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C3T3 fibroblasts by platelet derived growth factor
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DOWN REGULATION OF IL-1 RECEPTOR BXPRESSION ON PROGENITOR CELLS BY TRANSFORMING GROWTH FACTOR-BETA (TGF-,9).C.M. Dub&'. F.W. Ruscetti'. E.W. Palaszvnski3. L.A. Fa1k'J.J. ODoenheim' and J.R. Kelle#. 1,2,4*5wI, BRMP, DCT, NCI-FCRF, 3Child. H., Wash., D.C.. USA, and 6BCDP, PRI, NCI-FCRF, Frederick, ND, 21701-1013, USA. We have previously show" that TGF-fi is a potent negative regulator of immature hematopoietic progenitor cells including the high proliferative potential colony forming cells, that respond to IL-1 plus CSFs. To investigate the mechanism of TGFB inhibition, we examined the regulation of IL-1R ex2;ession on murine IL-3-dependent progenitor cell lines. Using P -I-IL-la, we found specific binding sites on three factor-dependent cell AC-Z, NFS-58 and NFS-60. The specific binding of '25-Ilines, IL-1 to NFS-60 cells was inhibited in a dose-dependent manner (IDS0 of 0.2 rig/ml) by treatment with TGF-p for 24 hrs. Fifty percent of IL-1R expression was inhibited by TGF-,4 in 4-6 hrs, and maximal inhibition occurs by 24 hrs. Scatchard analysis indicates at least one class of IL-1R on NFS-60 with approximately 400 R per cell and a" affinity of 1.0 f.2 X 10."M. Treatment with TGF-8 decreased the number of IL-1Rs to 50/tell without significant reduction in affinity (Kd- 1.3 f.4 X 10."M). Cross-linking studies revealed that TGF-0 treatment inhibited the Similarly TGF-8 expression of a 75-80 Kd IL-l-binding protein. inhibited IL-J-induced IL-1R on normal mouse bone marrow cells. These data suggest that the inhibitory effects of TGF-8 on early hematopoietic cells could be related to decreased IL-1R expression. Possible implications of these finding on other IL1 driven biological effects are under investigation.
PURIFICATION AND CHARACTERIZATION OF A TUMOR NECROSIS FACTOR a INHIBITOR (TNF~ INH). P. Seckinper. P. WinEfield, G. Turcatti. S. Isaaz and J.-M. Daver, Div. of Immunology & HBpital cant. univers. and Glaxo Allergy, Dept. of Medicine, IMB, 1211 Geneva, Switzerland. Urine of some febrile patients contains a TNFa INH [J. Exp. Med. 167:1511, 19881 when tested in the standard L929 cytotoxicity assay in the presence of actinomycin D. This inhibitor binds to TNFa, resulting in the inhibition of ligand binding [J. Biol. Chem. (1989)]. The TNFa INH was separated from urine and purified in successive steps by TNFa affinity, FPLC cation exchange (p1 5.5 - 6.1) and FPLC reverse-phase chromatographies. SDS-PAGE analysis followed by silver staining of the inhibitory fraction from the last step of purification yielded a single polypeptide with an Mr of = 33 kD, both under reducing grid non-reducing conditions. The inhibitor was purified -8 with a recovery of 3.3% and a specific activity of U/me (1 U corresnondine to 50% of inhibition of 0.2 rig/ml TNthyinhuced cytotdxicit;). NH2 terminal amino acid shows no homology with any other sequence (DSVXPQGKYIHPQXNSI) known protein. Glycanase treatment of the purified material reduces the mol wt of the TNFrr INH by -8 kD. The inhibitor does not share antigenicity with TNFa when tested by Western blot. These data demonstrate that TNFa activity is regulated by a glycoprotein with a novel amino acid sequence.
