- Email: [email protected]
Transforming growth factor-beta (TGFβ) gene expression in rat cardiac transplants during allograft rejection
THIRD
INTERNATIONAL
WORKSHOP
ON
CYTOKINES
/ 493
259
262
INTERLEUKIN6 (IL-6) IS PRESENTIN COLOSTRUMOF HEALTHYWOMEN. L. Paulesu, K. Van Bremen. f. Corradeschi, E. Luzzi, A. Oi Stefano and V.Bocci
AN INTERLEUKIN 1 BETA GENE RFLP REFLECTS IL-1 BETA SECRETION IN VITRO -A NEW SUSCEPTIBIUTY MARKER FOR NON DR3 AND NON DR4 IDDM PATIENT. F. Pociot. and J. Neruo. Steno Diabetes Cent& DK-2820 Gentofle, Denmark.
Institute of General Physiology, University of Siena, Siena, Italy. Cytokines such as IL-loL, IL-10. TNF (1.2) are present in traces in human colostrum and recently we have reported that also IFN f is measurable in physiological amounts. both by immunoassay and bioassay. We nw report that also IL-6 is readily detectable. particularly when crude colostrum is delipidated by centrifugation and separated by molecular sieving into several fractions. The procedure *as carried out using Centricon microconcentrators (Amicon) at 30 and 10 KD fractions. About 25 samples of colostrum were obtained from 14 women at the 2nd and 3rd day after parturition. Colostral cells were also incubated in RPM1 1640 at 1.5~10~ cells/ml for 24 hours. All the samples were tested by enzyme amplified sensitivity system (EASIATM. Hedgenix). Most of It-6 activity (55-190 pg/ml) was present in the Retentate 30, that is IL-6 seemedto be bound to a carrier protein. Little activity (24-30 pg/ml) was detected in the crude colostrum as well as in the filtrate 10, i.e., IL-S present in free form. Upon incubation. colostral cells released considerable amount of It-6 in the medium. In the whole these findings support the well known fact that "omen can undergo fever upon milk retention. Moreover it is possible that traces of cytokines in colostrum exert an important activation of the oropharyngeal and gut- associated lymphoid system of the newborn. 1) Seder 0. Int. Arch. Aller. Appl. Immunol. 83:1’3,1987; 2) Goldman A.S., Gold blum R.M. Ann. New York Acad. SC. 587:236,1990. Work supported by "Ricerca sanitaria finalizzata dalla Regione Toscana".
IL-1 may be the major Hector molecule in the initiation phase of the immunemediated destruction of beta-cells preceding overl IDDM. We have previously demonstrated
that
stable
interindivklual
differences
in in vitro
LPS-stimulated
monocyte (MO) IL-1 secretion exist. We hypothesized that such dmerences 1 response capacity are genetically determined and may be a contributory
in MO ILfactor in
IDDM pathogenesis. We identified a diallelic RFLP in the 5th exon of the human IL-1 gene with Taql consisting of fragments of 9.4 kb and 13.4 kb. The genotype frequencies in a control population (n=93) were: 57% (9.4/9.4 kb), 35% (9.4/13.4
kb), and 8% (13.4/13.4 kb). The corresponding
frequencies in a random IDDM group
(n=46) were not significantly different (p=O.3). However, when studying IDDM patients with no known major genetic marker, Le. IDDM patients negative for DR3 and DR4 (n=lO). significantly more IL-1 13.4 kb positive individuals were identified (p= 0.05). The functional impact of this RFLP was studied by analysing in vitro LPSstimulated (250 pg/ml) MO (3xlOE5) IL-1 secretion. An IL-1 allele dosage effect on secretory capacity was observed. 9.4/9.4 kb individuals (n=lO) had the lowest reponse: 3.76kO.35 rig/ml, 9.4/13.4 kb individuals (n=lO): 4.42tO.47 rig/ml and 13.4/13.4 kb individuals (n=4): 5.16+0.66 mg/mi (test for trend ~~0.05, p=O.13 for 9.419.4 vs 9.4113.4, p=O.O7 for 9.4jl3.4 vs 13.4/13.4, p=O.O2 for 9.4j9.4 vs 13.4/13.4). Conclusions: 1) The 13.4 kb allele represents a “high secretor’ phenotype. 2) The observed RFLP may be an additional susceptibility marker for IDDM in non DR3 and non DR4 individuals, and 3) high IL-1 production a pathogenetic factor for IDDM in these patients.
