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DEMONSTRATION OF CYTOKINES IN BIOLOGICAL MEDICINES PRODUCED IN MAMMALIAN CELL LINES
1011 IL-6 LEVELS
DEMONSTRATION OF CYTOKINES IN BIOLOGICAL MEDICINES PRODUCED IN MAMMALIAN CELL LINES ANDREW J. H. GEARING* CHRISTOPHER R. BIRD RICHARD PRIEST
(UIT11IN VIRAL VACCINES OR RECOMBINANT DNA PROTEIN PREPARATIONS
JUDITH E. CARTWRIGHT MEENU WADHWA ROBIN THORPE
Division of Immunobiology, National Institute for Biological Standards and Control, South Mimms, Potters Bar,
Hertfordshire EN6 3QG
Summary
Commercially
produced
biological
medicines may contain cytokines secreted by mammalian cell lines. Several such cell lines were found to produce interleukin 6 and, after stimulation, to secrete interleukin 1, tumour necrosis factor, granulocyte colonystimulating factor, and granulocyte-macrophage colonystimulating factor. High levels of interleukin 6 were detected in several vaccines and rDNA-derived proteins, and certain vaccines contained interleukin 1, granulocyte colonystimulating factor, and granulocyte-macrophage colonystimulating factor. Some preparations of human monoclonal antibodies were also found to contain interleukin 1 and tumour necrosis factor. Cytokines may contribute to certain types of adverse reactions to these
products. Introduction MANY cell lines of mammalian origin are used in the commercial production of conventional biological medicines and of new products derived by biotechnology, including vaccines, monoclonal antibodies, and recombinant DNA-derived proteins. Considerable attention has been given to ensure the safety and purity of these preparations in relation to freedom from extraneous microbial agents, proteins of host-cell origin and, for products made in transformed cells, DNA of host-cell origin.! We have demonstrated that many murine monoclonal antibodies produced from hybridoma cells contain inflammatory cytokines, including interleukin 1 (IL-1), interleukin 6 (IL-6), and tumour necrosis factor (TNF).2 These cytokines are potent mediators involved in local and systemic inflammation, including fever and acute phase responses.3 In view of these findings, we have investigated the levels of cytokines in vaccines, rDNA proteins, or human monoclonal antibodies produced in such cell lines. Materials and Methods Before assay, the vaccines were diluted to their recommended final dosage volume. Tissue plasminogen activator, erythropoietin, hepatitis B surface antigen, and HIV envelope glycoprotein (gpl20) were prepared in RPMI 1640 medium before assay. Cell lines producing monoclonal antibodies were grown to confluence or to about 106 cells per ml (for nonadherent lines). The supernatants were harvested and filter sterilised before assay. IL-6 levels were measured in the B9 cell line proliferation assay (detection limit 0-005 U/ml).4 Samples were calibrated with a National Institute for Biological Standards and Control (NIBSC) IL-6 reference reagent (88/514), where 1 U corresponds to about 200 pg human recombinant DNA-derived IL-6. For measurement of human IL-6, an indirect assay with an Epstein-Barr virus (EBV)
