In the rat, tumor necrosis factor (TNF-α) decreases the transplacental passage of amino acids
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504 / THIRD INTERNATIONAL WORKSHOP ON CYTOKINES 325 328 IDENTIFICATION OF TUMOR NECROSIS BLOCKING FACTOR IN SERUM AND TUWR TISSUE FROM PATIENTS WITH RECURRENT GLIOBLASTOMAS. Robert S. Yamamoto, Gene loli, Skip IN THE RAT, TUMOR NECROSIS FACTOR (TNF-a) DECREASES THE TRANSPLACENTAL PASSAGE OF AMINO ACIDS. J.M. AmllOs. N. Carb6 and F.J. L6pez-Soriano Bioquimica B, Unf versi tat de Barcelona, Barcelona, Spain Jacques, Yancy Beamer, Edward Jeffes, and Gale A. Granger. University of California Irvine, Memorial Cancer Institute, Long Beach Memorial Medical Center, Healthcare Medical Center Tustin and Good Samaritan Hospital of Los Angeles. About 30,000 adults each year are affected by brain tumors and a large majority of these tumors are glio- nla5. The prognosis for recurrent high grade glie blastomas after traditional therapies is dismal. In our studies we have made a new discovery, and that is brain tumor tissue may have the ability to depress endogenous host anti-tumor activity by releasing inhi- bitors to TNF/LT which block the biologic activity of these cytokines in vitro and in viva. These inhibitors, which have been ~rmedlockingfactors (BF), have been found in the serums and cerebrospinal fluids (CSF) in 4 of 5 tumor bearing patients and in 7 of 10 supernb tants from freshly cultured tumor tissues. The BF have not been identified in the serum or CSF of normal ic- dividuals. Because TNF and LT are key cytokines in the host cell-mediated anti-tumor mechanisms, factor(s) which inhibit these cytokines could have profound ef- fects on the tumor host interaction and their presence in viva should be considered before designing clinical -- trails that involve the use of these cytokines in viva. -- The implantation of a rapidly-growing tumour during the gestational period of the rat is associated with reduced fetal growth and development. Besides the fact that it is cytotoxic to certain types of tumour cells, TNF induces important metabolic and hormonal changes in the host, such as anorexia, lipid mobilization... It was the aim of the present study to ascertain whether TNF was responsible for the delayed fetal growth observed during tumour growth. Late pregnant rats were 1.v. injected with 20 pg of rhTNF-ol and 60 min. later they were given a” intragastrlc load of [ “Claminoisobutyrate (AIB), a non-metabolizable analogue of alanine. 90 minutes later, the tissue uptake of the amino acid was assessed in both maternal and fetal tissues. The results show that TNF administration lowered the uptake of the amino acid analogue by the feto-placental unit, both expressed ae a percentage of the absorbed dose or es the ratio tissue/plasma. It can be tentatively suggested that the cytokine may be reponsible for the delayed fetal growth observed in tumour-bearing pregnant rats. This would partially be the result of a reduced availability of amino acids for fetal growth. 326 MOLECULAR CLONING OF THE HUMAN INTERLEUKIN-1 RECEPTOR TYPE I (IL-1RtI) GENOMIC GENE K. Ye. C.A. Dinarello and B.D. Clark Tufts University School of Medicine and New England Medical Center Hospitals, Boston, MA 02111. 329 INTERLEUKIN-6 IS NOT THE MEDIATOR OF THE ENHANCED MUSCLE PROTEOLYSIS ASSOClATED WITH SEPSIS J.M. Amil~s. C. Garcia-Martinez and F.J. L6oez-Soriano BioqufmIca E, Unlversitat de Barcelona, Barcelona, Spain Both in viva and fn vftro studies show that during sepsis in the rat there Is a clear negative nitrogen balance which is mainlv a conseauence of a” aunmented release of amino Using a murine IL-lRt1 cDNA probe. we isolated a 1 kb cDNA from a human fibroblast cDNA library which encodes the transmembrane and cytoplasmic regions of the human IL-lRt1. We employed this 1 kb cDNA fragment to screen a human placental genomic library. Several overlaping but non-identical clones have been isolated. Synthetic oligonucleotides which correspond to 5’ non-coding, coding and 3’ non-coding region of the published cDNA sequences were made to verify these clones do contain specific IL-lRt1 sequences. The approximate length of the human IL-1RtI gene is estimated to spa” a 30 Kb region. Southern and partial sequence analysis of the putative genomic clone show that the non-coding region of the receptor is coded by two exons. Data will be presented on the 5’ promoter and putative regulatory sequences and the entire exon-intro” organization of the gene. RECOMBINANT HUMAN MONOCYTE-DERIVED INTERLEUKIN-1 RECEPTOR ANTAGONIST COUNTERACTS IL-1 EFFECTS ON a-CELL FUNCTION BUT NOT ON S-CELL FUNCTION IN ISOLATED RAT PANCREATIC ISLETS. U. 7umstea. F. Pock& .