458
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MODULATION OF IL-l ACTIVITIES AND IL-1 RECEPTOR GENE TRANSCRIPTION IN BALBK3T3 FIBROBLASTS BY PLATELET DERIVED GROWTH FACTOR. J.P. Sinqh, P.D.Bonin, L.D. Huqhes, D.B.CarterandW.J.Chiou The Upjohn Company,Kalamazoo,MI 49001 Our studiesshowthat platelet derived growth factor (PDGF) modulates IL-l activities (as determined by the stimulation of DNAsynthesisand PGE2 production)and IL-1 receptor expression in Balb/c3T3 fibroblasts. In cells incubated in medium containing platelet poor plasma(lacking PDGF),IL-1 alone failedtostimulate DNA synthesisand PGE2 production. PGE2 and DNA synthesis in response to IL-1 was restored when PDGF was included in the culture medium. Stimulation was dependent on the concentration of both IL-1 and PDGF. PDGF treatment of Balb/c3T3 cells produced 20-30 fold stimulation of IL-1 receptor mRNA with a concomitant increase in cell surface 125l-IL-l binding. Nuclear run-on transcription experiments show that PDGFtreatmentinducestranscriptionalactivat~onofIL-1 receptor gene. Thesestudiessuggestthat PDGF may play lmportantrolein modulation of IL-1 responsesin connectivetissuecells.
soulBLEw-Mela tiovick,~arlnut
AEtEPREsqIN-~~ Ehgehann,
David
Wall&
and
~~r%rnl~ , Weipnann Institute of Science, R&xx&, Israel +1nterpharm laboratories Science-based Ir&atrial Park Nes-Zima, Israel Analysis of urine fnm rrnxaldcncas was performed in order to address the cpsstim whether release of soluble cytokiz-ptorsintobcdyfluidsisagrzeral~. Affi"itychmnatcgYa@lyofcmdshLmla"urinaIyproteins~ either imnobilizad huna" rIL-6, huna" rIFN-8 or nanoclo"al a&i IFN-B receptor antibxlyyielded tha reqxtiva ti soluble racq&rs in ai9nificant quar~tities. Asiqle saquence of 30 a.a. residues was obtained by N-ten&ml nicrosequencin9ofthapmtai"peakpnified intisteps (affinitychromatcqraphY 01 IL-6 ~01~ and RP-HIJLC). This sequence was identical to the predicted N-terminal sequence of IL-6 recaptar as pravicualy reported by Yamasaki et al. Science, 241, 825. 1988. AnaPsis of the eluted oroteins fmn b3th IFN-B and'snti IFN-r &eptor colunma by inhibitionof sRIA, ELISA, SDS-PAGE, and Wastarn blottirg proved tha axistar%x of soluble IFN-r receptor in rxxmal wina. Gurfirdingtqethar with tha alreadypmsence ofuri."aqTNFbindiqpmteins and a soluble IL-2 receptor b3th in pland in urine, indicates that release of soluble cyt&ina receptors iritobodyfluidsisagenaralphenanerrmwhichoccursurxlar normalphyaiolcgical ccx&tias.
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IL-lj3 AND IL-1 INHIBITOR (IL-11) PRODUCTION IN HUMAN MONOCYTES ARE REGULATED SEPARATELY. William P. Arend,l Fenneke G. Jo&n,* Charles H. Hannum, Evie L. Verderber and Stephen p. Eisenberg. University of Colorado HSC,* Denver, CO, 80262 and Synergen, Inc., Boulder, CO 80301. Human monocytes cultured on adherent IgG produce a 22 kD IL-li while the supernatants of cells stimulated with LPS exhibit IL-1 bioactivity. These studies compared regulation of IL-la and IL-li production in human monocytes using cDNA probes to detect specific mRNA's (IL-li, Eisenberg, et al., this volume). Specific antibodies were utilized in ELISA's to measure IL-la and IL-11 protein levels. Cells cultured on plain plastic exhibited little to no transcription or production of either protein. Monocytes stimulated with 500 "g/ml LPS transcribed the IL-1s and IL-11 genes and synthesized and secreted both IL-la and IL-H: total production over 24 hr for IL-l@ protein was 1.5-8.0 "g/ml while IL-li protein levels were 36.0-39.5 "g/ml. In contrast, monocytes cultured on adherent IgG exhibited transient and low levels of IL-lp mRNA with little to no IL-10 pmtein production. However, the IgGstimulated cells showed transcription of the IL-11 gene and produced over 300 ng/mlIL-li protein over 48 hrs. The results of preliminary experiments indicate that IL-lp and IL-li production are regulated in a reciprocal fashion when monocytes are cultured on adherent IgG in the presence of increasing concentrations of LPS. Data on the kinetics of IL-la and IL-H steady-state mRNA levels will be presented. Thus, IL-lp and IL-Ii are produced together but regulated separately.
