INF-α induces expression of the CD69 activation antigen in human resting NK cells, while INF-γ and...
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THIRD INTERNATIONAL WORKSHOP ON CYTOKINES / 459 55 58 INF-a INDUCES EXPRESSION OF THE CD69 ACTIVATION ANTIGEN IN PULMONARY MACROPHAGES SECRETE THE THREE ISOFORMS OF PDGF HLIMAN RESTING NK CELLS, WHILE INF-), AND TNF-a ARE INEFFECTIVE. WHICH INDUCE DIFFERENTIAL MITOGENIC AND CHEMOTACTIC RESPONSES C. Benati, M. Tomnasi, G. Carra and F. Gerosa, Istituto di BY LUNG FIBROBLASTS. A. Brodv. A. Osorniw. J. BpnneL Lab. Scienze Immunologiche e Istituto di Chimica Biologica, Verona. Pulm. Pathobiol., Nail. Inst. Environ. Hlth. Sci., Res. Tri. Pk., NC 27709 The effect of rINF-U, rINF-y and rTNF-Uon induction of ex pression of CD69 early activation antigen in purified resting NK cells was analyzed. It was found that incubation of NKcells for 18 hr with rINF-cz induced CD69 antigen expression in the large majority of NK cells to an extent comparable with the ex pression of CD69 antigen induced by rIL-2. In contrast, rINF-j or rTNF-U did not induce CD69 antigen expression. rINF-Udid not induce expression of CD69 antigen on T cells. Phosphoryla tion of CD69 antigen occured after stimulation of NK cellswith rINF-(I. Finally, the effect of rINF-u, rINF-y and rTNF-Uwas evaluated on the cytolytic activity of NK cells againsttheK562 target cell line. It was found that both rINF-y and rTNF-U iz creased the cytolytic activity of NK cells, although less effi ciently than rINF-U. Since expression of CD69 antigen is depey! dent on the acquisition of an activation state, our data demon stratr that rINF-U, as well as rIL-2, selectively induced acti vation of resting NK cells. In contrast, it remains to be elu cidated the activatory role of rINF-y and rTNF-U, that, incr& sing the cytolytic activity of NK cells, did not increase ex pression of activation antigens such as CD69. Macrophages are believed 10 play a central role in mediating lung inflammation and fibrosis through elaboration of a wide variety of cytokines. We have shown lhat rat lung macrophages (RLMs) synthesize and secrete a homologue of platelet-derived growth factor (PDGF) which is known to be a potent mitogenic and chemotactic stimulus lor fibroblasts and smooth muscle cells. Now we have found that this homologue produced by RLMs is comprised of the three isoforms of PDGF, i.e. the AA, AB and BB dimers. Quantitative enzyme immunoassay5 were established using monoclonal antibodies to the AA and BB homodimers and a polyclonaf Ab 10 the AB heterodimer. We found that the BB homodimer constitutes -50% of the macrophage-derived (MD) PDGF, the AB heterodimer -37% and the AA -13%. PDGF-BB was the most polenl inducer of mitogenesis (>150% increase al 1 rig/ml) by early passage rat lung fibroblasts (RLFs), while the AA was the weakest (~25% at 20 nglml). Fibroblast chemotaxis exhibited a similar pattern, with the BB lar more potent. A radioreceptor assay showed that the RLFs possess high affinity receptors (specific binding -90%) for the BB homodimer, whereas Ihe AA was a weak compelitor. Thus, 10 establish the mechanisms of macrophage mediated inflammation and fibrogenesis, we must understand the biochemical nature of relevant growth laclors and their biological potential. 