1
92 / SECOND INTERNATIONAL WORKSHOP ON CYTOKINES 115 IMMUNOGENICITY OF CYTOKINES IS ENHANCED BY ENCAPSULATING INTO LIPOSOMES. LJin Kim and Do ad w. Dept. of Immunology Research, Genentech, Inc. SouthnSin Francisco, CA. Monoclonal antibodies have become useful reagents to define the biological and biochemical characteristic of new cytokines. However using conventional immunization methods! it has often been difficult to obtain sufficient quantities of a new cytokme to induce a significant antibody response in mice. In this study, we explored various adjuvants to enhance the immunogenicity of recombinant human IL-1fl (rHuIL-lp). Antibody titers (determined by ELISA) was compared after BALB/c mice were immunized twice, i.p., with 25 ug of rHuIL-lb emulsified in Freund’s adjuvant or with various concentrations (28-448ng) of rHuIL-lp encapsulated in Iiposomes and resuspended in monophosphoryl lipid- A/TrehaIose dimycolate (MPLfTDM). All groups of mice immunized with rHuIL-1 encapsulated in Iiposomes gave surprisingly higher titers than group using antigens in Freund’s adjuvant. Antibody titers did not change whether rHu IL-1 was encapsulated in liposome injected either in saline or in MPL!IDM. Using this technique significant antibody responses were obtained by immunizing mice with as little as 10 pg ofrHuIL-1. We were able to show significant antibody response to other cytokines such as rHuIL-6 and rHuIL-8 after immunization with long of antigens encapsulated in liposomes. In aII these cases tested we were able to generate antigen specific monoclonal antibodies using spleen cells of mice immunizedwith theantigensencapsulatedinliposomes. 116 PURIFICATION AND CHARACTERIZATION OF A COMPETITIVE INHIBITOR INTERLELIKIN-1 Seckinger*, J”!. Dayer* and A R' Sh:;J'Gla::Z:;; & Geneva 24, Switzerland, and- Medicine *,‘Geneva University Hospital, 1211 Geneva 4, Switzerland. The urine of selected patients with fevers above 39OC contains an inhibitor of Interleukin-1. This in- hibitor has been show" previously to block the activity of both IL-la and IL-1R while having no effect on the activity of TNFa, a cytokine which shares many of the activities of IL-l. We have purified this protein to "ear homogeneity by ion exchange chromatography, hydrophobicity chromatography, gel filtration and nega- tive immunoabsorption. The purified protein has a molecular weight of 26,000, and this value is reduced to 24,000 upon treatment with Endoglycosidase F. Using radioiodinated protein we show that the IL-l inhibitor is a competitive inhibitor and acfs by binding to the IL-l receptor on murine EL4.6.1 thymoma cells. Deglycosylation has no effect on the receptor blocking aciivity. Chemical cross-linking studies suggest that the inhibitor binds to the 80.000 molecular weiaht major component of the IL-l recepto;. - 117 ANTI-1FN-y MONOCLONAL ANTIBODIES INHIBIT THE PROLIFERATION AND DIFFERENTIATION OF HUNAN T LYMPH0CYTES.P. Novelli'. C, Carotta2. G. Forni'. and M. Giovarelli3. 'Inst. of Microbial., Univ. of Turin, 'Dept. of Exp. Med., Univ. of L'Aquile, Italy. and 'Control Roo. Unit, Hoffman-LaRoucbe, Basle, Switzerland. Increased expression of the mRNA for IFNy is one of the first events in lymphocyte activation and is associated with the initial accumulation of mRNA for IL-3. Our previous findings with a murine anti-IFNY Noah (ANlS) suggested that denletion of 1FN-r in cultures inhibits the T-cell response to aiioantigens, the generation of specific CTL. and the- generation of LAK cells. This paper investigated the role of 1FN-f in induction and activation of lymphocyte cytotoxicity in the human system, using MoAbs directed against several native and recombinant epitopes. The presence in a culture of NoAbs (IgGl) neutralizing human IFN7 (769, ~73, 7123) inhibited the proliferation and generation of MHC-specific cytotoxic lymphocyte, and significantly reduced both the IL-2 and anti- T3 antibody induced proliferation and the generation of IAK CellS. By contrast, the presence of non-neutralizing MoAbs directed against the aminoterminal portion (MoWN2) and against a specific carboxyterminal sequence (7127) of rIFNy did not result in any inhibitory effect. Taken as a whole, these data offer support for the findings that IFN7 plays a central role in the induction and activation of the lymphocyte cytotoxic program. 