*Sample numbers refer to individual manufacturers; a and b refer to different lots of same material. tAlso contained (U/ml): IL-1, 7-2; GCSF, 30 000; and GMCSF, 4. also contained (U/ml): GCSF, 2000; and GMCSF, 05. §CHO preparations not tested for GCSF and GMCSF because immunoassays do not detect these cytokines from hamsters. ND not detectable, CHO Chinese hamster ovary cell derived recombinant B surface materials, Hep = hepatitis antigen, EPO erythropoietin, and TPA tissue plasminogen activator. =
=
=
transformed lymphoblastoid cell line (CESS) was also used.s CESS cells, when stimulated with IL-6, produce IgG, which was measured in an immunoradiometric assay in which two different monoclonal antibodies specific for IgG were used (M. W. and R. T., unpublished). IL-1 levels were measured with the EL4 NOB-1 thymoma bioassay (detection limit 0-05 U/ml);6 in which the NOB-1 cells are stimulated by IL-1to release a second cytokine interleukin 2 (IL-2) which is assayed by the IL-2 dependent CTLL cell line. Titration of the NIBSC reference reagent for IL-1 alpha (86/632), in which 1 U corresponds to approximately 10 pg of recombinant DNAderived human IL-1, was included in each assay. TNF was measured with a cytotoxicity assay with actinomycin D treated L929 cells (detection limit 0-2 U/ml).7 The NIBSC TNF alpha reference reagent (1 U corresponds to about 25 pg human TNF) was used to calibrate each assay. Granulocyte colony-stimulating factor (GCSF) was measured in an immunoradiometric assay, which can detect 10 U/ml. The assay standard was NIBSC GCSF reference reagent (88/502), in which 1 U corresponds to approximately 10 pg human GCSF. Granulocyte-macrophage colony-stimulating factor (GMCSF) levels were assayed in the ’Insight GM’ ELISA (Medical Resources Limited, Australia). This two-site ELISA can detect 01 U/ml. The assay was calibrated with the GMCSF recombinant reference reagent 88/646 (1U corresponds to about 100 pg human GMCSF).
Results Four cell lines producing human monoclonal antibodies rhesus D were investigated. Two lines were EBV transformed (ESDI and BRAD-3) and two lines were EBV transformed and then fused with murine myeloma cells to to
*Present address: British Biotechnology Ltd, Watlington Road, Cowley, Oxford OX4 SC7.
=
1012
produce heterohybridomas (GAD and FOG-1). Only ESD 1 and BRAD-3 produced IL-1 (72 and 05 U/ml, respectively) and TNF (125 and 16 U/ml, respectively). No IL-6, GCSF, or GMCSF was detected in any of the supernatants. Vaccines and recombinant proteins that were produced in mammalian cells contained detectable IL-6, with some containing up to 800 U/ml (160 ng/ml) (table). Only one of the preparations contained IL-1, none contained TNF and two contained GCSF and GMCSF. Discussion
Preliminary experiments showed that fibroblast, epithelial, and lymphoblastoid cell lines, which are used to produce pharmaceutical products, are capable of secreting cytokines under normal culture conditions. These results confirm previous reports showing either detectable levels of cytokine mRNA or secretion of cytokine proteins by similar cell types.9lo Since these cell lines are used for the production of biological medicinal products we measured cytokine levels in certain viral vaccines and recombinant proteins. Significant levels of IL-6 were found in several viral vaccines, especially rabies vaccines. Some of the rabies vaccines were also found to contain IL-1, GCSF, and GMCSF. No TNF could be detected in any of the vaccines. Of the recombinant proteins, only gpl20 contained IL-6. Thus many preparations contain cytokines, some in large amounts.
