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© 2001 Schattauer GmbH, Stuttgart Thromb Haemost 2001; 86: 1421–4

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 sponse, stimulating hepatocytes to secrete acute phase proteins such as


C-reactive protein or fibrinogen (12). A widely used experimental
Thrombopoietin, inflammation, reactive thrombocytosis, acute model to study inflammatory reactions and IL-6 activation in vivo is the
phase reaction, LPS injection of bacterial lipopolysaccharide (LPS) (12).
Here, we investigated whether acute inflammation leads to increased
Summary hepatic and renal TPO mRNA levels. To this aim, rats were treated with

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LPS, and TPO mRNA expression in the liver and the kidney was
The plasma concentration of thrombopoietin (TPO) in general is measured by reverse transcription and competitive polymerase chain
inversely related to the mass of platelets and megakaryocytes. How- reaction (PCR).
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 Materials and Methods
bacterial lipopolysaccharide (LPS). After 6 h, total RNA from liver and Animal experiments. All animal experiments were approved by the govern-
kidney was reverse transcribed and analyzed by competitive PCR mental committee and performed according to the law for the protection of
for TPO and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). animals. Rats were injected intraperitoneously with either LPS (0.1 mg/kg
LPS-treated rats showed a significant increase in hepatic TPO mRNA body weight; Sigma, Munich, Germany) dissolved in sterile NaCl solution
concentration. The ratio of TPO to GAPDH mRNA was 3.5 ± 0.6% in (0.9% w/v) or equal volumes of sterile NaCl solution (13). After 6 h, the
the livers of control rats and 8.3 ± 2.0% in the livers of LPS-treated rats abdomen was opened under Nembutal anesthesia, and animals were bled to
(mean ± SD). Thus, reactive thrombocytosis of inflammatory disease death via aortic puncture. Liver and kidneys were rapidly removed and snap-
frozen in liquid nitrogen.
might result from an increase in hepatic TPO production. Since plate-
RNA extraction, reverse transcription and qualitative PCR. Total RNA was
lets are involved in the immune reaction, reactive thrombocytosis may extracted by the acid guanidinium thiocyanate/phenol-chloroform method (14)
be a mechanism of host defense. and reverse transcribed into cDNA with murine Moloney leukemia virus rever-
se transcriptase (Promega, Madison, WI) and oligo-dT primers (15). Prior to the
Introduction quantitation by competitive PCR, qualitative PCRs for glyceraldehyde 3-phos-
phate dehydrogenase (GAPDH) and TPO cDNAs were performed under pre-
Thrombopoietin (TPO), the primary regulator of thrombopoiesis and viously published conditions (15, 16). Primers were: 5’GAPDH, 5’-ATC ATC
megakaryopoiesis (1), is mainly produced by the hepatocytes in the CCT GCC TCT ACT GG-3’; 3’GAPDH, 5’-TGG GTG TCG CTG TTG AAG
liver (2). TPO mRNA has also been detected in kidney, bone marrow, TC-3’; 5’rTPO, 5’-TTT CTG GTC AGG TTC GCC TC-3’; 3’rTPO, 5’-GTT
lung, brain, intestine, and other organs (2, 3). The rate of the hepatic CGT GTG TCC CGT TCA GG-3’. For GAPDH cDNA 27 PCR cycles with
formation of TPO mRNA is independent of the concentration of circu- 55° C annealing temperature, and for TPO cDNA 32 PCR cycles with 60° C
annealing temperature were used. The expected PCR product lengths were
lating platelets (4). The inverse relationship between TPO and platelet
257 bp for GAPDH cDNA and 420 bp for TPO cDNA. Comparison of the rat
concentration in blood is probably achieved by binding, internalization, TPO cDNA sequence (17) (GenBank accession No. D32207) with the human
and degradation of TPO via the TPO receptor on platelets, mega- TPO gene (3) (GenBank accession No. S76771) showed that the primers bind
karyocytes and their progenitors (4-6). to regions corresponding to exons 5 and 6 of the human TPO gene.
The reactive thrombocytosis in patients with inflammatory or Competitive PCR. The TPO competitor DNA was constructed by PCR with
malignant diseases typically associates with a relatively high level of the 5’rTPO-composite primer, 5’-TTT CTG GTC AGG TTC GCC TCC CCT
circulating TPO (7-9). This might be due to decreased TPO clearance or CTG TGT CAG ACG GAC C-3’, and the 3’rTPO primer. The GAPDH
increased TPO production. Recent studies have shown that in vitro competitor DNA was constructed by restriction digestion of the GAPDH PCR
TPO production of the human hepatic cell line HepG2 can be increased product, ligation, and subsequent PCR amplification. The correct fragment
by interleukin-6 (IL-6) treatment while several other cytokines were lengths of 213 bp for GAPDH and 272 bp for TPO competitors were confirmed
without effect (10, 11). IL-6 is produced during systemic inflammation by agarose gel electrophoresis. The competitor fragments were purified with
the Wizard PCR Preps DNA Purification System (Promega), and the concen-
and plays an important role in the induction of the acute phase re-
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
Correspondence to: Dr. E.-M. Wolber, Institute of Physiology, Medical ratio of TPO mRNA to GAPDH mRNA was determined.
University of Luebeck, Ratzeburger Allee 160, D-23538 Luebeck, Germany – Statistical analysis. Results are given as mean and standard deviation (SD).
Tel.: +49 451 500 4167; Fax: +49 451 500 4171; E-mail: wolber@physio. Statistical significance was assigned if the two-tailed P value calculated by
mu-luebeck.de Student’s t-test was < 0.05.

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Thromb Haemost 2001; 86: 1421–4

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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Results increase was statistically significant (P = 0.004). The kidneys of
LPS-treated rats also showed higher TPO mRNA expression (9.3 ± 4.5
GAPDH and TPO mRNAs were detectable in the livers and kidneys versus 5.4 ± 2.2 in control rats), but this difference was not significant
of all rats. There was no difference in the GAPDH PCR signal between (P = 0.2).
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 Discussion
distinguishable by their size. The main PCR product in rat liver with a
size of 420 bp corresponds to human TPO-1. Hepatic TPO mRNA formation has been considered a constitutive
Quantitation of the TPO and GAPDH gene expression was per- process, as the TPO mRNA content in the liver is unaffected by plate-
formed by competitive PCR (Fig. 1). The results revealed an about let counts (2, 4). However, recent reports indicate that the level of cir-
twofold increase in TPO mRNA concentration related to the GAPDH culating TPO can be altered under pathophysiologic conditions. First,
mRNA concentration 6 h after LPS treatment (Fig 2). The ratio of TPO thrombocytopenic patients with liver cirrhosis show decreased hepatic
mRNA per 100 molecules of GAPDH mRNA was 3.5 ± 0.6 in the livers TPO mRNA expression (16, 18). Second, patients with reactive throm-
of control rats and 8.3 ± 2.0 in the livers of LPS-treated rats. This 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,
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 while transplantation of the liver of TPO knockout mice into normal
bars) and kidney (open bars) were measured by reverse transcription and com- mice nearly abolishes platelet production (23). The thrombocytopenia
petitive PCR. TPO expression is given as molecules TPO mRNA per 100 mole- of patients with liver cirrhosis shows that other organs cannot compen-
cules of GAPDH mRNA. Data are mean ± SD, n = 4, * P < 0.05 (Student’s sate for a loss of hepatic TPO production (16). Our data suggest that the
t-test versus control) liver is not only the main production site of TPO under normal condi-

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Wolber et al.: Increased TPO mRNA in Inflammation

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