56 FAIL.UREOFTCELLSFROMIL-~MJNUSMUTANTSTOHELPINIGMSE- CRETIONOFANTI-~F(AB,)2ACTIVAT!ZD B CELLS. IBerberich.H. A him 1 Institut fiir Virologie und Immunbiologie, UniversitBt Wiirzburg, Versbacher Str. 7.8700 Wiirzburg, Germany B cells stimulated to proliferate by Lipopolysaccharide &I%) or Interleukin 5 (IL-5) fail to secrete IgM if their antigen specific receptor (BCR) is engaged by crosslinking anti-IgM F(ab’h fragments (anti-~). At the molecular level this phenomenon is characterized by a failure to efficiently process the p precursor towards ps and to express the J-chain. Attempts to revert the negative signal show that inhibition of secretion depends on 1) the dose of BCR crosslinking antibodies and 2) the doses and the nature of lymphokines present in the culture. Thus, in the IL-S/anti-p driven system, both IL-2 and yIFN are needed for IgM secretion. The importance of JL-2is borne out by the use of T cells from normal and IL-2 deficient mice: In the absence of exogenous IL-2 only normalT's will induce&M secretion,while theIL2deficientcellsprovide help only ifIL-2is added to the cultures.The experiments demonstrate a directorindirectobligatoryroleforIL-2in overcoming the negative signal for IgM secretion mediatedaftermassiveBCRrrossliikage. 57 AMPLIFICATION OF CYTOKINE RELEASE FOLLOWING CONTACT BETWEEN LPS-MACROPHAGES AND VASCULAR TISSUE. C. Y. Gillaux. R. Meryal and A. Tedwi. INSERM U141, hBpital LariboisiBre, 75010 Paris. France. Cytokines play a key role in inflammatory processes as a result of interactions between macrophages (MO) and vascular cells. Precise regu- lation is essential to preserve the host from cytokine-mediated injury. To investigate the secretory cooperation between Ma and vascular cells, eli- cited peritoneal rabbit M@ were incubated for 18h at 5.10e/ml with LPS(iwg/ml) and then exposed for 4h to a challenge dose of LPS (ipg/ml) in flasks, or in the lumen of perfused and pressurised excised rabbit carotids. Supernatants from 18h LPS-activated Ma and 4h reincubated MO. as well as cell-free intraluminal (i.1) solutions were bioloaicallv as- sayed for IL-1 and TNF. using DlO.G4~icell clone proliferation-and-L929 murine fibroblasts cvtotoxicitv. resoectivelv. Results fmean+SEMj were: 18h’Ma .. hh rein&b. Ma ~ 4h i_l_ sol&on IL-I (U/ml) 360 + 19 (n=l6) 120 f 8 (n=7) 4400 f 1400 (n=4) TNF (U/ml) 29505 750 (n=4) 27 + 12 (n=3) 340 f 82 (n=6) Sequential exposure of M0 to LPS in flasks led to an effective down regulation of TNF release. Yet, IL-1 production seemed to be less affected by the early phase of LPS tolerance. in support of differential regulation of IL-1 and TNF secretions. Strikingly, a marked increase in TNF, and even more, in IL-1 concentrations was found in i.l. solutions. This suggests that contact between MQ and vascular tissue led to an amplification of the cytokine release, resulting from modification of MQ phenotype and/or from stimulation of the vessel wall self-production. These results highlight the critical role of cell-to-cell interactions in the production of potentially deleterious monokines involved in several vascular diseases. 59 EARLY TARGET CELL DNA DEGRADATION DUE TO A SYNERGISTIC EFFECT OF EXTRACELLULAR ATP AND TNF. V. Bronte, A. Zambon. S. Mandruzzato. A. Rosato. P. Pizza, P. Zanovello and D. Collavo. Inst. Oncology and Inst. General Patolosv, Univ. Padova, 35128 Padova, Italy. __. Recent evidence allow ATP, to be considered as a possible mediator of cell-mediated lysis. ATP causes a concentration-dependent cytotoxicity an8 DNA fragmentation of different targets. We demonstrated DNA fragmentation by means ye1 electrophoresis and quantified it by 24 agarose 51UdR or H-TdR release, to avoid problems of nuclear radiotoxicity and cell-cycle restricted labelling. We found that ATP,, can synergize with other lytic molecules endowed with DNA degrading activity, such as TNF or LT; however, these effects were cell-type dependent. Treatment of P815 and L1210 cells with