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Comparison of TNF-α and TNF-β gene expression in primary T lymphocytes
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MORE THAN ONE CYTOKINE IS SECRETED THROUGH AN ALTERNATIVE PATHWAY OF SECRETION A. Rubartelli”, G. Alkwena”, A. Bajetto”, F. Izmcia” and R. Sitian*. '1st. Naz.pcrlaRiarcasulCancm,Genova and Qt. Scimtifim S. Raffaele, Milmo,Italy. A number of proteins with a defined extracellular faction but lacking secretory signal sequences have been recently described. They include, among others, IL- lg and ADFAhioredotin. We studied the secretory pathway of IL- 1p. Our data indicate that IL-I 0 is secreted by activated monocytes through a novel pathway of secretion independent from fhe ER-Golgi classical one. Different hydrophobic drugs, affecting different cell compartments, increase secretion of IL- 1p, suggesting the possible involvement of members of the MDR family in alternative secretion. Trypsin-protected IL- 1B molecules are found only in those cells which actively secrete IL- lg suggesting that IL &containing vesids are part of the secretory route. Thioredoxin is a cytosolic enzyme involved in several reactions of oxide-reduction: in spite of its intracellular function, its presence has been demonstrated in supematants of transformed T and B cell lines. Studies on secretion of thioredoxin confirmed the existence of a novel pathway of secretion, modulated by several drugs. Thioredoxin is secreted by different cell types: interestingly, while normal resting B and T lymphocytes synthetize but do not secrete thioredoxin, activated B and T cells actively release thioredoxin extracelhdarly. The demonstration of an alternative secretory route followed by different cytokines lacking a secretory signal sequence suggests the existence of a common mechanism of secretion, for proteins that, for some reasons, must reach the extracellular space avoiding ER and Golgi. The role of thii alternative pathwayofsecretionwillbe discussedinthelightofsomestructuml(presence of free SH groups) and functional (role as autocrine growth factors) characteristics sharedbythisclassofsecretoryproteins.
POLYUNSATURATED FATTY ACIDS MODULATE SYNTHESIS OF TNF-a AND INTERLEUKIN-18 BY HUMAN MNC IN VITRO.
44
47
XANTHINE SUPPRESS ELEVATION
DERIVATIVES: COMPARISON OF THE POTENCY TO TNF-a SYNTHESIS WITH THE EXTENT OF cAMP-
and S. f=Mm
Medizinische Klinik, Klinikum Innenstadt, and ‘Institut fijr Prophylaxe und Epidemiologie der Kreislaufkrankheiten, UniversitP Miinchen, Miinchen, FRG. Metabolites of arachidonic acid (AA) regulate cytokine production. We studied the effects of in vitro-addition of AA and eicosapentaenoic acid (EPA, an active compound of fish oil) on the synthesis of IL-1 8 and TNF-a. We preincubated human blood mononuclear cells for 20 h with 10 pM, 20 HIM or 40 pM sodium salts of AA or EPA and stimulated the cells with heat-inactivated Staph. epidermidis (S. epi., 20 bacteria/cell) or LPS (10 rig/ml) for another 20 h. Cytokines were determined by specific RIA. AA dosedependently enhanced S. epi.-induced IL-l 6 synthesis to 300% of control at 40 pM and LPS-induced IL-1 6 production to 165% of control. The effect of AA on TNF-a depended on the stimulus: S. epi.-induced TNF-a synthesis was increased to 190%, while LPSinduced TNF-a synthesis was decreased to 50% of control. EPA exhibited weaker effects: S. epi.-induced IL-1 8 synthesis was suppressed at 40 pM EPA to 70%. TNF-a synthesis was not affected by EPA, regardless of the stimulus use. Enhancement of cytokine production by exogenous AA may result from increased formation of a lipoxygenase metabolite that upregulates cytokine synthesis.
Interleukin M. Svenson, St. Univ.
18 - an MB. Hosp.
unstable
monomer.
Hansen, P. Heegaard & K. and Univ. of Copenhagen,
Bendtzen. Denmark.