A SOLUBLE IL-4-BINDING PROTEIN IS PRESENT IN MOUSE SERUM AND ASCITES: A POTENTIAL ROLE IN IEMUNOREGULATION. R. Fernandez-Botran and E.S. Vitettq. Department of Microbiology, U.T. Southwestern Medical Center at Dallas, Dallas, Texas. Most T cell-derived lymphokines are highly pleiotropic and exert multiple activities on their target cells in vitro. It is unclear how these activities are regulated in viva. IL-4 is a lymphokine that plays a central role in the generation of humoral imnune responses and in isotype switching. Using a soluble phase [1zI]-IL-4-binding assay, we have detected an IL-4-binding protein (IL-4-BP) in amuse serum and ascites and also in supernatants of activated murine lymphocytes; its binding activity is specific and saturable. Binding of ['"II-IL-4 to the IL-4-BP can be inhibited with an excess of unlabeled IL-4 but not IL-Z. The IL-4-BP is species-specific, since sera from other species (e.g., rat, rabbit, human) show no activitv. The molecule resoonsible for this activity has a Mr of -36 Kda and binds IL-4 with a dissociation constant (Kd) similar to that of the cellular IL-4 receptor (Kd: 8 x lo-"). Although the IL-l-BP is present in the serum of normal and imnune mice, its levels are low in serum from SCID mice, suggesting that activated lymphoid cells might be the primary source of this protein. The presence of IL-4-BP results in the inhibition of binding of ['"II-IL-l to T or B cells, indicating that the IL-4-BP might function in viva by preventing IL-4 from acting on bystander cells located at sites distal from its generation in the lymphoid organ.
WORKSHOP
ON CYTOKINES
/ 149
457
460
DOWN REGULATION OF IL-1 RECEPTOR BXPRESSION ON PROGENITOR CELLS BY TRANSFORMING GROWTH FACTOR-BETA (TGF-,9).C.M. Dub&'. F.W. Ruscetti'. E.W. Palaszvnski3. L.A. Fa1k'J.J. ODoenheim' and J.R. Kelle#. 1,2,4*5wI, BRMP, DCT, NCI-FCRF, 3Child. H., Wash., D.C.. USA, and 6BCDP, PRI, NCI-FCRF, Frederick, ND, 21701-1013, USA. We have previously show" that TGF-fi is a potent negative regulator of immature hematopoietic progenitor cells including the high proliferative potential colony forming cells, that respond to IL-1 plus CSFs. To investigate the mechanism of TGFB inhibition, we examined the regulation of IL-1R ex2;ession on murine IL-3-dependent progenitor cell lines. Using P -I-IL-la, we found specific binding sites on three factor-dependent cell AC-Z, NFS-58 and NFS-60. The specific binding of '25-Ilines, IL-1 to NFS-60 cells was inhibited in a dose-dependent manner (IDS0 of 0.2 rig/ml) by treatment with TGF-p for 24 hrs. Fifty percent of IL-1R expression was inhibited by TGF-,4 in 4-6 hrs, and maximal inhibition occurs by 24 hrs. Scatchard analysis indicates at least one class of IL-1R on NFS-60 with approximately 400 R per cell and a" affinity of 1.0 f.2 X 10."M. Treatment with TGF-8 decreased the number of IL-1Rs to 50/tell without significant reduction in affinity (Kd- 1.3 f.4 X 10."M). Cross-linking studies revealed that TGF-0 treatment inhibited the Similarly TGF-8 expression of a 75-80 Kd IL-l-binding protein. inhibited IL-J-induced IL-1R on normal mouse bone marrow cells. These data suggest that the inhibitory effects of TGF-8 on early hematopoietic cells could be related to decreased IL-1R expression. Possible implications of these finding on other IL1 driven biological effects are under investigation.