Cytokine levels were not directly related to the low levels produced by the unstimulated host-cell lines. Endotoxin contamination of the cultures is an insufficient stimulus to explain these levels." Viruses such as measles and rubella can directly stimulate fibroblasts to produce IL-6 and colony-stimulating activity (probably a mixture of different colony-stimulating factors).1z It is therefore likely that viral growth in the bulk cultures is causing release of the cytokines which we detected in the final products. We have found high levels of IL-6 in the supernatants of some cell lines cultured with measles, mumps, or poliomyelitis virus. Vero cells alone produced 1-2 U/ml and when cultured with measles virus produced 44-5 U IL-6 per ml, and with mumps virus 25-5 U IL-6 per ml, whereas Hep 2C alone produced 02 U/ml and cells cultured with type 1 poliovirus produced 8-5 U/ml, type 2 poliovirus 1 -7 U/ml, and type 3 poliovirus 4-8
U/ml. Cytokine content seems to be a particular problem with the rabies vaccines that are not extensively purified during manufacture. Assays for relevant cytokines might therefore be useful in quality assessment of biological products produced by mammalian cells. The human monoclonal antibody containing supernatants derived from lymphoblastoid lines had high levels of TNF and IL-1. These cytokines have synergistic toxicity in animal models;13 purification procedures for these products should therefore be validated for removal of TNF and IL-1. The supernatants derived from heterohybridomas did not contain IL-1and TNF, indicating that the fusion procedure resulted either in loss of the human chromosomes (2 and 6) carrying genes coding for IL-1 and TNF or possibly of other chromosomes carrying regulatory elements. Although IL-6 may act beneficially as an adjuvant, potentiating the immune response to vaccines, the possible adverse effects of these cytokines on patients must be considered. Neither IL-6 nor GCSF have been reported to have inflammatory effects at sites of injection, although
systemic administration of large (ptg/kg) doses of IL-6 can give rise to febrile responses and production of acute phase proteins in laboratory animals .4,14 IL-1, however, is a potent inflammatory stimulus15 and as little as 10 U of IL-1 given intradermally causes striking erythematous lesions in volunteers.16 However, of the vaccine preparations we examined, only certain rabies vaccines contained concentrations of cytokines that would be predicted to cause inflammatory reactions. A further potential consequence of the presence of cytokines in biological medicines is the induction of anti-cytokine antibodies in recipients. Repeated therapeutic administration of high doses of human cytokines to patients has resulted in the development of neutralising antibodies." Where non-human cells are used to make vaccines, the cytokines produced are likely to differ to some extent from their human counterparts and hence should be more antigenic. Thus CHO cell products can contain high levels of hamster IL-6 which, although not biologically active in human cells, could still initiate the development of anti-IL-6 antibodies. We emphasise, however, that the amounts of cytokines present in most of the vaccines are below the level required for most antigens to generate an immune response.
We thank Dr J. P. Jacobs for some of the cell lines used, the Blood Products Laboratory, Elstree, for FOG-1 and BRAD-3, the University Medical School, Edinburgh, for ESD-1, Dr N. Hughes-Jones for GAD, and Dr L. Petrin for the gift of GMCSF assay kits. We also acknowledge Dr P. Minor and his staff of the Virology Division of NIBSC for their help and discussions, and Miss L. Hudson and Miss S. Ashbrook for secretarial assistance.
Correspondence should be addressed to A. J. H.
G.
REFERENCES 1. World Health
Organisation. Acceptability of cell substrates for production of biologicals. WHO Tech Rep Ser 747, 1987. 2. Gearing AJH, Leung H, Bird CR, Thorpe R. Presence of the inflammatory cytokines IL-1, TNF, and IL-6 in preparations of monoclonal antibodies, Hybridoma 1989; 8: 361-67. 3. Andus T, Geiger T, Hirano T, Kishimoto T, Heinrich PC. Action of recombinant human interleukin 6, interleukin 1 &bgr; and tumor necrosis factor &agr; on the mRNA induction of acute-phase proteins. Eur J Immunol 1988; 18: 739-46. 4. Helle M, Boeije L, Aarden LA Functional discrimination between interleukin 6 and interleukin 1. Eur J Immunol 1988; 18: 1535-40. 5. Muraguchi A, Kishimoto T, Miki Y, et al. T-cell replacing factor (TRF) induced IgG secretion in a human B blastoid cell line and demonstration of a receptor for TRF J Immunol 1981; 127: 412-21. 6. Gearing AJH, Bird CR, Bnstow A, Poole S, Thorpe R. A simple sensitive bioassay for interleukin-1 which is unresponsive to 103 U/ml of interleukin-2. J Immunol Methods 1987; 99: 7-11. 7. Matthews N, Neale ML. Cytotoxicity assays for tumour necrosis factor and lymphotoxin. In: Clemens MJ, Morris AG, Gearing AJH, eds. Lymphokines and interferons. a practical approach. Oxford: IRL Press, 1987. 8. Wadhwa M, Thorpe R, Bird CR, Gearing AJH. Production of polyclonal and monoclonal antibodies to human granulocyte colony-stimulating factor (GCSF) and development of immunoassays. J Immunol Methods (in press) 9. Kronke M, Hensel G, Schluter C, Scheurich P, Schutze S, Pfizenmaier K. Tumor necrosis factor and lymphotoxin gene expression in human tumors. Cancer Res
1988; 48: 5417-21. FC, Shields J, Smith SH, et al. B cell growth and differentiation induced by supernatants of transformed epithelial cell lines. Eur J Immunol 1986; 16: 1017-19 Seelentag W, Mermod JJ, Vassalli P. Interleukin 1 and tumor necrosis factor-a