118 PHARMACOLOGICAL REGULATION OF THE SYNOVIAL INTERLEUKIN l-LIKE CARTILAGE RESORBING ACTIVITY. K.D.RAINSFORD, McMaster Univ. Hamilton, CANADA. Synovial IL-l like activity (ILA) released from pig synovium in organ culture was found [1,2] to be (a) modestly inhibited by corticosteroids and NSAIDs, (b) stimulated by 5-lipoxygenase (LO) inhibitors and antimalarials, (c) unaffected by D-penicillamine and gold thiomalate. Present studies were designed to investigate the structure activity requirements for the effects of the steroids and NSAIDs and explore further the mechanisms of IL-1 release by pharmacol- -ogical probes. The results showed that steroids without glucocorticoid or anti-inflammatory activi- -ties (e.g. cortexolone, lo-100uM) were potent inhibitors of ILA and exerted effects at earlier times than agents such as dexamethasone. Etiochol- -anolone, a steroid previously shown to stimulate IL-l in man did not influence ILA and neither were a variety of inflammagenic agents (e.g.PAF,MDP). Some NSAIDs (e.g.azapropazone) were found to inhibit ILA at concentrations lower than for inhibition of prost- -aglandin(PG) production. These and related studies show that the drug effects on ILA production are ind- ependent of influences on production of mediators (eg PGs) or classical actions of anti-inflammatory agents Refs:[l] H.Sheppeard et a1,(1982) Ann.Rheum.Dis., u,463.[2] K.D.Rainsford (1987)Agents Act.a,338. 119 Modulation of experimental Anti Glomerular Basement Membrane Antibody (GBM.Ab) induced neuttophils by TNF. Koshino Y, Cashman SJ, Meager A, Rees AJ. Royal Postgraduate Medical School, London National Institute of Biological Standards and Control, South Mimms. We have show" previously that intravenous injections of TNF and LPS both aggravate injury in experimental anti glomerular basement membrane antibody (GBM Ab) induced nephritis (.I Immunol:142. 3083-90. 1989). We have now investigated the involvement of TNF in this model further by measuring serum and glomerular TNF concentrations after injection of anti-GBM antibodies, and by pretreatment of nephritic rats with anti-TNF antibodies. The results show that injection of anti- GBM antibodies is associated with transient increase in serum TNF concentration measured by the 1929 assay with specific inhibition by anti TNF antibodies (max at 2 hours, gone by 4 hours) and a later increase in glomerular TNF (max 4-8 hours). Despite this anti TNF antibodies do not decrease proteinuria after injection of high dose (20mg) anti-GBM antibodies. HOWeVer pretreated with anti TNF antibodies completely abolished the aggravating effect of LPS on low dose (10mg) anti-GBM antibodies; Albuminuria decreased from 82 + 20 to 21 + 6 /24 hours; mea" + SE. P < .Ol Wilcoxin. These results show the important modulating effect of TNF on this model of nephritis. 120 TUMOR NECROSIS FACTOR (I (TNFu)-INDUCED PGE2 CLASS I AND II ANTIGEN EXPRESSION IS REGULATED BY A TNFa INHIBITOR (TNFa INH). P. Seckineer. E. Vev. and J.-M. Daver, Div. of Immunology & Allergy, Dept. of Medicine, HBpital cant. univers., 1211 Geneva 4, Switzerland. TNFu regulates, among other biological activities, cytoto- xicity, inflammation and immune functions. A natural urine- derived TNFol inhibitor (TNFa INH; mol wt 33 kD; p1 5.5-6.1) was puri led to homogeneity revealing a specific activity of 1.4 x 10 F U/mg in the L929 cytotoxicity assay, in the presence of actinomycin D. One unit represents 50% inhibition of 0.2 rig/ml TNFa-induced cytotoxicity. We investigated the ef- fects of this inhibitor on (1) TNFa-induced PGE2 production by human dermal fibroblasts, and (2) MHC class I (W6/32) and II (anti-HLA-DR) antigen expression in the human carcinoma cell line Co10 205. - PGE2 production induced by TNFa (1 "g/ml) was blocked (100%) by 10 U/ml of TNFa INH. Increasing TNFu concen- trations overcame this effect. Class I HL&A,B,C antigen expression induced by TNFa (1 "g/ml) was blocked (78%) by 10 U/ml of TNFa INH. Besides, the inhibitor blocked (86%) class II HLA-DR antigen expression when induced by interferon- y (IFN-y; 10 U/ml) in the presence of TNFcr (10 "g/ml). How- ever, when induced by IFN-1 alone, expression of class I and II antigen was not affected by the inhibitor. Thus, in addi- tion to its anticytoroxic effect, TNFa INH can inhibit inflam- matory and immune functions; it may therefore be considered a novel "anticytokine".

