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The synthesis of tumor necrosis factor by human monocytes in vitro
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REGULATION OF c-jun GENE EXPRESSION BY TUMOR NECROSIS FACTOR. l%aLLhew L. Sherman. mnald w. Km. Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02115. The effects of tumor necrosis factor (TNF) on the regulation of c-jun gene expression have been studied in HL60 cells during monocytic differentiation. LOW levels of cjun transcripts were detectable in uninduced HL-60 cells, reached maximal levels by 30 min of exposure to TNF and returned to that of control cells by 3 h. Inhibition of de nova protein synthesis by cycloheximide alone had little HOWeVer, detectable effect on the accumulation of c-jun RNA. TNF treament in the presence of cycloheximide was associated with a superinduction of c-jun transcripts. Thus, treatment of HL-60 cells with cycloheximide and TNF for 3 h increased levels of c-jun WA 20-fold compared to treatment by TNF alone. In order to study the posttranscriptional regulation of TNF-induced c-jun mRNA levels, RL-60 cells were treated with TNF for 30 min and then exposed to actinomycin-D for The half-life of cvarious times to inhibit transcription. jun mRNA as determined by densitometric scanning was 30 min. In contrast, the half-life of c-jun mRNA in TNF-treated HL-60 cells exposed to cycloheximide and actinomycin-D was prolonged to 1 h. Taken together, these findings suggested that the increase in c-jun RNA observed during TNF treatment is mediated, at least in part, by posttranscriptional mechanisms involving the synthesis of a labile protein. Furthermore, these findings also sugqest that the c-jun product, AP-1, may be involved in the regulation of certain other gene expression by TNF.
THk SYNTHEL'IS OF TUMOR fJECROSls FACTOR BY HllMAN MONOCYTES IN VITRO. N. Voitenokx . N. Mlsu~ro. __-T-___ Kolesnikova A. Panvutich . A. Sudari.c.ke. --1 G. Idelson*. 0. Osipovich. Inst. of Exp. Hematol. & Riotechn"1.r. 125167 MOSCOW. Rvelorussian Inst. of Hematol.. 223059 Minsk. USSR. It was shown previously that pretreatment of human blood monocytes (MO) bv' actinomycin-D (ArD) did not block subsequent production of TNF in vitr". Posttranscrlptional induction of TNF synthesis was suggested.To study the mechanism of TNF production by human Ho we have measured the level of 1NF mRNA uSinU DNA probe. and the level of intracellular and secreted TNF bY ELISA and bs L-929 bioassav in the cultures of MO isolated hy Pet-co11 separation. It was shown that freshly isolated MO contained no intracellular ~""1 of TNF hut were capable to secrete nanograms of TNF per mg of MO after stimulation. Non or traces Of TNF mRNA could he detected in fr-eshlv isolated MO comparing with hiqh level of TNF mRNA in stimulated MO. It was shown that TNF mRNA synthesis in MO as well as in human lvmphocytes is relativelv resistant to the action of AcD. The data indicate that TNF Pr"duction by No is the result of de n"v" mRNA and protein synthesis.
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EPITOPE MAPPING DF IL-16: IDENTIFICATION OF A DISCONTINUOUS EPITOPE OF HUMAN IL-l ASSOCIATED WITH NEUTRALIZATION OF BIOLOGICAL ACTIVITY. P. L. Sinw. U. Feoderson. S. Locastro, J. SilvisVi. J. Lillquist. P. Young, and P. Bbatoagar. '5 -th Kl d trench Laboratories, King ot Prussia, P%sylv:~~aan194D6. Antibodies to synthetic peptides of human IL-16. and antibodies to rectiinant human IL-16 were used to identify epitopes associated with neutralization of biological activity (induction of IL-2 production by EL-4 cells). Four out of seventeen synthetic peptides induced the production of antibodies that were both illnunoprecipitating and neutralizing: 6-15. 49-80. 92-101, and 120-133 (where 1 = amino terminal AJ-A of mature IL-16). The predominant epitopes recognized by rabbit anti-IL-16 were within residues 41-47, 85-93. and 136-143. A non-neutralizing monoclonal antibody was mapped to residues 139-147. while a neutralizing monoclonal antibody mapped to two distinct regions, 6-11. and 87-95. suggesting a discontinuous epitope. Point nutations to several residues within these regions each separately resulted in lowered binding of the mutants to both the antibody and to the IL-l receptor. These results suggest a discontinuous epitope of IL-16 may be involved in receptor binding. Furthermore, antibodies directed against either colrponent of the epitope can block biological activity.