10. Rawle 11.
additively increase the levels of granulocyte colony-stimulating factor (CSF) mRNA m human fibroblasts. Eur J Immunol 1989; 19: 209-12. 12. Van Damme J, Schaafsma MR, Fibbe WE, Falkenburg JHF, Opdenakker G, Bilhau A. Simultaneous production of interleukin 6, interferon-&bgr; and colony-stimulating activity by fibroblasts after viral and bacterial infection. Eur J Immunol 1989; 19: 163-68. 13.
Waage A, Espevik
T. Interleukin 1
potentiates the
lethal effect of tumor necrosis
factor-&agr;/cachectin in mice. J Exp Med 1988; 167: 1987-92. 14. Geiger T, Andus T, Klapproth J, Hirano T, Kishimoto T, Heinrich PC Induction of rat acute-phase proteins by interleukin-6 in vivo. Eur J Immunol 1988; 18: 717-21 15. Dowd PM, Camp RDR, Greaves MW. In vivo effects of human recombinant interleukin-1 &agr; in normal skin. J Invest Dermatol 1987, 88: 485. 16. Fincham NJ, Camp RDR, Ross J, Gearing AJH, Bird CR. Potent pro-inflammatory properties of an IL-1 like material purified from the stratum comeum of normal skin. Br J Pharmacol (in press). 17. Figlin RA, Itri LM. Anti interferon antibodies: a perspective. Semin Hematol 1988, 25: 9-15.
DEMONSTRATION OF CYTOKINES IN BIOLOGICAL MEDICINES PRODUCED IN MAMMALIAN CELL LINES ANDREW J. H. GEARING* CHRISTOPHER R. BIRD RICHARD PRIEST
(UIT11IN VIRAL VACCINES OR RECOMBINANT DNA PROTEIN PREPARATIONS
JUDITH E. CARTWRIGHT MEENU WADHWA ROBIN THORPE
Division of Immunobiology, National Institute for Biological Standards and Control, South Mimms, Potters Bar,
Hertfordshire EN6 3QG
Summary
Commercially
produced
biological
medicines may contain cytokines secreted by mammalian cell lines. Several such cell lines were found to produce interleukin 6 and, after stimulation, to secrete interleukin 1, tumour necrosis factor, granulocyte colonystimulating factor, and granulocyte-macrophage colonystimulating factor. High levels of interleukin 6 were detected in several vaccines and rDNA-derived proteins, and certain vaccines contained interleukin 1, granulocyte colonystimulating factor, and granulocyte-macrophage colonystimulating factor. Some preparations of human monoclonal antibodies were also found to contain interleukin 1 and tumour necrosis factor. Cytokines may contribute to certain types of adverse reactions to these
products. Introduction MANY cell lines of mammalian origin are used in the commercial production of conventional biological medicines and of new products derived by biotechnology, including vaccines, monoclonal antibodies, and recombinant DNA-derived proteins. Considerable attention has been given to ensure the safety and purity of these preparations in relation to freedom from extraneous microbial agents, proteins of host-cell origin and, for products made in transformed cells, DNA of host-cell origin.! We have demonstrated that many murine monoclonal antibodies produced from hybridoma cells contain inflammatory cytokines, including interleukin 1 (IL-1), interleukin 6 (IL-6), and tumour necrosis factor (TNF).2 These cytokines are potent mediators involved in local and systemic inflammation, including fever and acute phase responses.3 In view of these findings, we have investigated the levels of cytokines