260
263
INTRAOPERATIVE CYTOKINE PRODUCTION DURING LIVER TRANSPLANTATION. J. Pirenne. F. Pirenne. D. Dezzroot. Y. Vrindts. P. Honor& P. Franchimont. Univ. Libge, 4 000, Belgium. Non-immunological inaaoperative events during liver transplantation (LTx) are likely 6 trigger cytokine production: surgery induced tissue injury, endotoxinemia, and kupffer cell activation after repcrfusion. Aim of this study was to assess inaaoperative cytokine production during LTx. Using Enzyme Amplified Sensitivity Immunoassay (Medgenix), cytokines ILID, tL2, IL6, TNF*, andrIF were measured in serum of 4 LTx patients, preoperatively, before anhepatic phase (AP) and at day 1 post TX. Cytokines were also measured in bile. Preop AP Day 1 serum serum serum serum bile serum bile
Coneluslon: High levels of monokines IL 6, TNF, and IL 1 appeared in serum and bile during LTx. In contrast, lvmphokines IL 2 and IF could not be detected. This-demonstrates a major &mulation of monocyte cell lineage in the absence of lymphocyte activation. The finding that maximum levels of monokines IL1 and IL6 were detected >AP and presence of these monokines in the bile suggests that the TX liver might be a major site of monokines synthesis. Since monokines exert important effects on hepamcyte metabolism, this might have significant implications with regards to function of the TX liver.
DEVELOPMENT OF A CHEMILUMINESCENT ENZYME-LINKED IMMUNOSORBANT ASSAY (ELISA) FOR THE QUANTITATION 0~ RECOMBINANT HUMAN INTERLEUKIN-4 (RHUIL-4) IN HUMAN SERUM AND COMPARISON OF ASSAY SENSITIVITY WITH COLORIMETRIC METHODS. Nicholas Ronca, Karen Trudeau, Marian Kelley, Gary Stevens, and Lottie m. Sterling Drug Inc., Rensselaer, NY and Malvern, PA. A chemiluminescent sandwich ELISA has been developed for the quantitation of rhulL-4 in human serum. An immobilized rabbit polyclonal antibody is used as the capture antibody; bound rhuIL-4 is detected by sequential exposure tn the biotinylated rabbit antibody followed by streptavidin alkaline phosphatase. Light produced by enzymatic dephosphorylation of the adamantyl 1,2-dioxetane phosphate (AMPPD) substrate is measured in a microplate luminnmeter. ASSSY sensitivity is approximately 20 (0.13 Pg/“l f”oles/well). The assay is approximately ten times “ore sensitive than a first generation SSSSY employing p-nitrophenyl phosphate (PNPP) as substrate and twice as sensitive as when nicotinamide adenine dinucleotide phosphate (NADP) is used for signal amplification. The accuracy and precision of this assay were determined fro” nine separate analyses performed in pooled human serum. Percent CV ard relative bias (%I ranged fro” 0.9 to 7% and from -2.2 to 3.57%, respectively. These results indicate that the chemiluminescent second generation assay, by virture of its enhanced sensitivity, should be useful in quantitating rhuIL-4 in human serum when levels of less than 200 pg/mL are anticipated.
261
264
TRANSFORMING GROWTH FACTOR-BETA (TGFB) GENE EXPRESSION IN RAl l CARDIAC TRANSPLANTS DURING ALLOGRAFT REJECTION. T.T.
BETA-2 MICROGLOBULIN, IL 2 AND 1L 2 RECEPTOR IN THE DZAGNOSlS AND THE FOLLiXJ UP OF THE HIV INFECTION.
