hepatic
TPO mRNA expression (16, 18). Second, patients with reactive throm-
bocytosis have high levels of circulating TPO when their increased
platelet counts are taken into account (7-9). Third, in vitro studies with
human hepatoma cells have shown that IL-6 stimulates and that inter-
feron- decreases TPO mRNA and protein production (10, 19). Still,
these in vitro findings are not generally accepted. Using Northern blot,
unaltered TPO mRNA levels in human hepatoma cells of the line
HepG2 were found following a 48 h period of treatment with various
cytokines, including hepatocyte growth factor (HGF) and IL-6 (20).
Other investigators reported increased TPO secretion despite unaltered
TPO mRNA levels in HepG2 cultures treated with IL-6 for 24 h (11).
IL-6 had no effect on TPO mRNA levels in primary cultures of rat
hepatocytes (21, 22). HGF stimulated TPO mRNA expression in one of
these studies (21), but not in the other (22). The conflicting in vitro fin-
dings may have been partly due to differences in the culture conditions,
regarding cell lines and incubation periods, as well as in the sensitivity
of the assay methods. The present in vivo investigation, which was
based on a quantitative RT-PCR assay, showed that LPS-induced acute
inflammation rapidly increased the TPO mRNA concentration in the
liver of rats.
The primary importance of hepatic TPO production has been de-
monstrated in several studies. In human fetuses the liver accounts for
94% of the total body TPO mRNA (15). In TPO knockout mice, trans-
plantation of a normal liver can restore TPO and platelet production,
while transplantation of the liver of TPO knockout mice into normal
mice nearly abolishes platelet production (23). The thrombocytopenia
of patients with liver cirrhosis shows that other organs cannot compen-
sate for a loss of hepatic TPO production (16). Our data suggest that the
liver is not only the main production site of TPO under normal condi-

tions, but furthermore reacts to systemic inflammation with increased
TPO mRNA expression.
Because TPO mRNA levels rise in HepG2 cells on IL-6 treatment
and the promoter sequences flanking the TPO gene contain 13 putative
IL-6 response elements (CTGGGA) (10), TPO can be considered an
acute phase protein. Folman et al. (11) have pointed out that increases
in both gene expression and mRNA stability could be responsible for
TPO mRNA accumulation in response to IL-6. Irrespective of the
molecular mechanisms controlling TPO mRNA levels, excess of TPO
and the resulting reactive thrombocytosis appear to be part of the acute
phase response. This notion is supported by data showing that concen-
trations of circulating IL-6 are elevated in reactive thrombocytosis (8)
and correlated with the concentration of TPO (9). The alterations of the
level of circulating TPO in response to inflammation resemble those of
acute phase reactants like IL-6 and C-reactive protein (24). It has been
earlier demonstrated that LPS induces IL-6 mRNA expression in many
organs and a rise in circulating IL-6 protein peaking 1 to 2 h after a
single intravenous injection (25).
In the present short-term experiments, no attempt was made to
measure plasma TPO concentrations, because circulating TPO is a
relatively long-lived glycoprotein whose level is primarily regulated by
receptor binding (4). Based on pharmacokinetic data in mice (5), it can
be calculated that the plasma level of TPO may maximally increase by
20% if TPO protein production is doubled for 6 h. Furthermore, the
alleged stimulation of TPO synthesis should be retarded, as it follows
the processes of the induction of IL-6 and TPO gene transcription.
In addition, LPS injections cause an initial drop in platelet numbers
(5, 26), which poses difficulties regarding the interpretation of changes
in the plasma TPO concentration. The level of circulating TPO may
increase under this condition irrespective from a change in production
rate, because of the slowed TPO metabolism at the reduced platelet
mass. Whether the rate of TPO clearance is influenced by inflammatory
processes remains unanswered from the present investigations.
When LPS is infused in healthy humans, peripheral platelet counts
recover after 24 h and exceed baseline values after 7 days (26, 27). Con-
sidering the biological role of reactive thrombocytosis one has to keep
in mind that bleeding disorders do not occur unless the platelet counts
drop to <10% of normal. Teleologically, the role of the increased TPO
production in response to inflammation is not only to be seen in main-
tenance of hemostasis. In addition to their function in blood coagula-
tion, platelets are important transporters of immune mediators (28).
Activated platelets initiate an inflammatory response of endothelial
cells (29). In inflammation, the increased TPO in blood can directly
stimulate platelets to facilitate their activation (30). Recently, it has
been shown that in vitro activated platelets release TPO (31). The
stimulation of thrombopoiesis by TPO results in elevated numbers of
platelets to support the immune reaction. The reactive thrombocytosis
of inflammatory disease may represent a mechanism to control
microbial infections.
Acknowledgements
We thank Gabriela Fletschinger for preparation of the figures and Dorothea
Brennecke for secretarial help. This work was supported by a grant from the
Deutsche Forschungsgemeinschaft (SFB367-C8, Luebeck).
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Received January 3, 2001 Accepted after resubmission September 4, 2001
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