in vaccines, rDNA proteins, or human monoclonal antibodies produced in such cell lines. Materials and Methods Before assay, the vaccines were diluted to their recommended final dosage volume. Tissue plasminogen activator, erythropoietin, hepatitis B surface antigen, and HIV envelope glycoprotein (gpl20) were prepared in RPMI 1640 medium before assay. Cell lines producing monoclonal antibodies were grown to confluence or to about 106 cells per ml (for nonadherent lines). The supernatants were harvested and filter sterilised before assay. IL-6 levels were measured in the B9 cell line proliferation assay (detection limit 0-005 U/ml).4 Samples were calibrated with a National Institute for Biological Standards and Control (NIBSC) IL-6 reference reagent (88/514), where 1 U corresponds to about 200 pg human recombinant DNA-derived IL-6. For measurement of human IL-6, an indirect assay with an Epstein-Barr virus (EBV)
*Sample numbers refer to individual manufacturers; a and b refer to different lots of same material. tAlso contained (U/ml): IL-1, 7-2; GCSF, 30 000; and GMCSF, 4. also contained (U/ml): GCSF, 2000; and GMCSF, 05. §CHO preparations not tested for GCSF and GMCSF because immunoassays do not detect these cytokines from hamsters. ND not detectable, CHO Chinese hamster ovary cell derived recombinant B surface materials, Hep = hepatitis antigen, EPO erythropoietin, and TPA tissue plasminogen activator. =
=
=
transformed lymphoblastoid cell line (CESS) was also used.s CESS cells, when stimulated with IL-6, produce IgG, which was measured in an immunoradiometric assay in which two different monoclonal antibodies specific for IgG were used (M. W. and R. T., unpublished). IL-1 levels were measured with the EL4 NOB-1 thymoma bioassay (detection limit 0-05 U/ml);6 in which the NOB-1 cells are stimulated by IL-1to release a second cytokine interleukin 2 (IL-2) which is assayed by the IL-2 dependent CTLL cell line. Titration of the NIBSC reference reagent for IL-1 alpha (86/632), in which 1 U corresponds to approximately 10 pg of recombinant DNAderived human IL-1, was included in each assay. TNF was measured with a cytotoxicity assay with actinomycin D treated L929 cells (detection limit 0-2 U/ml).7 The NIBSC TNF alpha reference reagent (1 U corresponds to about 25 pg human TNF) was used to calibrate each assay. Granulocyte colony-stimulating factor (GCSF) was measured in an immunoradiometric assay, which can detect 10 U/ml. The assay standard was NIBSC GCSF reference reagent (88/502), in which 1 U corresponds to approximately 10 pg human GCSF. Granulocyte-macrophage colony-stimulating factor (GMCSF) levels were assayed in the ’Insight GM’ ELISA (Medical Resources Limited, Australia). This two-site ELISA can detect 01 U/ml. The assay was calibrated with the GMCSF recombinant reference reagent 88/646 (1U corresponds to about 100 pg human GMCSF).
Results Four cell lines producing human monoclonal antibodies rhesus D were investigated. Two lines were EBV transformed (ESDI and BRAD-3) and two lines were EBV transformed and then fused with murine myeloma cells to to
*Present address: British Biotechnology Ltd, Watlington Road, Cowley, Oxford OX4 SC7.