1 0 ihi 36-88 TNF 29-88 IL2 lo
IF 10.
O-3 65-8845 16-109 lo
10
04 100-12645 14-93 lo
I”
. Molinowsko”.
060 1184:10738
I
36-166
0
I
0
3 4:00 26
13-0267 31-64
101
IoIoIo
0 4 l&70 O-16
0 IO
l #aosLA
.&&&@, Department of Cell Biology. Neurobiology, and Anatomy* and Department of Medicin&, Loyola Untvenity of Chicago. Stritch School of Medicine. Maywood. IL 60153. The mRNA expression of the multifunctional molecule, transforming growth facto&eta CIGFB), has been investigated in rat cardiac allografts during graft rejection. Total cellular RNA was prepared from newborn donor Buffalo rot hearts transpkmted into the rat ear pinnae of aduii Lewis recipients. The average time to rejection of the cardiac allografts. measured by the absence of detectable ECG activity. has been determined to be on Day 7-8 post-transplantation (post-tx). Cardiac grafts were retrieved on Days 1 through 10 post-tx, and RNA was examined for TGFP gene expression by Northern and slot blot analysis. TGFB mRNA expression appeared during the later time points of ollograft rejection TGFp transcripts were first detected on Day 5 post-tx and peaked by Day 7 posttx. coinciding with ollograft rejection as measured by ECG activity. TGFp mRNA levels were diminished by Day 10 post-tx. We have previously’ shown that TNFa mRNA expression demonstrated a biphasic pattern during rat cardiac allcgraft rejection, with peak levels first displayed on Day 3 past-tx. and then again on Day 7-8 post-tx. Since TGFP has reported antagonlstlc actions, opposing those of TNFa. we postulate that TGFB may be induced to moderate the damaging effects brought on earlier by TNFa during cardiac allograft rejection. We conclude that mRNA expression of TGFB is induced later during rat cardiac allogrcift rejection and may be a resutt of earlier TNFa gene expression. TGFp may therefore serve to counteract the deleterious effects of TNFa durlng graft rejection.
J.C.RENVERSEZ, S.ROUSSEL, A.DURUN. Labomto&e de 8iochimie A - Ph.EM.CHmgAZ-A.J.HAVJlAN CHRUG DE GRENOBLE -38043 GRENOBLE CEVW 9 - FRANCE. Beta-2
lB2ml 0 a Uehy .t?ow moeecukm moAn phosxphesned OM the ~wr@ce membmne 06 u-y a&Z the nutieated ce&Q. Raised 6emni concent%a.tiom mont o&ten heAt& 6hOtTI the inchsaAed Kuhn OVeh 06 dhebe cefi. IntehLeufh 2 (1L 21 and inteheeukin 2 heceptih [IL 2 RI ahe ai20 membmte and AokbCe cytokiti which ahe synthesized by a&ivatedT cek%. We have devtioped del6 madeifmnmomzymtic abhayb (ELlSA) to ev&ate thebe pammeL&tb ab mmkeh6 06 petiutbtiti 06 the hune henpome tig the 6oUow up 06 .thc human immunode6iciency V~LA (HIV) in6ection. 239 patieti have been inveutigated &king a pehiod 06 4 yemh. Elemted Aa”,?i &we,& 06 BZm appecvr 20 be highby phedictiue 06 .the deve~opmeti 06 acquihcd inmnode&iciency hyfldAOt?Ie [AIDS) in hwnan timunode6ioiency vti (HTV) bekohopObi,titiUe pa.&in
.tieti.
06
micmgCobtin
12 kUa,
A med
which
~a.&
0
06 5 mg/L
Aem
Zo be phed.itive
06
the
~i&$&~noicLtion between ~eLOpOhi.tiVe and Aehonegtive patienti. Sawn IL 2 and IL 2 R a/re L&O he@ztLve 06 the &~~ii+cLti~n 06 HIV induced diseases. A& men&, which have in the enx7uwce high uduti 06 8Zm !a? mg/tl,IL 2 +ZOO ng/Ll and IL 2R {*SO0 Ul/mLJ had Oh devtiopped AIDS in the 6ollowing tie.