Purification and characterization of a competitive inhibitor of interleukin-1

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92 / SECOND INTERNATIONAL WORKSHOP ON CYTOKINES

115

IMMUNOGENICITY OF CYTOKINES IS ENHANCED BY ENCAPSULATING INTO LIPOSOMES. LJin Kim and Do ad w. Dept. of Immunology Research, Genentech, Inc. SouthnSin Francisco, CA.

Monoclonal antibodies have become useful reagents to define the biological and biochemical characteristic of new cytokines. However using conventional immunization methods! it has often been difficult to obtain sufficient quantities of a new cytokme to induce a significant antibody response in mice. In this study, we explored various adjuvants to enhance the immunogenicity of recombinant human IL-1fl (rHuIL-lp). Antibody titers (determined by ELISA) was compared after BALB/c mice were immunized twice, i.p., with 25 ug of rHuIL-lb emulsified in Freund’s adjuvant or with various concentrations (28-448ng) of rHuIL-lp encapsulated in Iiposomes and resuspended in monophosphoryl lipid- A/TrehaIose dimycolate (MPLfTDM). All groups of mice immunized with rHuIL-1 encapsulated in Iiposomes gave surprisingly higher titers than group using antigens in Freund’s adjuvant. Antibody titers did not change whether rHu IL-1 was encapsulated in liposome injected either in saline or in MPL!IDM. Using this technique significant antibody responses were obtained by immunizing mice with as little as 10 pg ofrHuIL-1. We were able to show significant antibody response to other cytokines such as rHuIL-6 and rHuIL-8 after immunization with long of antigens encapsulated in liposomes. In aII these cases tested we were able to generate antigen specific monoclonal antibodies using spleen cells of mice immunizedwith theantigensencapsulatedinliposomes.

116

PURIFICATION AND CHARACTERIZATION OF A COMPETITIVE INHIBITOR INTERLELIKIN-1 Seckinger*, J”!. Dayer* and A R' Sh:;J'Gla::Z:;; & Geneva 24, Switzerland, and- Medicine *,‘Geneva University Hospital, 1211 Geneva 4, Switzerland.

The urine of selected patients with fevers above 39OC contains an inhibitor of Interleukin-1. This in- hibitor has been show" previously to block the activity of both IL-la and IL-1R while having no effect on the activity of TNFa, a cytokine which shares many of the activities of IL-l. We have purified this protein to "ear homogeneity by ion exchange chromatography, hydrophobicity chromatography, gel filtration and nega- tive immunoabsorption. The purified protein has a molecular weight of 26,000, and this value is reduced to 24,000 upon treatment with Endoglycosidase F. Using radioiodinated protein we show that the IL-l inhibitor is a competitive inhibitor and acfs by binding to the IL-l receptor on murine EL4.6.1 thymoma cells. Deglycosylation has no effect on the receptor blocking aciivity. Chemical cross-linking studies suggest that the inhibitor binds to the 80.000 molecular weiaht major component of the IL-l recepto;.

-

117 ANTI-1FN-y MONOCLONAL ANTIBODIES INHIBIT THE PROLIFERATION AND DIFFERENTIATION OF HUNAN T LYMPH0CYTES.P. Novelli'. C, Carotta2. G. Forni'. and M. Giovarelli3. 'Inst. of Microbial., Univ. of Turin, 'Dept. of Exp. Med., Univ. of L'Aquile, Italy. and 'Control Roo. Unit, Hoffman-LaRoucbe, Basle, Switzerland.

Increased expression of the mRNA for IFNy is one of the first events in lymphocyte activation and is associated with the initial accumulation of mRNA for IL-3. Our previous findings with a murine anti-IFNY Noah (ANlS) suggested that denletion of 1FN-r in cultures inhibits the T-cell response to aiioantigens, the generation of specific CTL. and the- generation of LAK cells. This paper investigated the role of 1FN-f in induction and activation of lymphocyte cytotoxicity in the human system, using MoAbs directed against several native and recombinant epitopes. The presence in a culture of NoAbs (IgGl) neutralizing human IFN7 (769, ~73, 7123) inhibited the proliferation and generation of MHC-specific cytotoxic lymphocyte, and significantly reduced both the IL-2 and anti- T3 antibody induced proliferation and the generation of IAK CellS. By contrast, the presence of non-neutralizing MoAbs directed against the aminoterminal portion (MoWN2) and against a specific carboxyterminal sequence (7127) of rIFNy did not result in any inhibitory effect. Taken as a whole, these data offer support for the findings that IFN7 plays a central role in the induction and activation of the lymphocyte cytotoxic program.