Medizinische Klinik, Klinikum lnnenstadt der Universittit Miinchen, and Hoechst AG, Werk Albert, Dep. of Clin. Res., Wiesbaden, FRG. The beneficial effect of xanthine derivatives in animal models of septic shock may be mediated, in part, by suppression of tumor necrosis factor-a (TNF) synthesis. We compared the in v&&effect of the following xanthine derivatives on the lipopolysaccharid (LPS) induced TNF-production in freshly isolated human mononuclear cells: Pentoxyfylline, penthydroxifylline, theophylline and the related agents A 80 2715, HWA 138 and HWA 448. All substances suppressed TNFproduction in a dose-dependent manner at concentrations ranging from 4 to 1000 PM. Suppression of TNF-production was paralleled by a dose-dependent elevation of total (i.e. intra- and extracellular) CAMP levels. The most potent substances in raising the CAMP levels were penthydroxifylline, theophylline and HWA 138 (CAMP concentration increased to around 200% at an effector dose of 250 FM). A 80 2715, which exhibited the strongest TNF-suppressive effect, only slightly raised CAMP (to 118% at 250 PM). In conclusion, we observe a concomitant rise in CAMP level parallel to TNF suppression by xanthine derivatives in our system. However, since we find no strict correlation in the potency of these two effects, elevation of CAMP may not be the only mechanism by which xanthine derivatives induce TNF suppression.
Molecular size chomatography of human XZsI-rIL-lfi preincubated at 37-C showed the appearance of high molecular size complexes and dimerization of monomeric XasI-rIL-1J3. When detected by a rabbit anti-rIL-lp RIA selective for monomeric lzsI-rILlp, preincubation of human recombinant and native IL-lp showed a transition in immunoreactivity when incubated between 30°C and 45V. The reduced immunoreactivity of IL-lj3 correlated with reduction of the bioactivity detected by the NOB-l, CTLL-2 assay. In the presence of serum the thermal inactivation at 370C of IL-lj3 was increased showing 50% conversion at 2-3 h. In contrast to at 4=C, the main binding of 12sI-rIL-1fi to serum at 370C was non saturable at 1 pg/ml. There was no signs of thermal inactivation at 37-C when using human recombinant or native IL-la. It is concluded that thermal denaturation of IL-lb, even at physiological temperature, may constitute a high capacity inhibitory process, which can be accelerated in body fluids by the presence of factors that bind the denaturated forms of IL-lo.
45
48
COMPARISON OF TNF-0. AND TNF-P GENE EXPRESSION IN PRIMARY T LYMPHOCYTES. & N. Sl.u.M~v and C.V.Jm Ludwig Inst. for cancer es ., CH-1066 EPALINGES, Switzerland.
GENETIC POLYMORPHISM IN THE HUMAN GENES TNFs AND THEIR RECEPTORS. R.L. Turetskava.
In vivo,macrophages are the most abundant source for TNF-a while lymphocytes produce mostly TNF-P. We have previously shown that in macrophages, nuclear factors of the NF-KB/rel family are essential for the LPS-mediated activation of the TNF-(X promoter. In an attempt to understand how the two genes are differentially regulated in lymphocytes, we started to investigate their expression in ConA-stimulated lymph node cells IT blasts). Northern blot analysis showed that TNF-a and TNF-P mRNAs have different kinetics of appearance in T blasts after activation by PNA and ionomycin: the level of TNF-a mPNA increased only after 1.5 h after activation, while TNF-P mP.NA was fully induced within 5 to 10 min. we performed bandshift analyses of nuclear proteins using probes derived from the KB3 site Of the TNF-a promoter or from the Ig KB enhancer. The 65kd/50kd NF-KB heterodimer, which has been shown to be involved in TNF-a activation in macrophages, appeared after 1.5 h of activation, concomitant with the induction of TNF-a mP.NA. Another factor with a slower electrophoretic mobility and a related binding specificity appeared with the same kinetics as TNF-P mRNA. We are in the process of characterizing this factor and its role in differential expression of TNF-aand -p genes.
ENCODING
I.A. Udalova. D.V. Kuorash, G.C. Webb, D.D. Chaolin g@ S.A. Nedos asov. Engelhardt Inst. Molec. Blol. USSR Acad. Sci;, Moscow-117984, USSR: Washington Univ. and HHNI, St. Louis, 63110 MO, USA. The genes human encoding TNFa and TNF,¶ (lymphotoxin) are closely linked between HLA-B and class III region of the MHC. Detecting genetic variability at TNF loci may give insights into pathogenesis of MHC-linked diseases, in particular, those characterized by altered TNF/LT production. To identify markers of TNF polymorphism we mapped and studied by PCR 5 microsatellites surrounding TNF genes. Use of only two markers identified more than 30 TNF alleles. Six polymorphic TNF markers, including the Nco I RFLP, have been used to type a panel of HLA workshop cell lines. Our data indicate strong association between TNF "haplotypes" and some MIX alleles. We have also cloned and characterized the human genes encoding 55kd and 75kD receotors for TNF and LT. and have studied several polymorphic microsatellites in the introns of TNF-R (both type I and type II) genes. Again, these polymorphisms may be relevant for studies of genetic predisposition to TNF related diseases and inherited susceptibility or resistance to infections.