INTERNATIONAL
WORKSHOP
ON
CYTOKINES
/ 493
259
262
INTERLEUKIN6 (IL-6) IS PRESENTIN COLOSTRUMOF HEALTHYWOMEN. L. Paulesu, K. Van Bremen. f. Corradeschi, E. Luzzi, A. Oi Stefano and V.Bocci
AN INTERLEUKIN 1 BETA GENE RFLP REFLECTS IL-1 BETA SECRETION IN VITRO -A NEW SUSCEPTIBIUTY MARKER FOR NON DR3 AND NON DR4 IDDM PATIENT. F. Pociot. and J. Neruo. Steno Diabetes Cent& DK-2820 Gentofle, Denmark.
Institute of General Physiology, University of Siena, Siena, Italy. Cytokines such as IL-loL, IL-10. TNF (1.2) are present in traces in human colostrum and recently we have reported that also IFN f is measurable in physiological amounts. both by immunoassay and bioassay. We nw report that also IL-6 is readily detectable. particularly when crude colostrum is delipidated by centrifugation and separated by molecular sieving into several fractions. The procedure *as carried out using Centricon microconcentrators (Amicon) at 30 and 10 KD fractions. About 25 samples of colostrum were obtained from 14 women at the 2nd and 3rd day after parturition. Colostral cells were also incubated in RPM1 1640 at 1.5~10~ cells/ml for 24 hours. All the samples were tested by enzyme amplified sensitivity system (EASIATM. Hedgenix). Most of It-6 activity (55-190 pg/ml) was present in the Retentate 30, that is IL-6 seemedto be bound to a carrier protein. Little activity (24-30 pg/ml) was detected in the crude colostrum as well as in the filtrate 10, i.e., IL-S present in free form. Upon incubation. colostral cells released considerable amount of It-6 in the medium. In the whole these findings support the well known fact that "omen can undergo fever upon milk retention. Moreover it is possible that traces of cytokines in colostrum exert an important activation of the oropharyngeal and gut- associated lymphoid system of the newborn. 1) Seder 0. Int. Arch. Aller. Appl. Immunol. 83:1’3,1987; 2) Goldman A.S., Gold blum R.M. Ann. New York Acad. SC. 587:236,1990. Work supported by "Ricerca sanitaria finalizzata dalla Regione Toscana".
IL-1 may be the major Hector molecule in the initiation phase of the immunemediated destruction of beta-cells preceding overl IDDM. We have previously demonstrated
that
stable
interindivklual
differences
in in vitro
LPS-stimulated
monocyte (MO) IL-1 secretion exist. We hypothesized that such dmerences 1 response capacity are genetically determined and may be a contributory
in MO ILfactor in
IDDM pathogenesis. We identified a diallelic RFLP in the 5th exon of the human IL-1 gene with Taql consisting of fragments of 9.4 kb and 13.4 kb. The genotype frequencies in a control population (n=93) were: 57% (9.4/9.4 kb), 35% (9.4/13.4
kb), and 8% (13.4/13.4 kb). The corresponding
frequencies in a random IDDM group
(n=46) were not significantly different (p=O.3). However, when studying IDDM patients with no known major genetic marker, Le. IDDM patients negative for DR3 and DR4 (n=lO). significantly more IL-1 13.4 kb positive individuals were identified (p= 0.05). The functional impact of this RFLP was studied by analysing in vitro LPSstimulated (250 pg/ml) MO (3xlOE5) IL-1 secretion. An IL-1 allele dosage effect on secretory capacity was observed. 9.4/9.4 kb individuals (n=lO) had the lowest reponse: 3.76kO.35 rig/ml, 9.4/13.4 kb individuals (n=lO): 4.42tO.47 rig/ml and 13.4/13.4 kb individuals (n=4): 5.16+0.66 mg/mi (test for trend ~~0.05, p=O.13 for 9.419.4 vs 9.4113.4, p=O.O7 for 9.4jl3.4 vs 13.4/13.4, p=O.O2 for 9.4j9.4 vs 13.4/13.4). Conclusions: 1) The 13.4 kb allele represents a “high secretor’ phenotype. 2) The observed RFLP may be an additional susceptibility marker for IDDM in non DR3 and non DR4 individuals, and 3) high IL-1 production a pathogenetic factor for IDDM in these patients.
