The plasma half-life of recombinant human interleukin-6 (rhIL-6) was determined in rats by measuring the disappearance of the biological activity as well as of ...
Eur. J. Biochem. 177, 357-361 (1988) 0FEBS 1988
Plasma clearance, organ distribution and target cells of interleukin-6/hepatocyte-stimulating factor in the rat Jose V. CASTELL I , Thomas GEIGER’, Volkcr GROSS2, Tilo ANDUS’, Eicke WALTER’, Toshio HIRANO’, Tadamitsu KISHlMOT03 and Peter C. HEINRICH’ Biochemisches Institut and Mcdizinische Klinik I 1 der Universitat Freiburg Institute for Molecular and Cellular Biology, Osaka University (Received March 18/June 24, 1988)
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EJB 88 0319
The plasma half-life of recombinant human interleukin-6 (rhIL-6) was determined in rats by measuring the disappearance of the biological activity as well as of the radioactivity of ‘251-rhIL-6 from the circulation. The kinetics of clearance were biphasic. It consisted of a rapid initial disappearance corresponding to a half-life of 3 min, and of a second slow one corresponding to a half-life of about 55 min. By cellulose-acetate electrophoresis it was shown that rhIL-6 binds to a plasma protein resulting in a complex migrating in the p-y region; 20 min after intravenous injection, about 80% of the 12’I-rhIL-6 that had disappeared from the circulation was found in the liver. ‘251-rhIL-6was exclusively localized on the surface of parenchymal cells suggesting the existence of an interleukin-6 receptor on the hepatocytes. Interleukin-6 is a novel cytokine produced by monocytes, fibroblasts and several cell lines and exhibits a pleiotropic action in the organism. It was described as interferon-P2, an antiviral agent synthesized by fibroblasts [l,21, as hybridoma plasmacytoma growth factor [3, 41, and as B-cell-stimulatory factor-2, synthesized and secreted by virus-transformed T-cells [5, 61. Recent work has shown that IL-6 is one of the major mediators of the acute-phase response in the liver. It induces the expression of acute-phase proteins in rat hepatocytes [7, 81, rat hepatoma cells [9], in the rat in vivo [lo], in the human hepatoma HepG2 cells [7] and in human hepatocytes in primary culture [lOa]. The question of the major target tissues and/or organs in the rat was particularly important considering the pleiotropic action of IL-6. 1251rhIL-6 was therefore intravenously injected into rats and the fate of the label was followed. It will be shown in the present paper that the liver is the target organ for IL-6. Within the liver the radioactive label is solely bound to the surface of hepatocytes, indicating the existence of an IL-6 receptor on these cells.
and Weiss [ I l l , were kindly provided by Dr F. Wiebel, Munich (FRG). Recombinant interleukin-6/BSF-2 (5 x lo6 units/mg protein) was prepared as described [6, 121. Animuls
Male Wistar rats of about 200 g body mass had free access to water and a carbohydrate-rich, 20%-protein diet (Altromin, Lage, FRG). lodination of rhIL-6
5 pg rhIL-6 was iodinated according to the procedure described by Markwell [13] using iodobeads as oxidant. The reaction was allowed to proceed for 20 min and stopped by the removal of the catalyst. Bovine serum albumin (BSA) was added to a final concentration of 0.5%. The reaction mixture was subsequently gel-filtered through a Sephadex G-50 column equilibrated with 2 YOBSA. The specific activity of the iodinated rhIL-6 was 50 kBq/pg. I2’I-rhIL-6, subsequently analyzed by paper chromatography [14], was found to contain less than 3% [1251]iodide. After sodium dodecyl sulfate/ MATERIALS AND METHODS polyacrylamide gel electrophoresis under reducing conditions and autoradiography, there was only a single band with an Chemicals apparent molecular mass of 20 kDa (see Fig. 4, left lane). The Carrier-free NaI2’1 (559 MBq/pg) and [ U - ~ ~ P I ~ C Tbiological P activity of the ‘”I-rhIL-6 was indistinguishable (110 TBq/mmol) were purchased from Amersham Inter- from that of rhIL-6. national (Braunschweig, FRG). Polystyrene- bound N-chlorobenzene sulfonamide (Na; iodobeads) was from Pierce Chemical Company (Rockford, IL, USA). Cellulose-acetate sheets Determinution of IL-6 activity were purchased from Serva (Heidelberg, FRG). Rat fl-fibrinorhIL-6 activity was measured by its capability to induce gen cDNA was a generous gift from Dr A. Mitchell, Parkville, p-fibrinogen mRNA in the rat hepatoma cell line Fao. The Australia. Fao cells, originally established by Deschatrette protocol for the cytoblot and the mRNA hybridization has __ been described previously [9].
