Thrombopoietin production in wild-type and interleukin-6 knockout mice with acute inflammation.

Clinical and laboratory studies indicate that thrombopoietin (TPO) gene expression increases during inflammation. To clarify the role of interleukin 6 (IL-6) in this process, blood cell counts, plasma TPO concentrations, and hepatic and renal TPO mRNA levels were investigated in wild-type and IL-6 knockout mice, with sterile abscesses produced by subcutaneous injection of turpentine oil. Treatment did not cause a change in blood cell counts during the 72 h period of observation. The numbers of thrombocytes and erythrocytes were slightly lower in the IL-6 knockout mice than in the wild-type littermates under all conditions. Plasma IL-6 and TPO concentrations increased on turpentine injection only in the wild-type mice. In addition, turpentine treatment of these caused an increase in hepatic TPO mRNA levels as assessed by competitive polymerase chain reaction (RT-PCR) and real-time PCR, whereas renal TPO mRNA levels were unaltered. TPO mRNA levels did not increase in the livers of IL-6 knockout mice on turpentine treatment. These results support the concept that TPO behaves like an acute-phase protein in that its synthesis is induced by IL-6 in the liver.

Expression of angiopoietin 1 and 2 in ectopic endometrium on the chicken chorioallantoic membrane.

OBJECTIVE: To investigate the role of angiopoietin 1 and 2 (ANGPT1/ANGPT2) in angiogenesis of the ectopic endometrium as a crucial step in the development of an endometriotic lesion, we analyzed their expression patterns in an experimental model of endometriosis. DESIGN: Experimental prospective study. SETTING: University hospital. PATIENT(S): Endometrium samples obtained from healthy, ovulating women undergoing hysterectomy for benign gynecologic conditions. INTERVENTION(S): Endometrial fragments were transplanted to the chicken chorioallantoic membrane (CAM) and cultivated for 0, 24, 48, and 72 hours. MAIN OUTCOME MEASURE(S): Expression of ANGPT1 and ANGPT2 mRNA was quantified by competitive reverse transcriptase polymerase chain reaction (RT-PCR) and normalized to expression of the housekeeping gene human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. The expression of ANGPT1 and ANGPT2 protein was analyzed by immunohistochemical staining. RESULT(S): All grafts expressed ANGPT1 and ANGPT2 mRNA. The mRNA concentration of both factors decreased after cultivation, but the ANGPT2/ANGPT1 ratio increased considerably during the first 24 hours of cultivation. The immunohistochemical investigation for ANGPT1 and ANGPT2 revealed presence of both proteins at all the times but no obvious correlation with the duration of cultivation. CONCLUSION(S): The ratio of ANGPT2/ANGPT1 mRNA in endometrial grafts increased after 24 hours of cultivation on the chicken chorioallantoic membrane and shows a shift toward a more dominant role of ANGPT2. This agrees with the current model of angiopoietin action in angiogenesis and might indicate angiogenic activity in the endometrial graft. The angiopoietins are likely to play an important role in the pathogenesis of endometriosis.

Thrombopoietin gene expression in the developing human central nervous system.

Thrombopoietin gene expression in the human adult central nervous system (CNS) appears to be locally restricted. The aim of this study was to identify areas of thrombopoietin expression in the developing human CNS, and to compare the thrombopoietin mRNA content in the CNS to that in liver and kidneys as major sites of thrombopoietin production. Thrombopoietin protein concentrations in the cerebrospinal fluid (CSF) were measured by ELISA. In 14 fetuses and neonates with perinatal death, thrombopoietin mRNA expression was measured by competitive RT-PCR. Thrombopoietin mRNA was expressed in 29 of 32 specimens taken from the CNS. The following ranking of the intensity of expression in the CNS was possible: Spinal cord=cerebellum=cortex>>pituitary gland>>>brain stem=corpora amygdala=hippocampus. Whereas in the latter three tissues only trace amounts of thrombopoietin transcripts were detectable, thrombopoietin mRNA levels in the spinal cord were comparable to levels in liver and kidney. Thrombopoietin protein concentrations in CSF ranged between 41 and 75 pg/ml. In the developing human CNS, the thrombopoietin gene is abundantly expressed. Considering that thrombopoietin contains a neurotrophic sequence, it may well play a role in neuronal cell biology.