PURIFICATION AND CHARACTERIZATION OF A TUMOR NECROSIS FACTOR a INHIBITOR (TNF~ INH). P. Seckinper. P. WinEfield, G. Turcatti. S. Isaaz and J.-M. Daver, Div. of Immunology & HBpital cant. univers. and Glaxo Allergy, Dept. of Medicine, IMB, 1211 Geneva, Switzerland. Urine of some febrile patients contains a TNFa INH [J. Exp. Med. 167:1511, 19881 when tested in the standard L929 cytotoxicity assay in the presence of actinomycin D. This inhibitor binds to TNFa, resulting in the inhibition of ligand binding [J. Biol. Chem. (1989)]. The TNFa INH was separated from urine and purified in successive steps by TNFa affinity, FPLC cation exchange (p1 5.5 - 6.1) and FPLC reverse-phase chromatographies. SDS-PAGE analysis followed by silver staining of the inhibitory fraction from the last step of purification yielded a single polypeptide with an Mr of = 33 kD, both under reducing grid non-reducing conditions. The inhibitor was purified -8 with a recovery of 3.3% and a specific activity of U/me (1 U corresnondine to 50% of inhibition of 0.2 rig/ml TNthyinhuced cytotdxicit;). NH2 terminal amino acid shows no homology with any other sequence (DSVXPQGKYIHPQXNSI) known protein. Glycanase treatment of the purified material reduces the mol wt of the TNFrr INH by -8 kD. The inhibitor does not share antigenicity with TNFa when tested by Western blot. These data demonstrate that TNFa activity is regulated by a glycoprotein with a novel amino acid sequence.
458
461
MODULATION OF IL-l ACTIVITIES AND IL-1 RECEPTOR GENE TRANSCRIPTION IN BALBK3T3 FIBROBLASTS BY PLATELET DERIVED GROWTH FACTOR. J.P. Sinqh, P.D.Bonin, L.D. Huqhes, D.B.CarterandW.J.Chiou The Upjohn Company,Kalamazoo,MI 49001 Our studiesshowthat platelet derived growth factor (PDGF) modulates IL-l activities (as determined by the stimulation of DNAsynthesisand PGE2 production)and IL-1 receptor expression in Balb/c3T3 fibroblasts. In cells incubated in medium containing platelet poor plasma(lacking PDGF),IL-1 alone failedtostimulate DNA synthesisand PGE2 production. PGE2 and DNA synthesis in response to IL-1 was restored when PDGF was included in the culture medium. Stimulation was dependent on the concentration of both IL-1 and PDGF. PDGF treatment of Balb/c3T3 cells produced 20-30 fold stimulation of IL-1 receptor mRNA with a concomitant increase in cell surface 125l-IL-l binding. Nuclear run-on transcription experiments show that PDGFtreatmentinducestranscriptionalactivat~onofIL-1 receptor gene. Thesestudiessuggestthat PDGF may play lmportantrolein modulation of IL-1 responsesin connectivetissuecells.
soulBLEw-Mela tiovick,~arlnut
AEtEPREsqIN-~~ Ehgehann,
David
Wall&
and
~~r%rnl~ , Weipnann Institute of Science, R&xx&, Israel +1nterpharm laboratories Science-based Ir&atrial Park Nes-Zima, Israel Analysis of urine fnm rrnxaldcncas was performed in order to address the cpsstim whether release of soluble cytokiz-ptorsintobcdyfluidsisagrzeral~. Affi"itychmnatcgYa@lyofcmdshLmla"urinaIyproteins~ either imnobilizad huna" rIL-6, huna" rIFN-8 or nanoclo"al a&i IFN-B receptor antibxlyyielded tha reqxtiva ti soluble racq&rs in ai9nificant quar~tities. Asiqle saquence of 30 a.a. residues was obtained by N-ten&ml nicrosequencin9ofthapmtai"peakpnified intisteps (affinitychromatcqraphY 01 IL-6 ~01~ and RP-HIJLC). This sequence was identical to the predicted N-terminal sequence of IL-6 recaptar as pravicualy reported by Yamasaki et al. Science, 241, 825. 1988. AnaPsis of the eluted oroteins fmn b3th IFN-B and'snti IFN-r &eptor colunma by inhibitionof sRIA, ELISA, SDS-PAGE, and Wastarn blottirg proved tha axistar%x of soluble IFN-r receptor in rxxmal wina. Gurfirdingtqethar with tha alreadypmsence ofuri."aqTNFbindiqpmteins and a soluble IL-2 receptor b3th in pland in urine, indicates that release of soluble cyt&ina receptors iritobodyfluidsisagenaralphenanerrmwhichoccursurxlar normalphyaiolcgical ccx&tias.