GENETIC VARIABILITY AT THE HUMAN TUMOR NECROSIS FACTOR 0: AND 6 GENE LOCI. G. Webb and D. Chaplin. Howard Hughes Med. Inst. and Washington Univ. Sch. of Med., St. Louis, HO 63110. The human tumor necrosis factor (TNF) CLand B genes are closely linked to each other near Hh-B.in the &ajor histocompatibility complex (HBC). They encode potent immunomodulatory cytokines produced primarily by macrophages and lymphocytes. Because polymorphism is important in the function of many HHC genes, we have studied the nature of variability at the TNF loci. RFLP analysis using TNP a and B hybridization probes identified variant restriction patterns in genomic DNA-digested with the restriction enzymes-Nco I and Act I. Nco I dinestion showed two restriction fragment alleles of 10.4 kb and 5.4 + 5.0 kb reflecting the absence or presence of an Nco I site in the first intro" of the TNF f3 gene. In contrast, digestion of genomic DNA with Act I demonstrated complex restriction fragment patterns that could not be reconciled with a simple allelic system. Studies using the polymerase chain reaction to examine the structure of the TNF genes and studies using 5-azacytidine indicate that the different Act I restriction fragment patterns are the result of differential DNA methylation. The variably methylated Act I sites are located in the 5'-flanking sequence and fourth exon of the TNF B gene. Further studies have shown that T cells, B cells, monocytes, and neutrophils purified from peripheral blood exhibit different Act I restriction patterns at the TNF f3 locus. This differential methylation may be significant to the expression of TNF in different cell types.
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MODULATION OF LPS INDUCED MONOKING PRODUCTION BY PARTIAL STRUCTURES OF LIPID A. A.J.Ulmer, W.Feist. H.Brade, S.Kusumoto. and Ii.-D.Flad. Forschungsinstitut Borstel, D-2061 Borstel, FRG. The aim of our study was to investigate the relation between the chemical structure of LPS and its biological activity in vitro. With regard to this we have measured the production of TNF, IL-l, and IL-6 by human monocytes stimulated with LPS, lipid A, or synthetic partial structures thereof (precursor Ia and lipid X) alone, and analysed the concert action of these structures. We found that the minimal structure inducing TNF. IL-l, and IL-6 release, is the hexaacylated lipid A, whereas the synthetic precursor Ia and synthetic lipid X are not active. When investigating the modulation by precursor Ia and lipid X we found that both partial structures can inhibit LPS and lipid A induced monokine production. This suppression was also found on the level of intracellular occurrence of the monokines. Beside an inhibitory we also found a stimulatory effect of precursor Ia on the response of human monocytes to LPS. However, this phenomenon was only observed when the precursor was added to the cultures lh before LPS. The nature of the different modulatory activities of lipid A partial structures is not known and is under investigation now.
ACIVICIN-INDUCED MONOCYTOID DIFFERENTIATION OF HL-60 CELLS: CORRELATION WITH INDUCTION OF TNF-a AND IL-@ mRNA. JB. VA and Duke, Durham, NC 27705 We have previously noted that the glutamine antagonist acivicin induces monocytoid differentiation of HL-60 cells. This study was to determine the effects of acivicin on the levels of I-IL-60 cell mRNA transcripts of several cytokines/factors implicated in the control of hematopoietic cell proliierationdifferentiation. Cells were grown + 10 ug/ml acivicin, or 16 nM PMA, the RNA extracted, and Northern blots performed. Acivicin and PMA caused no changes in the expression of mRNA for G-CSF, GM-CSF, IL-3, or IL-6. PMA reduced expression of c-myc and HSWO, and enhanced that of M-CSF COlltrOl Aciv-24h PMA-24h Aciv-48h PMA48h A&-72h PMA-72h Prok 4+ 3+ c-myc 0 3+ 0 1+ 1+ 4+ 1+ 2+ 1+ 0 c-myb M-CSF o 0 3+ 0 4+ v-fin.5
0
IL-1p
0
TNFU.
0
0
2+
0
3+
-
-
3+ 0 1+ 0 4+ 0 l+ 4+ 2+ ) HSP70 1 3+ 1 2+ 1+ 1 3+ 1+ 13+ and v&s. Acivicin caused some decrease in expression of c-myc, and impressively increased mRNA for IL-lp, TNF-a, and c-myb. Studies of mRNA stabilitv and functional/antiaenic nrotein measurements will heln define these pr&esses further. Both?%%; and IL-lb have been shown tb influence myeloid leukemia cell differentiation, and the c-myb protein is known to bind DNA and possibly modulate gene expression. Our results demonsaate that acivicin modulates expression of TNF-a, IL-ID, c-myc, and c-myb, and suggests that some or all of these factors might (in an autocrine manner) be involved in the differentiation.