Correspondence to P. C. Heinrich, Biochemisches Institut, Universitat Freiburg, Hermann-Herder-Str. 7, D-7800 Freiburg, Federal Republic of Germany Abbreviations. rhl L-6, Recombinant human interleukin-6; RSA, rat serum albumin.
Cellulose-acetate electrophoresis
Samples were applied to cellulose-acetate sheets presoaked in electrophoresis buffer (37 mM Tris/HCI, 7.5 mM
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Fig. 1 . Clearance of'rhIL-6.from rat plasma. 4 x lo4 units rh -6 in 0.14 M NaC1/1% f e d calf serum were injected into the tail vein of a male Wistar rat (200g body mass). At the times indicated in the figure, 0.2-ml blood samples were taken from the catheterized vena cava inferior and IL-6 activity was determined in the plasma as described in Materials and Methods. IL-6 concentrations are expressed as a percentage of the maximum plasma level at 30 s after injection
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Fig. 3. Disappearance o j 'Z51-rhIL-6from rat plasma. 1 2 x lo6 cpm lz5I-rhTL-6(specific activity SO kBq/pg) in a total volume of 0.5 ml 50 mM phosphate buffer (Na/K), pH 7.4, containing 0.14 M NaCl and 0.05% bovine serum albumin were injected into the tail vein of a male Wistar rat (200 g body mass). At the Limes indicated, blood samples were taken from the catheterized vena cava inferior. Radioactivity was determined in plasma ( 0 )and in blood cells (A).Data are expressed as radioactivity per SO p1 plasma or blood cells contained in 50 pl blood, respectively, and represent the average SD of four independent animal experiments
abdominis were excised and the total tissue mass determined. Aliquots of the various organs were used for the determination of the radioactivity. Autoradiography oj'liver slices
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Fig. 2. Biological activities of rhI1,-ti bcfore and after iodination. RhIL-6 was iodinated to a specific activity of 50 kBq/pg and its biological activity ( 0 )assayed and compared to the activity of the unlabeled rhlL-6 (0)as described in Materials and Methods
sodium 5,5-diethylbarbiturate and 75 mM glycine, pH 8.9), allowed to drain and then electrophoresed for 2 h at 20 Vjcm. The sheets were stained for proteins with Ponceau red (0.2% in 3% trichloroacetic acid), made transparent with acetic acid, allowed to dry and subjected to autoradiography. Clearance and organ distribution qfrhIL-6 0.45 kg '251-rhIL-6(about 1.2 x 10' cpm) was injected intravenously into the tail vein of anesthetized rats ( n = 5). Prior to the injection the vena cava inferior was catheterized, and blood samples of 0.2-ml volume were taken through this catheter and transferred to heparinized vials. Blood cells were separated from the plasma and washed twice with 50 mM phosphate buffer (Na/K), pH 7.4 containing 0.14 M NaCl. Radioactivity was determined in whole blood, plasma and blood cells. 20 min after '251-rhIL-6 administration the animal was sacrificed and liver, spleen, kidneys, heart, lungs, small intestine, thymus, and a portion of the musculus rectus
7.5 x lo6 cpm of lz5I-rhIL-6 (specific activity 50 kBq/pg) were intravenously injected into 80-g male Wistar rats ( n = 3). 20 min later the liver was perfused with 50 mM phosphate (Na/K) buffer, pH 7.4 containing 0.14 M NaCl and subsequently fixed in situ with 4% glutaraldehyde in the same buffer. Liver pieces were dried in a graded series of ethanol, and embedded in paraffin. Sections, 5 pm thick, were cut from tissue blocks, deparaffinized by three 5-min washes in xylene followed by two 5-min washes in absolute ethanol. The slides were coated with Kodak NTB-2 nuclear track emulsion, dried, transferred to light-proof boxes and exposed at 4°C. Exposure time was 7 days. After exposure the slides were developed in Kodak D-19 developer followed by brief washing and fixation in Kodak fixer. After washing the slides were stained with hematoxylin. RESULTS The plasma half-life of rhIL-6 was studied in rats. After intravenous injection of 40000 units rhIL-6, blood samples were taken for the determination of the biological activity. rhIL-6 activity was measured by the induction of a-fibrinogen mRNA in the rat hepatoma cell line Fao. Fig. 1 shows that rhIL-6 activity disappeared very rapidly from the plasma. To study the fate of the rhIL-6 in the rat after intravenous injection, rhIL-6 was iodinated. Iodination did not lead to a reduction in biological activity of rhlL-6 as measured by /?-fibrinogen-mRNA stimulation in Fao cells (Fig. 2). Fig. 3 shows that the iodinated rhIL-6 disappeared in a biphasic manner. The clearance curve consisted of two exponential components: the initial, fast one, corresponded to an average