ATP and TNF/LT mainly induced DNA fragmentation that was similar to cytotoxicity mediated by intact effector cells in terms of kinetis and extent. WEHI cells responded to the same combination of molecules with an increase in DNA breakdown and cell lysis, as assessed by 51Cr release. Some cells, like YAC-1, showed only partial apoptosis. Moreover, in different cell types, ATP, and LTfTNF- induced DNA degradation w s f+. not affected by chelation of extracellular Ca 60 PHOSPHOL I P IDS CAN AFFECT THE CAPABILITY OF INTERFERON IN MC DULATING THE EXPRESSION OF TWO GENES IN DAUDI CELL NUCLE1.A. - Cataldi,R.Di Primio,‘O.Trubiani,S.Miscia.lstituto di Morfolo - qia Umana Normale.Universit~ di Chieti:“lstituto di Citomor folcgia del C.N.R.,Chieti. - Investigations on the metabolic response of nuclei isol ted from interferon treated Daudi celIs,have disclosed early and transient modifications of chromatin associated phospholl pids concomitant to changes of in vitro nucleic acid synthe sis.Since chromatin phospholipids seem to interact with gent mic DNA and affect in vitro RNA synthesis,we have studied the influence of phosphatidylcholine (PC) and phosphatidylserine (PS)on the regulatory action of interferon alpha on the trac scription of two genes (plFN-INDl - plFN-IND2) in nuclei iso lated from interferon treated Daudi ceiIs.The addition of PC Iiposomes to the reaction mixture appears to inhibit the sy! thesis of the r&NA of these two proteins,usually induced by interferon,while PS Iiposomes are not effective.These results along with findings of an early increase of nuclear PC level after interferon treatment,support the hypothesis that chrg matin associated lipids may play a role in the regulatory activity of interferon on cell growth.
Transcript of INF-α induces expression of the CD69 activation antigen in human resting NK cells, while INF-γ and...
THIRD INTERNATIONAL WORKSHOP ON CYTOKINES / 459
55 58
INF-a INDUCES EXPRESSION OF THE CD69 ACTIVATION ANTIGEN IN PULMONARY MACROPHAGES SECRETE THE THREE ISOFORMS OF PDGF HLIMAN RESTING NK CELLS, WHILE INF-), AND TNF-a ARE INEFFECTIVE. WHICH INDUCE DIFFERENTIAL MITOGENIC AND CHEMOTACTIC RESPONSES C. Benati, M. Tomnasi, G. Carra and F. Gerosa, Istituto di BY LUNG FIBROBLASTS. A. Brodv. A. Osorniw. J. BpnneL Lab. Scienze Immunologiche e Istituto di Chimica Biologica, Verona. Pulm. Pathobiol., Nail. Inst. Environ. Hlth. Sci., Res. Tri. Pk., NC 27709
The effect of rINF-U, rINF-y and rTNF-Uon induction of ex pression of CD69 early activation antigen in purified resting NK cells was analyzed. It was found that incubation of NKcells for 18 hr with rINF-cz induced CD69 antigen expression in the large majority of NK cells to an extent comparable with the ex pression of CD69 antigen induced by rIL-2. In contrast, rINF-j or rTNF-U did not induce CD69 antigen expression. rINF-Udid not induce expression of CD69 antigen on T cells. Phosphoryla tion of CD69 antigen occured after stimulation of NK cellswith rINF-(I. Finally, the effect of rINF-u, rINF-y and rTNF-Uwas evaluated on the cytolytic activity of NK cells againsttheK562 target cell line. It was found that both rINF-y and rTNF-U iz creased the cytolytic activity of NK cells, although less effi ciently than rINF-U. Since expression of CD69 antigen is depey! dent on the acquisition of an activation state, our data demon stratr that rINF-U, as well as rIL-2, selectively induced acti vation of resting NK cells. In contrast, it remains to be elu cidated the activatory role of rINF-y and rTNF-U, that, incr& sing the cytolytic activity of NK cells, did not increase ex pression of activation antigens such as CD69.