Differential induction of matrix metalloproteinase 1 and 2 in ectopic endometrium.

According to the transplantation theory, endometriosis develops from endometrial fragments that are retrogradely menstruated into the peritoneal cavity. In order to develop into endometriotic lesions, they have to connect to the vascular system by angiogenesis, probably involving matrix metalloproteinases (MMP) as key enzymes in extracellular matrix remodelling. A model of endometriosis using the chorioallantoic membrane (CAM) of chick embryos was established. Eutopic endometrium from healthy women was transferred to the CAM and cultivated ectopically for up to 3 days. Before transplantation and after 24, 48 and 72 h of culture on the CAM, total RNA was extracted and reverse transcribed. Human MMP-1 (interstitial collagenase) and MMP-2 (gelatinase A) mRNA expression was assessed by competitive PCR. Results were normalized to the content of human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. In eutopic endometrium, 0.29 amol MMP-1 mRNA and 0.42 fmol MMP-2 mRNA per fmol GAPDH mRNA were found. Relative MMP-1 mRNA concentrations increased strongly after culture on the CAM, while MMP-2 mRNA levels were nearly unaltered. This differential regulation suggests different roles of these enzymes in the angiogenesis of ectopic endometrial fragments and during the development of endometriosis.

The kidney as a second site of human C-reactive protein formation in vivo.

C-reactive protein (CRP) is the main acute phase reactant in humans. Its production is presumably restricted to the liver but extrahepatic expression by inflamed tissue has not been studied in detail. By real-time PCR and immunohistochemistry we here show that renal cortical tubular epithelial cells (TEC) express CRP mRNA and protein within 6 h after stimulation with conditioned medium (CM) or IL-6, but not IL-1alpha or TNF-alpha. Western blot analysis with monoclonal anti-CRP antibody that recognizes native CRP revealed protein secretion into supernatants of CM-stimulated TEC cultures. While hepatoma-derived Hep3B cells could be induced similarly, peripheral blood mononuclear cells could not. CRP mRNA transcripts were observed in nephrectomized renal allografts with severe acute rejection but not with chronic allograft nephropathy (CAN). Of 19 needle biopsies of acutely rejecting kidney transplants, 15 demonstrated CRP mRNA production with the relative expression levels increasing with the severity of rejection. On the other hand, none of 7 graft biopsies with acute tubular necrosis (ATN) or CAN showed CRP mRNA expression. By using monoclonal anti-CRP antibody, cortical tubules as well as glomerular cells were shown to locally express CRP in rejecting, but not in ATN kidneys. We conclude that inflamed kidneys represent a so far unknown site of CRP formation in vivo. These data shed new light on the acute phase reaction not merely representing a systemic inflammatory pathway but probably being part of the local immune response.

Thrombopoietin: the novel hepatic hormone.

The glycoprotein thrombopoietin (TPO) is the major stimulator of megakaryopoiesis and platelet production. Hepatocytes express TPO mRNA at a constant rate. The plasma TPO level is inversely correlated to the mass of megakaryocytes and platelets, which degrade the hormone following its binding to specific membrane receptors.

Thrombopoietin production in human hepatic cell cultures (HepG2) is resistant to IFN-alpha, IFN-beta, and IFN-gamma treatment.

Thrombocytopenia is an important complication of interferon (IFN) therapy for chronic viral hepatitis. To study whether IFN interferes with hepatic thrombopoietin (TPO) synthesis, we used the human hepatoma cell line HepG2. Our results show that IFN-alpha, IFN-beta, or IFN-gamma did not impair TPO mRNA expression, as determined by quantitative RT-PCR, even when high IFN doses (up to 5000 U/ml) or long-term incubations (up to 14 days) were applied. Neither was the rate of secretion of immunoreactive TPO reduced on IFN treatment. These findings support the concept that IFNs primarily mediate effects on megakaryocytic cells and platelets rather than on TPO-producing hepatocytes.