TRANSFORMING GROWTH FACTOR ,8 INDUCES THE PRODUCTION OF INTERLEUKIN 6 BY HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS Martin
Turner*,
David Chantry,
Marc Feldmann
Previous studies have indicated that the cytokine transforming growth factor & (TGFP,) has immunosuppressive properties and can inhibit the production of tumor necrosis factor (TNF) and Interleukin 1 (IL 1) by human peripheral blood mononuclear cells. In this study, we have examined the effects of TGF/3r on the production of Interleukin 6 (IL 6) by human peripheral blood mononuclear cells. Treatment with only TGFP, leads to the induction of IL 6, and this was both dose- and time-dependent. The effect of TGF& was evident at the level of IL 6 mRNA, suggesting TGFP,-induced de novo synthesis of IL 6. Induction of IL 6 by TGFP, was specific, as other cytokines made by mononuclear cells (TNF and IL 1) were not induced by TGFP,. Furthermore, when a panel of stimuli were compared for their ability to induce IL 1, TNF and IL 6 in the presence or absence of TGFP,, IL 6 levels were augmented in the presence of TGF&, while the induction of IL 1 and TNF was inhibited significantly. These results indicate that TGFP, has complex effects on the production of cytokines by peripheral blood mononuclear cells and that TGFP, is not inhibitory for all cytokine production. The ability of TGF/3, to induce IL 6 suggests that IL 6 may mediate some of the effects of TGFBr. o 1990 by W.B. Saunders Company.
Transforming growth factor pi (TGFP,), a member of a superfamily of growth and differentiation factors, has recently been shown to mediate a number of immunosuppressive effects.’ TGFP,-inhibited IL 2-driven lymphocyte proliferation,’ lymphokine activated killer (LAK) cell generation3 cytotoxic T lymphocyte (CTL) generation in mixed lymphocyte culture4 and natural killer (NK) activity.5 TGFP, has also been shown to inhibit B cell proliferation and IgG production.6 In addition to its effects on lymphoid cells, TGFP, has been shown to block H,O, production and other macrophage functions.7 Many of the effects of TGFP, on immune-celleffector-function may be secondary to the inhibition of cytokine production or responsiveness, which is essential for the generation of these activities. It has been shown that TGFP, blocks tumor necrosis factor (TNF) production by peripheral blood mononuclear cells (PBM), in
From
the Charing Cross Sunley Research Centre, Hammersmith, London W6 SLW UK *To whom reprint requests should be addressed. o 1990 by W.B. Saunders Company. 1043-4666/90/0202-0001$05.00/0 KEY
WORDS:
CYTOKINE,
TGF
P/IL-6/Human
Vol. 2, No.
3 (May),
PBM
1990: pp 211-216
Lurgan
Avenue,
response to lipopolysaccharide (LPS).**’ In addition phytohemagglutinin (PHA)-induced IFNr production by PBM was blocked by the pretreatment of human lymphocytes with TGFP,.’ The role of TGFP, in regulation of IL 1 production is less clear. TGF/3, has been shown to induce IL 1 mRNA,sV’o~ll but our study did not lead to the induction of detectable IL 1 protein.8 In contrast, other work has shown a biological activity consistent with the presence of IL 1 in murine thymocyte and fibroblast proliferation assays.lO~ll The ability of TGFP, to mediate inhibitory effects on immune cells depends on the nature of the inducing stimulus and the activation state of the responding cells. For example, IL l-driven thymocyte proliferation can be completely blocked by TGFP,, but thymocyte proliferation that is stimulated by IL 7 is considerably more resistant to TGFP, inhibition.12 Induction of IL lp and TNF by bacterial LPS is blocked by TGFP,, while induction of these cytokines by phorbol ester (PMA) is unaffected by TGFP,. This suggests different pathways of cytokine induction are differentially susceptible to the effects of TGF&.* We demonstrate, in this study the ability of TGFP, to induce production and secretion of interleukin 6 (IL 6) by peripheral blood mononuclear cells under circumstances where it can inhibit the production of IL l/3 and TNFcE. 211
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Chantry,
Feldmann
CYTOKINE,
RESULTS TGFP, has been shown to inhibit the release of a number of cytokines including TNFLu, IL 1, and IFNy.*” It was therefore of interest to examine the effects of TGFP, on the production of IL 6, a cytokine that is highly inducible by IL 1 and TNF.13 PBM were separated on Lymphoprep and cultured for 16 hr with various doses of TGFP,. The resulting supernatants were assayed for IL 6 using the IL 6-dependent cell line B9. TGFP, induced a dose-dependent increase in IL 6 activity in the conditioned medium (Fig. la). The kinetics of the induction of IL 6 activity by TGFP, were established by incubating PBM for up to 96 hr in the presence of 10 ng/mL TGFP,. TGFP, elicited an increase in IL 6 production as early as 4 hr, and IL 6 levels continued to accumulate in a time-dependent manner (Fig. lb). Next we determined the effect of TGFP, on IL 6-induced B9 proliferation, because the conditioned medium used for the IL 6 assay contained significant quantities of bioactive recombinant TGFP, remaining from its addition’ and it can interfere with some bioassays for cytokines.’ TGFP, alone had no stimulatory effect on the proliferation of B9 cells. Furthermore, TGFP, did not inhibit or enhance the proliferative response of B9 cells to exogenously added recombinant human IL 6 (Fig. 2a). Having shown that TGF/3, did not influence the IL 6 bioassay, we verified that the identity of the B9 stimulatory activity present in TGFP, conditioned medium was
Vol. 2, No. 3 (May
indeed IL 6. This was done by specific neutralization with goat antiserum that was raised against recombinant human IL 6, while normal goat serum, used at the same dilutions, was ineffectual (Fig. 2b). To characterize the mechanism of IL 6 induction by TGFP,, we extracted RNA from PBM treated at various times with TGFP, and performed northern blots with an IL g-specific probe. TGF/3, transiently induced IL 6 mRNA (Fig. 3). Levels of ribosomal RNA, which served as a control, were unchanged by treatment with TGFP,, and verified equal loading on each lane of the gel. Conditioned medium, collected from these cells, showed a continued time-dependent accumulation of IL 6 bioactivity (data not shown). We have previously shown that treatment of PBM with TGF/3, causes an transient increase in steady state levels of mRNA for IL la, IL l/3 and TNFa,’ and, as shown here, of IL 6. However no release of immunoreactive or bioactive IL 1 or TNFa was demonstrable, even using sensitive bioassays combined with anti-TGF& antibodies.8 Therefore, it was of interest to determine simultaneously the effects of TGF@, on the production of all three cytokines. We tested a panel of stimuli which have been shown to be potent inducers of cytokine production, both in the presence and absence of TGFP, (Fig. 4). PBM, cultured with medium alone, produced little IL l/3, TNFol or IL 6 protein, and there was no enhancement of IL I/? or TNFa production when TGFP, was included at 10 ng/mL. In contrast, IL 6 levels in the same supernatants were significantly elevated
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TGFp(ng/ml) Figure
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Dose response
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Time (Hours) induction
1990: 211-216)
of IL-6.