=
1012
produce heterohybridomas (GAD and FOG-1). Only ESD 1 and BRAD-3 produced IL-1 (72 and 05 U/ml, respectively) and TNF (125 and 16 U/ml, respectively). No IL-6, GCSF, or GMCSF was detected in any of the supernatants. Vaccines and recombinant proteins that were produced in mammalian cells contained detectable IL-6, with some containing up to 800 U/ml (160 ng/ml) (table). Only one of the preparations contained IL-1, none contained TNF and two contained GCSF and GMCSF. Discussion
Preliminary experiments showed that fibroblast, epithelial, and lymphoblastoid cell lines, which are used to produce pharmaceutical products, are capable of secreting cytokines under normal culture conditions. These results confirm previous reports showing either detectable levels of cytokine mRNA or secretion of cytokine proteins by similar cell types.9lo Since these cell lines are used for the production of biological medicinal products we measured cytokine levels in certain viral vaccines and recombinant proteins. Significant levels of IL-6 were found in several viral vaccines, especially rabies vaccines. Some of the rabies vaccines were also found to contain IL-1, GCSF, and GMCSF. No TNF could be detected in any of the vaccines. Of the recombinant proteins, only gpl20 contained IL-6. Thus many preparations contain cytokines, some in large amounts.
Cytokine levels were not directly related to the low levels produced by the unstimulated host-cell lines. Endotoxin contamination of the cultures is an insufficient stimulus to explain these levels." Viruses such as measles and rubella can directly stimulate fibroblasts to produce IL-6 and colony-stimulating activity (probably a mixture of different colony-stimulating factors).1z It is therefore likely that viral growth in the bulk cultures is causing release of the cytokines which we detected in the final products. We have found high levels of IL-6 in the supernatants of some cell lines cultured with measles, mumps, or poliomyelitis virus. Vero cells alone produced 1-2 U/ml and when cultured with measles virus produced 44-5 U IL-6 per ml, and with mumps virus 25-5 U IL-6 per ml, whereas Hep 2C alone produced 02 U/ml and cells cultured with type 1 poliovirus produced 8-5 U/ml, type 2 poliovirus 1 -7 U/ml, and type 3 poliovirus 4-8
U/ml. Cytokine content seems to be a particular problem with the rabies vaccines that are not extensively purified during manufacture. Assays for relevant cytokines might therefore be useful in quality assessment of biological products produced by mammalian cells. The human monoclonal antibody containing supernatants derived from lymphoblastoid lines had high levels of TNF and IL-1. These cytokines have synergistic toxicity in animal models;13 purification procedures for these products should therefore be validated for removal of TNF and IL-1. The supernatants derived from heterohybridomas did not contain IL-1and TNF, indicating that the fusion procedure resulted either in loss of the human chromosomes (2 and 6) carrying genes coding for IL-1 and TNF or possibly of other chromosomes carrying regulatory elements. Although IL-6 may act beneficially as an adjuvant, potentiating the immune response to vaccines, the possible adverse effects of these cytokines on patients must be considered. Neither IL-6 nor GCSF have been reported to have inflammatory effects at sites of injection, although
systemic administration of large (ptg/kg) doses of IL-6 can give rise to febrile responses and production of acute phase proteins in laboratory animals .4,14 IL-1, however, is a potent inflammatory stimulus15 and as little as 10 U of IL-1 given intradermally causes striking erythematous lesions in volunteers.16 However, of the vaccine preparations we examined, only certain rabies vaccines contained concentrations of cytokines that would be predicted to cause inflammatory reactions. A further potential consequence of the presence of cytokines in biological medicines is the induction of anti-cytokine antibodies in recipients. Repeated therapeutic administration of high doses of human cytokines to patients has resulted in the development of neutralising antibodies." Where non-human cells are used to make vaccines, the cytokines produced are likely to differ to some extent from their human counterparts and hence should be more antigenic. Thus CHO cell products can contain high levels of hamster IL-6 which, although not biologically active in human cells, could still initiate the development of anti-IL-6 antibodies. We emphasise, however, that the amounts of cytokines present in most of the vaccines are below the level required for most antigens to generate an immune response.
We thank Dr J. P. Jacobs for some of the cell lines used, the Blood Products Laboratory, Elstree, for FOG-1 and BRAD-3, the University Medical School, Edinburgh, for ESD-1, Dr N. Hughes-Jones for GAD, and Dr L. Petrin for the gift of GMCSF assay kits. We also acknowledge Dr P. Minor and his staff of the Virology Division of NIBSC for their help and discussions, and Miss L. Hudson and Miss S. Ashbrook for secretarial assistance.