INTERNATIONAL
WORKSHOP
ON CYTOKINES
/ 79
31
40
REGULATION OF c-jun GENE EXPRESSION BY TUMOR NECROSIS FACTOR. l%aLLhew L. Sherman. mnald w. Km. Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02115. The effects of tumor necrosis factor (TNF) on the regulation of c-jun gene expression have been studied in HL60 cells during monocytic differentiation. LOW levels of cjun transcripts were detectable in uninduced HL-60 cells, reached maximal levels by 30 min of exposure to TNF and returned to that of control cells by 3 h. Inhibition of de nova protein synthesis by cycloheximide alone had little HOWeVer, detectable effect on the accumulation of c-jun RNA. TNF treament in the presence of cycloheximide was associated with a superinduction of c-jun transcripts. Thus, treatment of HL-60 cells with cycloheximide and TNF for 3 h increased levels of c-jun WA 20-fold compared to treatment by TNF alone. In order to study the posttranscriptional regulation of TNF-induced c-jun mRNA levels, RL-60 cells were treated with TNF for 30 min and then exposed to actinomycin-D for The half-life of cvarious times to inhibit transcription. jun mRNA as determined by densitometric scanning was 30 min. In contrast, the half-life of c-jun mRNA in TNF-treated HL-60 cells exposed to cycloheximide and actinomycin-D was prolonged to 1 h. Taken together, these findings suggested that the increase in c-jun RNA observed during TNF treatment is mediated, at least in part, by posttranscriptional mechanisms involving the synthesis of a labile protein. Furthermore, these findings also sugqest that the c-jun product, AP-1, may be involved in the regulation of certain other gene expression by TNF.
THk SYNTHEL'IS OF TUMOR fJECROSls FACTOR BY HllMAN MONOCYTES IN VITRO. N. Voitenokx . N. Mlsu~ro. __-T-___ Kolesnikova A. Panvutich . A. Sudari.c.ke. --1 G. Idelson*. 0. Osipovich. Inst. of Exp. Hematol. & Riotechn"1.r. 125167 MOSCOW. Rvelorussian Inst. of Hematol.. 223059 Minsk. USSR. It was shown previously that pretreatment of human blood monocytes (MO) bv' actinomycin-D (ArD) did not block subsequent production of TNF in vitr". Posttranscrlptional induction of TNF synthesis was suggested.To study the mechanism of TNF production by human Ho we have measured the level of 1NF mRNA uSinU DNA probe. and the level of intracellular and secreted TNF bY ELISA and bs L-929 bioassav in the cultures of MO isolated hy Pet-co11 separation. It was shown that freshly isolated MO contained no intracellular ~""1 of TNF hut were capable to secrete nanograms of TNF per mg of MO after stimulation. Non or traces Of TNF mRNA could he detected in fr-eshlv isolated MO comparing with hiqh level of TNF mRNA in stimulated MO. It was shown that TNF mRNA synthesis in MO as well as in human lvmphocytes is relativelv resistant to the action of AcD. The data indicate that TNF Pr"duction by No is the result of de n"v" mRNA and protein synthesis.
38
41
EPITOPE MAPPING DF IL-16: IDENTIFICATION OF A DISCONTINUOUS EPITOPE OF HUMAN IL-l ASSOCIATED WITH NEUTRALIZATION OF BIOLOGICAL ACTIVITY. P. L. Sinw. U. Feoderson. S. Locastro, J. SilvisVi. J. Lillquist. P. Young, and P. Bbatoagar. '5 -th Kl d trench Laboratories, King ot Prussia, P%sylv:~~aan194D6. Antibodies to synthetic peptides of human IL-16. and antibodies to rectiinant human IL-16 were used to identify epitopes associated with neutralization of biological activity (induction of IL-2 production by EL-4 cells). Four out of seventeen synthetic peptides induced the production of antibodies that were both illnunoprecipitating and neutralizing: 6-15. 49-80. 92-101, and 120-133 (where 1 = amino terminal AJ-A of mature IL-16). The predominant epitopes recognized by rabbit anti-IL-16 were within residues 41-47, 85-93. and 136-143. A non-neutralizing monoclonal antibody was mapped to residues 139-147. while a neutralizing monoclonal antibody mapped to two distinct regions, 6-11. and 87-95. suggesting a discontinuous epitope. Point nutations to several residues within these regions each separately resulted in lowered binding of the mutants to both the antibody and to the IL-l receptor. These results suggest a discontinuous epitope of IL-16 may be involved in receptor binding. Furthermore, antibodies directed against either colrponent of the epitope can block biological activity.