260
263
INTRAOPERATIVE CYTOKINE PRODUCTION DURING LIVER TRANSPLANTATION. J. Pirenne. F. Pirenne. D. Dezzroot. Y. Vrindts. P. Honor& P. Franchimont. Univ. Libge, 4 000, Belgium. Non-immunological inaaoperative events during liver transplantation (LTx) are likely 6 trigger cytokine production: surgery induced tissue injury, endotoxinemia, and kupffer cell activation after repcrfusion. Aim of this study was to assess inaaoperative cytokine production during LTx. Using Enzyme Amplified Sensitivity Immunoassay (Medgenix), cytokines ILID, tL2, IL6, TNF*, andrIF were measured in serum of 4 LTx patients, preoperatively, before anhepatic phase (
Coneluslon: High levels of monokines IL 6, TNF, and IL 1 appeared in serum and bile during LTx. In contrast, lvmphokines IL 2 and IF could not be detected. This-demonstrates a major &mulation of monocyte cell lineage in the absence of lymphocyte activation. The finding that maximum levels of monokines IL1 and IL6 were detected >AP and presence of these monokines in the bile suggests that the TX liver might be a major site of monokines synthesis. Since monokines exert important effects on hepamcyte metabolism, this might have significant implications with regards to function of the TX liver.
DEVELOPMENT OF A CHEMILUMINESCENT ENZYME-LINKED IMMUNOSORBANT ASSAY (ELISA) FOR THE QUANTITATION 0~ RECOMBINANT HUMAN INTERLEUKIN-4 (RHUIL-4) IN HUMAN SERUM AND COMPARISON OF ASSAY SENSITIVITY WITH COLORIMETRIC METHODS. Nicholas Ronca, Karen Trudeau, Marian Kelley, Gary Stevens, and Lottie m. Sterling Drug Inc., Rensselaer, NY and Malvern, PA. A chemiluminescent sandwich ELISA has been developed for the quantitation of rhulL-4 in human serum. An immobilized rabbit polyclonal antibody is used as the capture antibody; bound rhuIL-4 is detected by sequential exposure tn the biotinylated rabbit antibody followed by streptavidin alkaline phosphatase. Light produced by enzymatic dephosphorylation of the adamantyl 1,2-dioxetane phosphate (AMPPD) substrate is measured in a microplate luminnmeter. ASSSY sensitivity is approximately 20 (0.13 Pg/“l f”oles/well). The assay is approximately ten times “ore sensitive than a first generation SSSSY employing p-nitrophenyl phosphate (PNPP) as substrate and twice as sensitive as when nicotinamide adenine dinucleotide phosphate (NADP) is used for signal amplification. The accuracy and precision of this assay were determined fro” nine separate analyses performed in pooled human serum. Percent CV ard relative bias (%I ranged fro” 0.9 to 7% and from -2.2 to 3.57%, respectively. These results indicate that the chemiluminescent second generation assay, by virture of its enhanced sensitivity, should be useful in quantitating rhuIL-4 in human serum when levels of less than 200 pg/mL are anticipated.
261
264
TRANSFORMING GROWTH FACTOR-BETA (TGFB) GENE EXPRESSION IN RAl l CARDIAC TRANSPLANTS DURING ALLOGRAFT REJECTION. T.T.
BETA-2 MICROGLOBULIN, IL 2 AND 1L 2 RECEPTOR IN THE DZAGNOSlS AND THE FOLLiXJ UP OF THE HIV INFECTION.
1 0 ihi 36-88 TNF 29-88 IL2 lo
IF 10.