INTERNATIONAL
WORKSHOP
ON
CYTOKINES
/ 457
43
46
MORE THAN ONE CYTOKINE IS SECRETED THROUGH AN ALTERNATIVE PATHWAY OF SECRETION A. Rubartelli”, G. Alkwena”, A. Bajetto”, F. Izmcia” and R. Sitian*. '1st. Naz.pcrlaRiarcasulCancm,Genova and Qt. Scimtifim S. Raffaele, Milmo,Italy. A number of proteins with a defined extracellular faction but lacking secretory signal sequences have been recently described. They include, among others, IL- lg and ADFAhioredotin. We studied the secretory pathway of IL- 1p. Our data indicate that IL-I 0 is secreted by activated monocytes through a novel pathway of secretion independent from fhe ER-Golgi classical one. Different hydrophobic drugs, affecting different cell compartments, increase secretion of IL- 1p, suggesting the possible involvement of members of the MDR family in alternative secretion. Trypsin-protected IL- 1B molecules are found only in those cells which actively secrete IL- lg suggesting that IL &containing vesids are part of the secretory route. Thioredoxin is a cytosolic enzyme involved in several reactions of oxide-reduction: in spite of its intracellular function, its presence has been demonstrated in supematants of transformed T and B cell lines. Studies on secretion of thioredoxin confirmed the existence of a novel pathway of secretion, modulated by several drugs. Thioredoxin is secreted by different cell types: interestingly, while normal resting B and T lymphocytes synthetize but do not secrete thioredoxin, activated B and T cells actively release thioredoxin extracelhdarly. The demonstration of an alternative secretory route followed by different cytokines lacking a secretory signal sequence suggests the existence of a common mechanism of secretion, for proteins that, for some reasons, must reach the extracellular space avoiding ER and Golgi. The role of thii alternative pathwayofsecretionwillbe discussedinthelightofsomestructuml(presence of free SH groups) and functional (role as autocrine growth factors) characteristics sharedbythisclassofsecretoryproteins.
POLYUNSATURATED FATTY ACIDS MODULATE SYNTHESIS OF TNF-a AND INTERLEUKIN-18 BY HUMAN MNC IN VITRO.
44
47
XANTHINE SUPPRESS ELEVATION
DERIVATIVES: COMPARISON OF THE POTENCY TO TNF-a SYNTHESIS WITH THE EXTENT OF cAMP-
and S. f=Mm
Medizinische Klinik, Klinikum Innenstadt, and ‘Institut fijr Prophylaxe und Epidemiologie der Kreislaufkrankheiten, UniversitP Miinchen, Miinchen, FRG. Metabolites of arachidonic acid (AA) regulate cytokine production. We studied the effects of in vitro-addition of AA and eicosapentaenoic acid (EPA, an active compound of fish oil) on the synthesis of IL-1 8 and TNF-a. We preincubated human blood mononuclear cells for 20 h with 10 pM, 20 HIM or 40 pM sodium salts of AA or EPA and stimulated the cells with heat-inactivated Staph. epidermidis (S. epi., 20 bacteria/cell) or LPS (10 rig/ml) for another 20 h. Cytokines were determined by specific RIA. AA dosedependently enhanced S. epi.-induced IL-l 6 synthesis to 300% of control at 40 pM and LPS-induced IL-1 6 production to 165% of control. The effect of AA on TNF-a depended on the stimulus: S. epi.-induced TNF-a synthesis was increased to 190%, while LPSinduced TNF-a synthesis was decreased to 50% of control. EPA exhibited weaker effects: S. epi.-induced IL-1 8 synthesis was suppressed at 40 pM EPA to 70%. TNF-a synthesis was not affected by EPA, regardless of the stimulus use. Enhancement of cytokine production by exogenous AA may result from increased formation of a lipoxygenase metabolite that upregulates cytokine synthesis.
Interleukin M. Svenson, St. Univ.
18 - an MB. Hosp.
unstable
monomer.
Hansen, P. Heegaard & K. and Univ. of Copenhagen,
Bendtzen. Denmark.