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Fig. 4. Sodiunr tiotlc.c:i~l~ ~ i i l f ~ ~ t ~ ~ gel ~ ~ rlc,c.trophoretic, ~ o l ~ ~ ~ ~unulysis ~ r ~ ~of l ~ "1-rhlL-6 ~ t ~ i i in~ plusmu t ~ ~ und liver uficr iniruvcvous inic>ction.As detailcd in the legend to Fig. 3, I .2x 10" cpm '251-rhIL-6were intravenously injected into male Wistar rats. At the times indicatcd (30 s to 20 min) 0.2-ml blood samples wcrc takcn and 2 4 aliquots of plasma were subjected to sodium dodecyl sulfate/polyacrylamidc gcl clectrophoresis and autoradiography. After 20 inin an aliquot of the liver was homogenized, centrifuged and 2 samples were also analyzed by sodium dodecyl sulfate/polyacrylamidc gcl clcctrophorcsis (two lancs on thc right). The left lane shows an aliquot of the '251-labcled rhlL-6 used for the injection into the animal
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Organ distribution of 11-6 200
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Fig. 5. Binding 0 f ~ ~ ~ I - r l i ltLo -( I6rc1tpl~st~iup~0ti~in,fi.u~ii0n. 1 pl 1251rhIL-6 (4 x lo4 cpm) in 50 m M phosphate buffer, pH 7.4, containing 0.14 M NaCl and 0.05% bovine scrum albumin (BSA) (lanes 1 and 4) or 1 p1 rat serum (lanes 3 and 6) or 1 p1 of a 1 : I mixture of Iz5I-rhIL-6 and rat scrum (lancs 2 and 5) wcrc clcctrophorctically separated on cellulose-acetatc papcr, and stained either for proteins (lanes 1-3) or subjcctcd to autoradiography (lancs 4-6). The arrow indicates the start
Fig. 6. Orgun distribution of 1251-rhlL-6.As described in the legend to Fig. 3, 1.2 x l o 6 cpm I2'1-rhlL-6 (specific activity 50 kBq/pg) was intravcnously injcctcd into malc Wistar rats. After 20 min, organs were excised and their wet masses and radioactivities were measured. Data are expressed as absolute radioactivity determined in each organ. The three groups of blocks relate to three different rats
half-life of about 3 min, whereas the half-life of the second slow component was about 55 min. N o 1251-rhIL-6 was bound to blood cells. From the comparison of Figs 1 and 3 we assume that the pharmacokinetics of rhIL-6 is not altered after iodination. To demonstrate that the radioactivity measured represents intact IL-6, aliquots of plasma samples were subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing conditions and subsequently to autoradiography. It can be seen from Fig. 4 that no partially degraded or processed forms of rhIL-6 were detectable up to 20 min. The radioactivity of the excised bands corresponding
to '251-rhIL-6 was determined and compared to the radioactivity in plasma. The ratio of radioactivity in plasma to radioactivity in the IL-6 band was constant at all time points (data not shown) suggesting that no degradation of rhIL-6 had occurred. In order to investigate whether IL-6 is associated with other serum proteins, we incubated 1251-rhIL-6with rat serum and separated the mixture by cellulose-acetate electrophoresis under non-denaturing conditions. The cellulose-acetate sheet was stained with Ponceau red to visualize proteins (Fig. 5, left part). To localize the 1251-rhIL-6the sheet was subsequently exposed to an X-ray film (Fig. 5 , right part). The band with
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Fig. 7. Distrihution uf' '2sI-rhlL-6in liver. '2sl-rhIL-6 (7.5 x lo6 cpm, 50 kBq/pg) were injected into the tail vein of malc Wistar rats. After 20 min the liver was perfuscd in .situ with 4% glutaraldehyde in 50 mM phosphate (Na/K) buffer, pH 7.4 containing 0.14 M NaCI. After fixation, liver pieces were embedded in paraffin. sliced and analyzed by autoradiography and light microscopy, as described in Materials and Methods. Magnification: A, 480 x ; B, 1400 x