O-3 65-8845 16-109 lo
10
04 100-12645 14-93 lo
I”
. Molinowsko”.
060 1184:10738
I
36-166
0
I
0
3 4:00 26
13-0267 31-64
101
IoIoIo
0 4 l&70 O-16
0 IO
l #aosLA
.&&&@, Department of Cell Biology. Neurobiology, and Anatomy* and Department of Medicin&, Loyola Untvenity of Chicago. Stritch School of Medicine. Maywood. IL 60153. The mRNA expression of the multifunctional molecule, transforming growth facto&eta CIGFB), has been investigated in rat cardiac allografts during graft rejection. Total cellular RNA was prepared from newborn donor Buffalo rot hearts transpkmted into the rat ear pinnae of aduii Lewis recipients. The average time to rejection of the cardiac allografts. measured by the absence of detectable ECG activity. has been determined to be on Day 7-8 post-transplantation (post-tx). Cardiac grafts were retrieved on Days 1 through 10 post-tx, and RNA was examined for TGFP gene expression by Northern and slot blot analysis. TGFB mRNA expression appeared during the later time points of ollograft rejection TGFp transcripts were first detected on Day 5 post-tx and peaked by Day 7 posttx. coinciding with ollograft rejection as measured by ECG activity. TGFp mRNA levels were diminished by Day 10 post-tx. We have previously’ shown that TNFa mRNA expression demonstrated a biphasic pattern during rat cardiac allcgraft rejection, with peak levels first displayed on Day 3 past-tx. and then again on Day 7-8 post-tx. Since TGFP has reported antagonlstlc actions, opposing those of TNFa. we postulate that TGFB may be induced to moderate the damaging effects brought on earlier by TNFa during cardiac allograft rejection. We conclude that mRNA expression of TGFB is induced later during rat cardiac allogrcift rejection and may be a resutt of earlier TNFa gene expression. TGFp may therefore serve to counteract the deleterious effects of TNFa durlng graft rejection.
J.C.RENVERSEZ, S.ROUSSEL, A.DURUN. Labomto&e de 8iochimie A - Ph.EM.CHmgAZ-A.J.HAVJlAN CHRUG DE GRENOBLE -38043 GRENOBLE CEVW 9 - FRANCE. Beta-2
lB2ml 0 a Uehy .t?ow moeecukm moAn phosxphesned OM the ~wr@ce membmne 06 u-y a&Z the nutieated ce&Q. Raised 6emni concent%a.tiom mont o&ten heAt& 6hOtTI the inchsaAed Kuhn OVeh 06 dhebe cefi. IntehLeufh 2 (1L 21 and inteheeukin 2 heceptih [IL 2 RI ahe ai20 membmte and AokbCe cytokiti which ahe synthesized by a&ivatedT cek%. We have devtioped del6 madeifmnmomzymtic abhayb (ELlSA) to ev&ate thebe pammeL&tb ab mmkeh6 06 petiutbtiti 06 the hune henpome tig the 6oUow up 06 .thc human immunode6iciency V~LA (HIV) in6ection. 239 patieti have been inveutigated &king a pehiod 06 4 yemh. Elemted Aa”,?i &we,& 06 BZm appecvr 20 be highby phedictiue 06 .the deve~opmeti 06 acquihcd inmnode&iciency hyfldAOt?Ie [AIDS) in hwnan timunode6ioiency vti (HTV) bekohopObi,titiUe pa.&in
.tieti.
06
micmgCobtin
12 kUa,
A med
which
~a.&
0
06 5 mg/L
Aem
Zo be phed.itive
06
the
~i&$&~noicLtion between ~eLOpOhi.tiVe and Aehonegtive patienti. Sawn IL 2 and IL 2 R a/re L&O he@ztLve 06 the &~~ii+cLti~n 06 HIV induced diseases. A& men&, which have in the enx7uwce high uduti 06 8Zm !a? mg/tl,IL 2 +ZOO ng/Ll and IL 2R {*SO0 Ul/mLJ had Oh devtiopped AIDS in the 6ollowing tie.