GENETIC VARIABILITY AT THE HUMAN TUMOR NECROSIS FACTOR 0: AND 6 GENE LOCI. G. Webb and D. Chaplin. Howard Hughes Med. Inst. and Washington Univ. Sch. of Med., St. Louis, HO 63110. The human tumor necrosis factor (TNF) CLand B genes are closely linked to each other near Hh-B.in the &ajor histocompatibility complex (HBC). They encode potent immunomodulatory cytokines produced primarily by macrophages and lymphocytes. Because polymorphism is important in the function of many HHC genes, we have studied the nature of variability at the TNF loci. RFLP analysis using TNP a and B hybridization probes identified variant restriction patterns in genomic DNA-digested with the restriction enzymes-Nco I and Act I. Nco I dinestion showed two restriction fragment alleles of 10.4 kb and 5.4 + 5.0 kb reflecting the absence or presence of an Nco I site in the first intro" of the TNF f3 gene. In contrast, digestion of genomic DNA with Act I demonstrated complex restriction fragment patterns that could not be reconciled with a simple allelic system. Studies using the polymerase chain reaction to examine the structure of the TNF genes and studies using 5-azacytidine indicate that the different Act I restriction fragment patterns are the result of differential DNA methylation. The variably methylated Act I sites are located in the 5'-flanking sequence and fourth exon of the TNF B gene. Further studies have shown that T cells, B cells, monocytes, and neutrophils purified from peripheral blood exhibit different Act I restriction patterns at the TNF f3 locus. This differential methylation may be significant to the expression of TNF in different cell types.
39
42
MODULATION OF LPS INDUCED MONOKING PRODUCTION BY PARTIAL STRUCTURES OF LIPID A. A.J.Ulmer, W.Feist. H.Brade, S.Kusumoto. and Ii.-D.Flad. Forschungsinstitut Borstel, D-2061 Borstel, FRG. The aim of our study was to investigate the relation between the chemical structure of LPS and its biological activity in vitro. With regard to this we have measured the production of TNF, IL-l, and IL-6 by human monocytes stimulated with LPS, lipid A, or synthetic partial structures thereof (precursor Ia and lipid X) alone, and analysed the concert action of these structures. We found that the minimal structure inducing TNF. IL-l, and IL-6 release, is the hexaacylated lipid A, whereas the synthetic precursor Ia and synthetic lipid X are not active. When investigating the modulation by precursor Ia and lipid X we found that both partial structures can inhibit LPS and lipid A induced monokine production. This suppression was also found on the level of intracellular occurrence of the monokines. Beside an inhibitory we also found a stimulatory effect of precursor Ia on the response of human monocytes to LPS. However, this phenomenon was only observed when the precursor was added to the cultures lh before LPS. The nature of the different modulatory activities of lipid A partial structures is not known and is under investigation now.
ACIVICIN-INDUCED MONOCYTOID DIFFERENTIATION OF HL-60 CELLS: CORRELATION WITH INDUCTION OF TNF-a AND IL-@ mRNA. JB. VA and Duke, Durham, NC 27705 We have previously noted that the glutamine antagonist acivicin induces monocytoid differentiation of HL-60 cells. This study was to determine the effects of acivicin on the levels of I-IL-60 cell mRNA transcripts of several cytokines/factors implicated in the control of hematopoietic cell proliierationdifferentiation. Cells were grown + 10 ug/ml acivicin, or 16 nM PMA, the RNA extracted, and Northern blots performed. Acivicin and PMA caused no changes in the expression of mRNA for G-CSF, GM-CSF, IL-3, or IL-6. PMA reduced expression of c-myc and HSWO, and enhanced that of M-CSF COlltrOl Aciv-24h PMA-24h Aciv-48h PMA48h A&-72h PMA-72h Prok 4+ 3+ c-myc 0 3+ 0 1+ 1+ 4+ 1+ 2+ 1+ 0 c-myb M-CSF o 0 3+ 0 4+ v-fin.5
0
IL-1p
0
TNFU.
0
0
2+
0
3+
-
-
3+ 0 1+ 0 4+ 0 l+ 4+ 2+ ) HSP70 1 3+ 1 2+ 1+ 1 3+ 1+ 13+ and v&s. Acivicin caused some decrease in expression of c-myc, and impressively increased mRNA for IL-lp, TNF-a, and c-myb. Studies of mRNA stabilitv and functional/antiaenic nrotein measurements will heln define these pr&esses further. Both?%%; and IL-lb have been shown tb influence myeloid leukemia cell differentiation, and the c-myb protein is known to bind DNA and possibly modulate gene expression. Our results demonsaate that acivicin modulates expression of TNF-a, IL-ID, c-myc, and c-myb, and suggests that some or all of these factors might (in an autocrine manner) be involved in the differentiation.