$t Helctvlst. C. Dinarello and J. NW&. Steno Diabetes Centre and Hagedorn Research Laboratory, DK-2820 Gentofte, Denmark, and Tufts University Schod of Medicine. Boston, Massachusetts. The cylokine and immune mediator peptide interteukin-10 (IL-l) is cytotoxlc to t3- cells in isolated islets of Langetins and may be the effector mdecule in the lmmundogical process leading to Insulin dependent diabetes mellitus. The ifluence of a recently cloned and normally by stimulated human monocytes produced IL-1 receptor antagonist (IL-lra, SYNERGEN) on insulin and glucagon release of IL-1 exposed rat islets was investigated. In the mouse and rat thymocyte costimulatory assay (LAF) IL-1 ra exerted a dose-dependent inhlbklon of IL-l-Induced BHthymidine incorporation. In rat islets a 24 hour exposure to 160 pg/ml authentic recombinant human IL-16 (biol. acthrity 400 IU/ng) decreased glucose-Induced insulin relsasa (0.39?0.07 vs 3.98+0.&l ng/lslet/2h in contrde): addition of IL-lra In a 100 fold excess did not orotect islets fO.53+0.09 na/islet/2h. n=6. o<O.O5\. In contrast. 24 hour IL-1 exf&ure stimulate islet glucagon &a& to &6?2B% of cont&but addkion of IL-ire resulted in a glucagon release of 117+ 12% of controls (ns vs contrde, p<O.O5 vs IL-1 group). A 6 day IL-1 incubation decreased islet insulin content to 35?4%, but coincubation with IL-lra dM not Inhibit this (39+6% of controls). Similar results have been found by using IL-1 and IL-lra on mouse and human islets. Thus IL-lra orotected a-cells aoainst IL-1 effects but not O-cells. These data suggest that the tyde of IL-1 receptor on thymocyles and ucells differs from the one on 6cells. This could explain at least partially why E-cells are extremely sensitive to IL-1 lnduc8cl cytotoxidty. I acids from skeletal muscle. Despire the efforts devoted to the identiffcation of the factor responsible for the enhanced protealysls, it has not yet bee” identified. Interleukin-6 (IL-61, produced and secreted by the immune system, is a cytokine with pleiotropic effects, being mainly involucrated in the acute-phase response associated with infectfon or trauma. It was the aim of the present study to ascertain If IL-6 could be the factor responsible for the enhanced proteolysfs observed in skeletal muscle during sepsis. Rat soleus and extensor digltorun longus (EDL) muscles were incubated in Krebs-tlenselelt physiologfcal saline containing 5 mM gl”CO.5.e and 1 mM ~“Clmethylaminoisobutyrete (MeAIB) in the presence or absence of IL-6 ( 2000 U/ml). The rate of MeAIB uptake and the tyrosine released to the medium were assessed. The results indicate that the addition of the cytokine did not cause any changes in either amino acid uptake or muscle proteolysfs. It is thus concluded that IL-6 is not responsible for the enhanced proteolysis observed when isolated rat muscles are incubated in the presence of septic plasma. 330 EFFECTS OF A WEEKLY DOSIS OF METHOTREXATE ON IL-l, TNF AND IL-6 IN PATIENTS WITH RHEUMATOID ARTHRITIS. P.Barrera. EMJanssen. AMTh.Boarbooms. LBA van de Putte. RW Sauewein. JWM van der Meer. University Hospital Nijmsgen, Postbox 9101. The Netherlands. The effectiveness of treatment wkh lowdose methotrexate (MTX) in rheumatoid arthriiis (RA) is well established. Recent reports have shown that MTX inhibiis interfeukln-1 (IL-l) activity in vitro. Because of the importance of proinflammatory cytokines (IL-l, TNF and 11-6) in RA we decided to measure the effect of a weekiv dosis MTX (7.5 ma/week) on circulating levels and sx- viva production of ihese cytokin&. S&en Ri patients were studied (4 of them in two occasions\. Blood samdes were collected before. 