Medizinische Klinik, Klinikum lnnenstadt der Universittit Miinchen, and Hoechst AG, Werk Albert, Dep. of Clin. Res., Wiesbaden, FRG. The beneficial effect of xanthine derivatives in animal models of septic shock may be mediated, in part, by suppression of tumor necrosis factor-a (TNF) synthesis. We compared the in v&&effect of the following xanthine derivatives on the lipopolysaccharid (LPS) induced TNF-production in freshly isolated human mononuclear cells: Pentoxyfylline, penthydroxifylline, theophylline and the related agents A 80 2715, HWA 138 and HWA 448. All substances suppressed TNFproduction in a dose-dependent manner at concentrations ranging from 4 to 1000 PM. Suppression of TNF-production was paralleled by a dose-dependent elevation of total (i.e. intra- and extracellular) CAMP levels. The most potent substances in raising the CAMP levels were penthydroxifylline, theophylline and HWA 138 (CAMP concentration increased to around 200% at an effector dose of 250 FM). A 80 2715, which exhibited the strongest TNF-suppressive effect, only slightly raised CAMP (to 118% at 250 PM). In conclusion, we observe a concomitant rise in CAMP level parallel to TNF suppression by xanthine derivatives in our system. However, since we find no strict correlation in the potency of these two effects, elevation of CAMP may not be the only mechanism by which xanthine derivatives induce TNF suppression.
Molecular size chomatography of human XZsI-rIL-lfi preincubated at 37-C showed the appearance of high molecular size complexes and dimerization of monomeric XasI-rIL-1J3. When detected by a rabbit anti-rIL-lp RIA selective for monomeric lzsI-rILlp, preincubation of human recombinant and native IL-lp showed a transition in immunoreactivity when incubated between 30°C and 45V. The reduced immunoreactivity of IL-lj3 correlated with reduction of the bioactivity detected by the NOB-l, CTLL-2 assay. In the presence of serum the thermal inactivation at 370C of IL-lj3 was increased showing 50% conversion at 2-3 h. In contrast to at 4=C, the main binding of 12sI-rIL-1fi to serum at 370C was non saturable at 1 pg/ml. There was no signs of thermal inactivation at 37-C when using human recombinant or native IL-la. It is concluded that thermal denaturation of IL-lb, even at physiological temperature, may constitute a high capacity inhibitory process, which can be accelerated in body fluids by the presence of factors that bind the denaturated forms of IL-lo.
45
48
COMPARISON OF TNF-0. AND TNF-P GENE EXPRESSION IN PRIMARY T LYMPHOCYTES. & N. Sl.u.M~v and C.V.Jm Ludwig Inst. for cancer es ., CH-1066 EPALINGES, Switzerland.
GENETIC POLYMORPHISM IN THE HUMAN GENES TNFs AND THEIR RECEPTORS. R.L. Turetskava.
In vivo,macrophages are the most abundant source for TNF-a while lymphocytes produce mostly TNF-P. We have previously shown that in macrophages, nuclear factors of the NF-KB/rel family are essential for the LPS-mediated activation of the TNF-(X promoter. In an attempt to understand how the two genes are differentially regulated in lymphocytes, we started to investigate their expression in ConA-stimulated lymph node cells IT blasts). Northern blot analysis showed that TNF-a and TNF-P mRNAs have different kinetics of appearance in T blasts after activation by PNA and ionomycin: the level of TNF-a mPNA increased only after 1.5 h after activation, while TNF-P mP.NA was fully induced within 5 to 10 min. we performed bandshift analyses of nuclear proteins using probes derived from the KB3 site Of the TNF-a promoter or from the Ig KB enhancer. The 65kd/50kd NF-KB heterodimer, which has been shown to be involved in TNF-a activation in macrophages, appeared after 1.5 h of activation, concomitant with the induction of TNF-a mP.NA. Another factor with a slower electrophoretic mobility and a related binding specificity appeared with the same kinetics as TNF-P mRNA. We are in the process of characterizing this factor and its role in differential expression of TNF-aand -p genes.
ENCODING
I.A. Udalova. D.V. Kuorash, G.C. Webb, D.D. Chaolin g@ S.A. Nedos asov. Engelhardt Inst. Molec. Blol. USSR Acad. Sci;, Moscow-117984, USSR: Washington Univ. and HHNI, St. Louis, 63110 MO, USA. The genes human encoding TNFa and TNF,¶ (lymphotoxin) are closely linked between HLA-B and class III region of the MHC. Detecting genetic variability at TNF loci may give insights into pathogenesis of MHC-linked diseases, in particular, those characterized by altered TNF/LT production. To identify markers of TNF polymorphism we mapped and studied by PCR 5 microsatellites surrounding TNF genes. Use of only two markers identified more than 30 TNF alleles. Six polymorphic TNF markers, including the Nco I RFLP, have been used to type a panel of HLA workshop cell lines. Our data indicate strong association between TNF "haplotypes" and some MIX alleles. We have also cloned and characterized the human genes encoding 55kd and 75kD receotors for TNF and LT. and have studied several polymorphic microsatellites in the introns of TNF-R (both type I and type II) genes. Again, these polymorphisms may be relevant for studies of genetic predisposition to TNF related diseases and inherited susceptibility or resistance to infections.