Correspondence should be addressed to A. J. H.
G.
REFERENCES 1. World Health
Organisation. Acceptability of cell substrates for production of biologicals. WHO Tech Rep Ser 747, 1987. 2. Gearing AJH, Leung H, Bird CR, Thorpe R. Presence of the inflammatory cytokines IL-1, TNF, and IL-6 in preparations of monoclonal antibodies, Hybridoma 1989; 8: 361-67. 3. Andus T, Geiger T, Hirano T, Kishimoto T, Heinrich PC. Action of recombinant human interleukin 6, interleukin 1 &bgr; and tumor necrosis factor &agr; on the mRNA induction of acute-phase proteins. Eur J Immunol 1988; 18: 739-46. 4. Helle M, Boeije L, Aarden LA Functional discrimination between interleukin 6 and interleukin 1. Eur J Immunol 1988; 18: 1535-40. 5. Muraguchi A, Kishimoto T, Miki Y, et al. T-cell replacing factor (TRF) induced IgG secretion in a human B blastoid cell line and demonstration of a receptor for TRF J Immunol 1981; 127: 412-21. 6. Gearing AJH, Bird CR, Bnstow A, Poole S, Thorpe R. A simple sensitive bioassay for interleukin-1 which is unresponsive to 103 U/ml of interleukin-2. J Immunol Methods 1987; 99: 7-11. 7. Matthews N, Neale ML. Cytotoxicity assays for tumour necrosis factor and lymphotoxin. In: Clemens MJ, Morris AG, Gearing AJH, eds. Lymphokines and interferons. a practical approach. Oxford: IRL Press, 1987. 8. Wadhwa M, Thorpe R, Bird CR, Gearing AJH. Production of polyclonal and monoclonal antibodies to human granulocyte colony-stimulating factor (GCSF) and development of immunoassays. J Immunol Methods (in press) 9. Kronke M, Hensel G, Schluter C, Scheurich P, Schutze S, Pfizenmaier K. Tumor necrosis factor and lymphotoxin gene expression in human tumors. Cancer Res
1988; 48: 5417-21. FC, Shields J, Smith SH, et al. B cell growth and differentiation induced by supernatants of transformed epithelial cell lines. Eur J Immunol 1986; 16: 1017-19 Seelentag W, Mermod JJ, Vassalli P. Interleukin 1 and tumor necrosis factor-a
10. Rawle 11.
additively increase the levels of granulocyte colony-stimulating factor (CSF) mRNA m human fibroblasts. Eur J Immunol 1989; 19: 209-12. 12. Van Damme J, Schaafsma MR, Fibbe WE, Falkenburg JHF, Opdenakker G, Bilhau A. Simultaneous production of interleukin 6, interferon-&bgr; and colony-stimulating activity by fibroblasts after viral and bacterial infection. Eur J Immunol 1989; 19: 163-68. 13.
Waage A, Espevik
T. Interleukin 1
potentiates the
lethal effect of tumor necrosis
factor-&agr;/cachectin in mice. J Exp Med 1988; 167: 1987-92. 14. Geiger T, Andus T, Klapproth J, Hirano T, Kishimoto T, Heinrich PC Induction of rat acute-phase proteins by interleukin-6 in vivo. Eur J Immunol 1988; 18: 717-21 15. Dowd PM, Camp RDR, Greaves MW. In vivo effects of human recombinant interleukin-1 &agr; in normal skin. J Invest Dermatol 1987, 88: 485. 16. Fincham NJ, Camp RDR, Ross J, Gearing AJH, Bird CR. Potent pro-inflammatory properties of an IL-1 like material purified from the stratum comeum of normal skin. Br J Pharmacol (in press). 17. Figlin RA, Itri LM. Anti interferon antibodies: a perspective. Semin Hematol 1988, 25: 9-15.