24 and 46 hours after MTX intake: IL-16 and iNF were determined by &dioimmuno- assay both in plasma and in a whole blood culture stimulated with LPS. For plasma IL-1 6 measurement chloroform extraction was perfoned. Serum IL6 was measured by bioassay (B9 cell line). RESULTS: Plasma IL-16 and TNF were low and did not significantly change during 46 h. Serum ILS levels ranged from 1 to 69 U/ml and did not change significantly. IL-1 6 and TNF after ex-viva Stimulation showed a trend to decrease after MTX which did not reach statistical significance: t=Oh t=24h t=46h mean range mean range mean range IL-la (“g/ml) 8.05 (0.06-20.04) 6.59 (0.26-19.46) 5.17 (1.66-7.72) TNF (“g/ml) 4.90 (1.06-13.06) 4.16 (O-9.6) 2.66 (0.46-7.66) IL-16 and TNF levels after ex-viva stimulation were strongly correlated (Spearman = 0.77). The three patients with highest valuas of stimulated IL-10 and TNF showed a decrease of more than 50% after MTX. Serum IL4 did not correlate with IL-10 or TNF production. In conclusion a lowdose MTX seems to induce changes in IL-10 and TNF prodution in some RA patients.
Transcript of In the rat, tumor necrosis factor (TNF-α) decreases the transplacental passage of amino acids
504 / THIRD INTERNATIONAL WORKSHOP ON CYTOKINES
325 328
IDENTIFICATION OF TUMOR NECROSIS BLOCKING FACTOR IN SERUM AND TUWR TISSUE FROM PATIENTS WITH RECURRENT GLIOBLASTOMAS. Robert S. Yamamoto, Gene loli, Skip
IN THE RAT, TUMOR NECROSIS FACTOR (TNF-a) DECREASES THE TRANSPLACENTAL PASSAGE OF AMINO ACIDS. J.M. AmllOs. N. Carb6 and F.J. L6pez-Soriano Bioquimica B, Unf versi tat de Barcelona, Barcelona, Spain Jacques, Yancy Beamer, Edward Jeffes, and Gale A.
Granger. University of California Irvine, Memorial Cancer Institute, Long Beach Memorial Medical Center, Healthcare Medical Center Tustin and Good Samaritan Hospital of Los Angeles. About 30,000 adults each year are affected by brain tumors and a large majority of these tumors are glio- nla5. The prognosis for recurrent high grade glie blastomas after traditional therapies is dismal. In our studies we have made a new discovery, and that is brain tumor tissue may have the ability to depress endogenous host anti-tumor activity by releasing inhi- bitors to TNF/LT which block the biologic activity of these cytokines in vitro and in viva. These inhibitors, which have been ~rmedlockingfactors (BF), have been found in the serums and cerebrospinal fluids (CSF) in 4 of 5 tumor bearing patients and in 7 of 10 supernb tants from freshly cultured tumor tissues. The BF have not been identified in the serum or CSF of normal ic- dividuals. Because TNF and LT are key cytokines in the host cell-mediated anti-tumor mechanisms, factor(s) which inhibit these cytokines could have profound ef- fects on the tumor host interaction and their presence in viva should be considered before designing clinical -- trails that involve the use of these cytokines in viva. --
The implantation of a rapidly-growing tumour during the gestational period of the rat is associated with reduced fetal growth and development. Besides the fact that it is cytotoxic to certain types of tumour cells, TNF induces important metabolic and hormonal changes in the host, such as anorexia, lipid mobilization... It was the aim of the present study to ascertain whether TNF was responsible for the delayed fetal growth observed during tumour growth. Late pregnant rats were 1.v. injected with 20 pg of rhTNF-ol and 60 min. later they were given a” intragastrlc load of [ “Claminoisobutyrate (AIB), a non-metabolizable analogue of alanine. 90 minutes later, the tissue uptake of the amino acid was assessed in both maternal and fetal tissues. The results show that TNF administration lowered the uptake of the amino acid analogue by the feto-placental unit, both expressed ae a percentage of the absorbed dose or es the ratio tissue/plasma. It can be tentatively suggested that the cytokine may be reponsible for the delayed fetal growth observed in tumour-bearing pregnant rats. This would partially be the result of a reduced availability of amino acids for fetal growth.