the highest electrophoretic mobility seen in lane 1 represents liver, kidney, spleen, heart, thymus, lungs, muscle, and small bovine serum albumin (BSA) used as carrier for '2'I-rhIL-6. intestine were excised and the radioactivities of whole organs Lane 3 shows the typical plasma-protein fractions obtained were determined. Fig. 6 shows clearly that liver is the major after cellulose-acetate electrophoresis, with rat serum albumin target organ for rhIL-6. In three independent experiments, (RSA) as the major band. RSA exhibits a lower elec- about 80% of 12'1-rhIL-6 that had disappeared from the trophoretic mobility than BSA. This is also evident in lane 2, circulation was found in this organ. Upon sodium dodecyl containing a mixture of IL-6 dissolved in BSA and rat plasma. sulfate/polyacrylamide gel electrophoresis, the radioactive material taken up by the liver was shown to be intact It is obvious from the autoradiography (lane 4) that 12'1rhIL-6 migrated more slowly than BSA (lane 1) and RSA '251-rhIL-6 (see Fig. 4, right lanes). (lane 3) indicating that it binds neither to BSA nor to RSA. To identify the target cells within the liver, we examined In the presence of rat serum, a n additional band of slower liver sections, taken 20 min after injection of lz5I-rhIL-6, electrophoretic mobility appeared (lane 5). Whereas free by light microscopy after autoradiography. Autoradiography rhIL-6 migrated in the a-globulin fraction, the additional band showed the radioactivity exclusively a t the surface of was found in the b-y-globulin fraction. When the radioac- hepatocytes and essentially not on non-parenchymal cells tivity of both bands was eluted and analyzed by denaturing (Fig. 7). N o internalization of the radioactively labeled sodium dodecyl sulfate/polyacrylamide gel electrophoresis, rhIL-6 was observed at this time. both bands were shown to contain intact rhIL-6 (data not shown). This finding suggests that IL-6 is bound to a plasma DISCUSSION protein. '251-rhIL-6 intravenously injected into a rat disappears After having demonstrated the short plasma half-life of rhIL-6, the question arose, which organs are involved in its from the circulation with biphasic kinetics. From the fact that clearance. 20 min after intravenous injection of 251-rhIL-6 the peak plasma concentration of 251-rhIL-6 accounted for
361 only 50% of the initially injected IL-6 (volume distribution 20 m1/200 g rat) it follows that lz51-rhIL-6 rapidly disappears from the circulation. Furthermore, we have found that 1251rhIL-6 forms a complex with a plasma protein (Fig. 5). The rapid disappearance from the circulation may probably represent a first-pass effect of removal by the liver. In addition, rhIL-6/plasma-protein complex formation may also be involved in the biphasic clearance kinetics of 1L-6. Interestingly, most of the radioactivity (80%) that disappeared from the blood was found in the liver. In spite of the fact that IL-6 has been described to act on B-cells [5, 61 and on plasmacytoma cells [ 3 , 41, its major site of action seems to be the liver. No significant uptake of IL-6 into spleen (3%) or thymus (0.4%) was found. Besides the liver, kidneys are also involved in the clearance of IL-6, but to a much lower extent (10-15%). It is known that proteins of low molecular mass are easily filtered by the kidneys. Therefore, it is not surprising that IL-6, exhibiting a molecular mass of about 20 kDa, is found in this organ. From the autoradiographic study of liver slices it became clear that among the liver cells only hepatocytes bind 1251rhIL-6 on their surface (Fig. 7). This strongly suggests the existence of an 1L-6 receptor on hepatocytes. Such a receptor has recently been identified and characterized by Coulie et al. [15] in lymphoblastoid cells. The finding that, within liver, hepatocytes are the target cells for IL-6 is in agreement with recent results showing IL-6 as a potent inducer of the acutephase response in hepatocytes, hepatoma cells and in rat liver in vivo [7 - 101. Under physiological conditions the IL-6 released by stimulated monocytes is most likely first bound to a specific plasma protein, transported to the liver, where it binds to its receptor on the plasma membrane of the hepatocytes and in turn stimulates acute-phase protein synthesis. J.V.C. is indebted to Conselleria de Culturu de la Generalitat de Vulencia. Insfituto Nacional de Iu Salud and Universidud de Valencia for facilitating this research. The authors thank M. David for excellent technical assistance and H. Gottschalk ior her help with preparation of the manuscript. This work was supported by grants from the
Note udded m proof (received September 23, 1988). Yamaski ct al.[Science (Wash. D C ) 241,825-828(1988)]succeededin thecDNA cloning and expression of the 1L-6 receptor.
Deutsche ~orschungsgemeinsc.haft, Bonn, and the Fonds der Chemischen lndustrie, Frankfurt.
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