459
462
IL-lj3 AND IL-1 INHIBITOR (IL-11) PRODUCTION IN HUMAN MONOCYTES ARE REGULATED SEPARATELY. William P. Arend,l Fenneke G. Jo&n,* Charles H. Hannum, Evie L. Verderber and Stephen p. Eisenberg. University of Colorado HSC,* Denver, CO, 80262 and Synergen, Inc., Boulder, CO 80301. Human monocytes cultured on adherent IgG produce a 22 kD IL-li while the supernatants of cells stimulated with LPS exhibit IL-1 bioactivity. These studies compared regulation of IL-la and IL-li production in human monocytes using cDNA probes to detect specific mRNA's (IL-li, Eisenberg, et al., this volume). Specific antibodies were utilized in ELISA's to measure IL-la and IL-11 protein levels. Cells cultured on plain plastic exhibited little to no transcription or production of either protein. Monocytes stimulated with 500 "g/ml LPS transcribed the IL-1s and IL-11 genes and synthesized and secreted both IL-la and IL-H: total production over 24 hr for IL-l@ protein was 1.5-8.0 "g/ml while IL-li protein levels were 36.0-39.5 "g/ml. In contrast, monocytes cultured on adherent IgG exhibited transient and low levels of IL-lp mRNA with little to no IL-10 pmtein production. However, the IgGstimulated cells showed transcription of the IL-11 gene and produced over 300 ng/mlIL-li protein over 48 hrs. The results of preliminary experiments indicate that IL-lp and IL-li production are regulated in a reciprocal fashion when monocytes are cultured on adherent IgG in the presence of increasing concentrations of LPS. Data on the kinetics of IL-la and IL-H steady-state mRNA levels will be presented. Thus, IL-lp and IL-Ii are produced together but regulated separately.
A SOLUBLE IL-4-BINDING PROTEIN IS PRESENT IN MOUSE SERUM AND ASCITES: A POTENTIAL ROLE IN IEMUNOREGULATION. R. Fernandez-Botran and E.S. Vitettq. Department of Microbiology, U.T. Southwestern Medical Center at Dallas, Dallas, Texas. Most T cell-derived lymphokines are highly pleiotropic and exert multiple activities on their target cells in vitro. It is unclear how these activities are regulated in viva. IL-4 is a lymphokine that plays a central role in the generation of humoral imnune responses and in isotype switching. Using a soluble phase [1zI]-IL-4-binding assay, we have detected an IL-4-binding protein (IL-4-BP) in amuse serum and ascites and also in supernatants of activated murine lymphocytes; its binding activity is specific and saturable. Binding of ['"II-IL-4 to the IL-4-BP can be inhibited with an excess of unlabeled IL-4 but not IL-Z. The IL-4-BP is species-specific, since sera from other species (e.g., rat, rabbit, human) show no activitv. The molecule resoonsible for this activity has a Mr of -36 Kda and binds IL-4 with a dissociation constant (Kd) similar to that of the cellular IL-4 receptor (Kd: 8 x lo-"). Although the IL-l-BP is present in the serum of normal and imnune mice, its levels are low in serum from SCID mice, suggesting that activated lymphoid cells might be the primary source of this protein. The presence of IL-4-BP results in the inhibition of binding of ['"II-IL-l to T or B cells, indicating that the IL-4-BP might function in viva by preventing IL-4 from acting on bystander cells located at sites distal from its generation in the lymphoid organ.