326
MOLECULAR CLONING OF THE HUMAN INTERLEUKIN-1 RECEPTOR TYPE I (IL-1RtI) GENOMIC GENE K. Ye. C.A. Dinarello and B.D. Clark Tufts University School of Medicine and New England Medical Center Hospitals, Boston, MA 02111.
329
INTERLEUKIN-6 IS NOT THE MEDIATOR OF THE ENHANCED MUSCLE PROTEOLYSIS ASSOClATED WITH SEPSIS J.M. Amil~s. C. Garcia-Martinez and F.J. L6oez-Soriano BioqufmIca E, Unlversitat de Barcelona, Barcelona, Spain
Both in viva and fn vftro studies show that during sepsis in the rat there Is a clear negative nitrogen balance which is mainlv a conseauence of a” aunmented release of amino
Using a murine IL-lRt1 cDNA probe. we isolated a 1 kb cDNA from a human fibroblast cDNA library which encodes the transmembrane and cytoplasmic regions of the human IL-lRt1. We employed this 1 kb cDNA fragment to screen a human placental genomic library. Several overlaping but non-identical clones have been isolated. Synthetic oligonucleotides which correspond to 5’ non-coding, coding and 3’ non-coding region of the published cDNA sequences were made to verify these clones do contain specific IL-lRt1 sequences. The approximate length of the human IL-1RtI gene is estimated to spa” a 30 Kb region. Southern and partial sequence analysis of the putative genomic clone show that the non-coding region of the receptor is coded by two exons. Data will be presented on the 5’ promoter and putative regulatory sequences and the entire exon-intro” organization of the gene.
RECOMBINANT HUMAN MONOCYTE-DERIVED INTERLEUKIN-1 RECEPTOR ANTAGONIST COUNTERACTS IL-1 EFFECTS ON a-CELL FUNCTION BUT NOT ON S-CELL FUNCTION IN ISOLATED RAT PANCREATIC ISLETS. U. 7umstea. F. Pock& .$t Helctvlst. C. Dinarello and J. NW&. Steno Diabetes Centre and Hagedorn Research Laboratory, DK-2820 Gentofte, Denmark, and Tufts University Schod of Medicine. Boston, Massachusetts.
The cylokine and immune mediator peptide interteukin-10 (IL-l) is cytotoxlc to t3- cells in isolated islets of Langetins and may be the effector mdecule in the lmmundogical process leading to Insulin dependent diabetes mellitus. The ifluence of a recently cloned and normally by stimulated human monocytes produced IL-1 receptor antagonist (IL-lra, SYNERGEN) on insulin and glucagon release of IL-1 exposed rat islets was investigated. In the mouse and rat thymocyte costimulatory assay (LAF) IL-1 ra exerted a dose-dependent inhlbklon of IL-l-Induced BHthymidine incorporation. In rat islets a 24 hour exposure to 160 pg/ml authentic recombinant human IL-16 (biol. acthrity 400 IU/ng) decreased glucose-Induced insulin relsasa (0.39?0.07 vs 3.98+0.&l ng/lslet/2h in contrde): addition of IL-lra In a 100 fold excess did not orotect islets fO.53+0.09 na/islet/2h. n=6. o<O.O5\. In contrast. 24 hour IL-1 exf&ure stimulate islet glucagon &a& to &6?2B% of cont&but addkion of IL-ire resulted in a glucagon release of 117+ 12% of controls (ns vs contrde, p<O.O5 vs IL-1 group). A 6 day IL-1 incubation decreased islet insulin content to 35?4%, but coincubation with IL-lra dM not Inhibit this (39+6% of controls). Similar results have been found by using IL-1 and IL-lra on mouse and human islets. Thus IL-lra orotected a-cells aoainst IL-1 effects but not O-cells. These data suggest that the tyde of IL-1 receptor on thymocyles and ucells differs from the one on 6cells. This could explain at least partially why E-cells are extremely sensitive to IL-1 lnduc8cl cytotoxidty.