Macrophages are believed 10 play a central role in mediating lung inflammation and fibrosis through elaboration of a wide variety of cytokines. We have shown lhat rat lung macrophages (RLMs) synthesize and secrete a homologue of platelet-derived growth factor (PDGF) which is known to be a potent mitogenic and chemotactic stimulus lor fibroblasts and smooth muscle cells. Now we have found that this homologue produced by RLMs is comprised of the three isoforms of PDGF, i.e. the AA, AB and BB dimers. Quantitative enzyme immunoassay5 were established using monoclonal antibodies to the AA and BB homodimers and a polyclonaf Ab 10 the AB heterodimer. We found that the BB homodimer constitutes -50% of the macrophage-derived (MD) PDGF, the AB heterodimer -37% and the AA -13%. PDGF-BB was the most polenl inducer of mitogenesis (>150% increase al 1 rig/ml) by early passage rat lung fibroblasts (RLFs), while the AA was the weakest (~25% at 20 nglml). Fibroblast chemotaxis exhibited a similar pattern, with the BB lar more potent. A radioreceptor assay showed that the RLFs possess high affinity receptors (specific binding -90%) for the BB homodimer, whereas Ihe AA was a weak compelitor. Thus, 10 establish the mechanisms of macrophage mediated inflammation and fibrogenesis, we must understand the biochemical nature of relevant growth laclors and their biological potential.
56
FAIL.UREOFTCELLSFROMIL-~MJNUSMUTANTSTOHELPINIGMSE- CRETIONOFANTI-~F(AB,)2ACTIVAT!ZD B CELLS. IBerberich.H. A him 1 Institut fiir Virologie und Immunbiologie, UniversitBt Wiirzburg, Versbacher Str. 7.8700 Wiirzburg, Germany
B cells stimulated to proliferate by Lipopolysaccharide &I%) or Interleukin 5 (IL-5) fail to secrete IgM if their antigen specific receptor (BCR) is engaged by crosslinking anti-IgM F(ab’h fragments (anti-~). At
the molecular level this phenomenon is characterized by a failure to efficiently process the p precursor towards ps and to express the J-chain.
Attempts to revert the negative signal show that inhibition of secretion depends on 1) the dose of BCR crosslinking antibodies and 2) the doses and the nature of lymphokines present in the culture. Thus, in the IL-S/anti-p driven system, both IL-2 and yIFN are needed for IgM
secretion. The importance of JL-2is borne out by the use of T cells from normal and IL-2 deficient mice: In the absence of exogenous IL-2 only normalT's will induce&M secretion,while theIL2deficientcellsprovide help only ifIL-2is added to the cultures.The experiments demonstrate a directorindirectobligatoryroleforIL-2in overcoming the negative signal for IgM secretion mediatedaftermassiveBCRrrossliikage.
57
AMPLIFICATION OF CYTOKINE RELEASE FOLLOWING CONTACT BETWEEN LPS-MACROPHAGES AND VASCULAR TISSUE. C. Y. Gillaux. R. Meryal and A. Tedwi. INSERM U141, hBpital LariboisiBre, 75010 Paris. France.