118 PHARMACOLOGICAL REGULATION OF THE SYNOVIAL

INTERLEUKIN l-LIKE CARTILAGE RESORBING ACTIVITY. K.D.RAINSFORD, McMaster Univ. Hamilton, CANADA. Synovial IL-l like activity (ILA) released from pig synovium in organ culture was found [1,2] to be (a) modestly inhibited by corticosteroids and NSAIDs, (b) stimulated by 5-lipoxygenase (LO) inhibitors and antimalarials, (c) unaffected by D-penicillamine and gold thiomalate. Present studies were designed to investigate the structure activity requirements for the effects of the steroids and NSAIDs and explore further the mechanisms of IL-1 release by pharmacol- -ogical probes. The results showed that steroids without glucocorticoid or anti-inflammatory activi- -ties (e.g. cortexolone, lo-100uM) were potent inhibitors of ILA and exerted effects at earlier times than agents such as dexamethasone. Etiochol- -anolone, a steroid previously shown to stimulate IL-l in man did not influence ILA and neither were a variety of inflammagenic agents (e.g.PAF,MDP). Some NSAIDs (e.g.azapropazone) were found to inhibit ILA at concentrations lower than for inhibition of prost- -aglandin(PG) production. These and related studies show that the drug effects on ILA production are ind- ependent of influences on production of mediators (eg PGs) or classical actions of anti-inflammatory agents

Refs:[l] H.Sheppeard et a1,(1982) Ann.Rheum.Dis., u,463.[2] K.D.Rainsford (1987)Agents Act.a,338.

119

Modulation of experimental Anti Glomerular Basement Membrane Antibody (GBM.Ab) induced neuttophils by TNF. Koshino Y, Cashman SJ, Meager A, Rees AJ. Royal Postgraduate Medical School, London National Institute of Biological Standards and Control, South Mimms. We have show" previously that intravenous injections of TNF and LPS both aggravate injury in experimental anti glomerular basement membrane antibody (GBM Ab) induced nephritis (.I Immunol:142. 3083-90. 1989). We have now investigated the involvement of TNF in this model further by measuring serum and glomerular TNF concentrations after injection of anti-GBM antibodies, and by pretreatment of nephritic rats with anti-TNF antibodies. The results show that injection of anti- GBM antibodies is associated with transient increase in serum TNF concentration measured by the 1929 assay with specific inhibition by anti TNF antibodies (max at 2 hours, gone by 4 hours) and a later increase in glomerular TNF (max 4-8 hours). Despite this anti TNF antibodies do not decrease proteinuria after injection of high dose (20mg) anti-GBM antibodies. HOWeVer pretreated with anti TNF antibodies completely abolished the aggravating effect of LPS on low dose (10mg) anti-GBM antibodies; Albuminuria decreased from 82 + 20 to 21 + 6 /24 hours; mea" + SE. P < .Ol Wilcoxin. These results show the important modulating effect of TNF on this model of nephritis.

120 TUMOR NECROSIS FACTOR (I (TNFu)-INDUCED PGE2 CLASS I AND II ANTIGEN EXPRESSION IS REGULATED BY A TNFa INHIBITOR (TNFa INH). P. Seckineer. E. Vev. and J.-M. Daver, Div. of Immunology & Allergy, Dept. of Medicine, HBpital cant. univers., 1211 Geneva 4, Switzerland.

TNFu regulates, among other biological activities, cytoto- xicity, inflammation and immune functions. A natural urine- derived TNFol inhibitor (TNFa INH; mol wt 33 kD; p1 5.5-6.1) was puri led to homogeneity revealing a specific activity of 1.4 x 10 F U/mg in the L929 cytotoxicity assay, in the presence of actinomycin D. One unit represents 50% inhibition of 0.2 rig/ml TNFa-induced cytotoxicity. We investigated the ef- fects of this inhibitor on (1) TNFa-induced PGE2 production by human dermal fibroblasts, and (2) MHC class I (W6/32) and II (anti-HLA-DR) antigen expression in the human carcinoma cell line Co10 205. - PGE2 production induced by TNFa (1 "g/ml) was blocked (100%) by 10 U/ml of TNFa INH. Increasing TNFu concen- trations overcame this effect. Class I HL&A,B,C antigen expression induced by TNFa (1 "g/ml) was blocked (78%) by 10 U/ml of TNFa INH. Besides, the inhibitor blocked (86%) class II HLA-DR antigen expression when induced by interferon- y (IFN-y; 10 U/ml) in the presence of TNFcr (10 "g/ml). How- ever, when induced by IFN-1 alone, expression of class I and II antigen was not affected by the inhibitor. Thus, in addi- tion to its anticytoroxic effect, TNFa INH can inhibit inflam- matory and immune functions; it may therefore be considered a novel "anticytokine".