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acids from skeletal muscle. Despire the efforts devoted to the identiffcation of the factor responsible for the enhanced protealysls, it has not yet bee” identified. Interleukin-6 (IL-61, produced and secreted by the immune system, is a cytokine with pleiotropic effects, being mainly involucrated in the acute-phase response associated with infectfon or trauma. It was the aim of the present study to ascertain If IL-6 could be the factor responsible for the enhanced proteolysfs observed in skeletal muscle during sepsis. Rat soleus and extensor digltorun longus (EDL) muscles were incubated in Krebs-tlenselelt physiologfcal saline containing 5 mM gl”CO.5.e and 1 mM ~“Clmethylaminoisobutyrete (MeAIB) in the presence or absence of IL-6 ( 2000 U/ml). The rate of MeAIB uptake and the tyrosine released to the medium were assessed. The results indicate that the addition of the cytokine did not cause any changes in either amino acid uptake or muscle proteolysfs. It is thus concluded that IL-6 is not responsible for the enhanced proteolysis observed when isolated rat muscles are incubated in the presence of septic plasma.
330
EFFECTS OF A WEEKLY DOSIS OF METHOTREXATE ON IL-l, TNF AND IL-6 IN PATIENTS WITH RHEUMATOID ARTHRITIS. P.Barrera. EMJanssen. AMTh.Boarbooms. LBA van de Putte. RW Sauewein. JWM van der Meer. University Hospital Nijmsgen, Postbox 9101. The Netherlands.
The effectiveness of treatment wkh lowdose methotrexate (MTX) in rheumatoid arthriiis (RA) is well established. Recent reports have shown that MTX inhibiis interfeukln-1 (IL-l) activity in vitro. Because of the importance of proinflammatory cytokines (IL-l, TNF and 11-6) in RA we decided to measure the effect of a weekiv dosis MTX (7.5 ma/week) on circulating levels and sx- viva production of ihese cytokin&. S&en Ri patients were studied (4 of them in two occasions\. Blood samdes were collected before. 24 and 46 hours after MTX intake: IL-16 and iNF were determined by &dioimmuno- assay both in plasma and in a whole blood culture stimulated with LPS. For plasma IL-1 6 measurement chloroform extraction was perfoned. Serum IL6 was measured by bioassay (B9 cell line).
RESULTS: Plasma IL-16 and TNF were low and did not significantly change during 46 h. Serum ILS levels ranged from 1 to 69 U/ml and did not change significantly. IL-1 6 and TNF after ex-viva Stimulation showed a trend to decrease after MTX which did not reach statistical significance:
t=Oh t=24h t=46h mean range mean range mean range
IL-la (“g/ml) 8.05 (0.06-20.04) 6.59 (0.26-19.46) 5.17 (1.66-7.72) TNF (“g/ml) 4.90 (1.06-13.06) 4.16 (O-9.6) 2.66 (0.46-7.66)
IL-16 and TNF levels after ex-viva stimulation were strongly correlated (Spearman = 0.77). The three patients with highest valuas of stimulated IL-10 and TNF showed a decrease of more than 50% after MTX. Serum IL4 did not correlate with IL-10 or TNF production. In conclusion a lowdose MTX seems to induce changes in IL-10 and TNF prodution in some RA patients.