Cytokines play a key role in inflammatory processes as a result of interactions between macrophages (MO) and vascular cells. Precise regu- lation is essential to preserve the host from cytokine-mediated injury. To investigate the secretory cooperation between Ma and vascular cells, eli- cited peritoneal rabbit M@ were incubated for 18h at 5.10e/ml with LPS(iwg/ml) and then exposed for 4h to a challenge dose of LPS (ipg/ml) in flasks, or in the lumen of perfused and pressurised excised rabbit carotids. Supernatants from 18h LPS-activated Ma and 4h reincubated MO. as well as cell-free intraluminal (i.1) solutions were bioloaicallv as- sayed for IL-1 and TNF. using DlO.G4~icell clone proliferation-and-L929 murine fibroblasts cvtotoxicitv. resoectivelv. Results fmean+SEMj were:
18h’Ma .. hh rein&b. Ma ~ 4h i_l_ sol&on IL-I (U/ml) 360 + 19 (n=l6) 120 f 8 (n=7) 4400 f 1400 (n=4) TNF (U/ml) 29505 750 (n=4) 27 + 12 (n=3) 340 f 82 (n=6)
Sequential exposure of M0 to LPS in flasks led to an effective down regulation of TNF release. Yet, IL-1 production seemed to be less affected by the early phase of LPS tolerance. in support of differential regulation of IL-1 and TNF secretions. Strikingly, a marked increase in TNF, and even more, in IL-1 concentrations was found in i.l. solutions. This suggests that contact between MQ and vascular tissue led to an amplification of the cytokine release, resulting from modification of MQ phenotype and/or from stimulation of the vessel wall self-production. These results highlight the critical role of cell-to-cell interactions in the production of potentially deleterious monokines involved in several vascular diseases.
59
EARLY TARGET CELL DNA DEGRADATION DUE TO A SYNERGISTIC EFFECT OF EXTRACELLULAR ATP AND TNF. V. Bronte, A. Zambon. S. Mandruzzato. A. Rosato. P. Pizza, P. Zanovello and D. Collavo. Inst. Oncology and Inst. General Patolosv, Univ. Padova, 35128 Padova, Italy.
__.
Recent evidence allow ATP, to be considered as a possible mediator of cell-mediated lysis. ATP causes a concentration-dependent cytotoxicity an8 DNA fragmentation of different targets. We demonstrated DNA fragmentation by means ye1 electrophoresis and quantified it by
24 agarose 51UdR or
H-TdR release, to avoid problems of nuclear radiotoxicity and cell-cycle restricted labelling. We found that ATP,, can synergize with other lytic molecules endowed with DNA degrading activity, such as TNF or LT; however, these effects were cell-type dependent. Treatment of P815 and L1210 cells with ATP and TNF/LT mainly induced DNA fragmentation that was similar to cytotoxicity mediated by intact effector cells in terms of kinetis and extent. WEHI cells responded to the same combination of molecules with an increase in DNA breakdown and cell lysis, as assessed by 51Cr release. Some cells, like YAC-1, showed only partial apoptosis. Moreover, in different cell types, ATP, and LTfTNF- induced DNA degradation w s
f+. not affected by
chelation of extracellular Ca
60
PHOSPHOL I P IDS CAN AFFECT THE CAPABILITY OF INTERFERON IN MC DULATING THE EXPRESSION OF TWO GENES IN DAUDI CELL NUCLE1.A. - Cataldi,R.Di Primio,‘O.Trubiani,S.Miscia.lstituto di Morfolo - qia Umana Normale.Universit~ di Chieti:“lstituto di Citomor folcgia del C.N.R.,Chieti.
-
Investigations on the metabolic response of nuclei isol ted from interferon treated Daudi celIs,have disclosed early and transient modifications of chromatin associated phospholl pids concomitant to changes of in vitro nucleic acid synthe sis.Since chromatin phospholipids seem to interact with gent mic DNA and affect in vitro RNA synthesis,we have studied the influence of phosphatidylcholine (PC) and phosphatidylserine (PS)on the regulatory action of interferon alpha on the trac scription of two genes (plFN-INDl - plFN-IND2) in nuclei iso lated from interferon treated Daudi ceiIs.The addition of PC Iiposomes to the reaction mixture appears to inhibit the sy!
thesis of the r&NA of these two proteins,usually induced by interferon,while PS Iiposomes are not effective.These results along with findings of an early increase of nuclear PC level after interferon treatment,support the hypothesis that chrg matin associated lipids may play a role in the regulatory activity of interferon on cell growth.
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