(A) PBM at 1 x lo6 cells/ml in RPM1 1640 containing 10% FCS were cultured for with varying doses of TGFB, and conditioned medium was collected after 20 hr and assayed for ability to support B9 cell proliferation as described in Materials and Methods. (B) Kinetics of IL 6 induction by TGFP,. Freshly separated PBM were cultured for varying lengths of time at 1 x lo6 cells/ml in RPM1 1640 containing 10% FCS and 10 ng/mL TGFP,. Conditioned media was collected and assayed for IL 6.
Transforming
I
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Figure
2.
2 4 IL-6 (U/ml) Effect
of TGFj3,
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Dilution of antisera assay.
(A) Serial dilutions of recombinant human IL-6 were added to B9 cells in the presence or absence of 0.1 ng/mL or 10 ng/mL TGFP,. Proliferation was determined by tritiated thymidine uptake, the results are expressed as mean counts per minute i standard error of the mean. (B) The IL 6 bioactivity is immunologically identical to IL 6. Medium that had been conditioned for 16 hr by treating PBM at 1 x 106/mL with 10 ng/mL TGFP, was incubated with varying dilutions of goat anti IL-6 serum or normal goat serum (NGS) and assayed for ability to stimulate B9 cell proliferation as described in Materials and Methods
above background. Combinations of stimuli which included the phorbol ester, PMA were potent inducers of all three cytokines and were not susceptible to inhibition by TGFP,, as shown in our previous findings.’ IFNy
IL -6L3
DISCUSSION
18S* time
3 8 16 (hours)
of IL-6
by TGFB,.
0 Figure
3.
PBM were FCS and harvested. denaturing under UV nitrocellulose
Northern
alone, or IFNy with TGFP, caused small but significant elevations in the production of all three cytokines. With the other stimuli tested, which included PHA, LPS, TNF, LPS + IFNy, TNF + IFNy and poly I:C, the production of IL l/3 and TNFa was inhibited in the presence of TGFP,. In contrast, there was a marginal increase in the amount of IL 6 produced, however, this effect was not synergistic (Fig. 4).
analysis
1 induction
cultured at 1 x lo6 cells/ml in RPM1 1640 containing 10% 10 ng/mL TGFP, and at the indicated times RNA was Twenty pg of total cellular RNA was separated on a agarose gel and the 18s ribosomal RNA photographed illumination (bottom panel). The RNA was transferred to a filter and hybridized with an IL 6 probe (top panel).
TGFP, is a member of a family of growth and differentiation factors, which have among their properties a number of immunosuppressive effects.2T7Because of the multiple effects of TGFP, in the cytokine network, it is conceivable that these immunosuppressive effects are manifested through inhibiting production of stimulatory cytokines, or through interfering with their biological functions. TGFP, has been shown to block the production of TNFa, IL 1 and IFNy,*” and cause inhibition of IL 2 and IFNa receptor expression.235 It was surprising to find that TGFP, was able to induce the production of IL 6. These observations extend further the number of cytokines that have been shown to induce IL 6, including platelet-derived growth factor (PDGF), IL 1, TNFa, lymphotoxin (also known as TNFP), granulocyte macrophage colony stimulating factor (GMCSF) (D. Chantry unpublished data) and IL 7.13,14A recent report has demonstrated the ability of TGFP, to inhibit IL l/3 and TNFa-induced IL 6 production in
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lNF (pglml f sem) Figure
4.
Influence
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production.
PBM were cultured at 1 x lo6 cells/ml in RPM1 1640 containing 10% FCS in the presence (closed bars) or absence (open bars) of 10 ng/mL TGF/3, and were further stimulated with the following agents: PMA 50 ng/mL, PHA 1 pg/mL, IFNr 1000 U/mL, LPS 10 rg/mL, TNFa 2500 U/mL, poly 1:C 100 wg/mL in the indicated combinations. Conditioned medium was collected and assayed for IL 6 using B9 cells, and IL l@ and TNFol using ELISA as described in Materials and Methods. Statistically significant values determined using the Students t test are denoted by asterisks: *P < 0.05, **P -c 0.01, ***p < 0.005.
Vol. 2, No. 3 (May
1990: 211-216)
cultures of primary human endothelial cells,15 suggesting that the stimulatory effects of TGFP, on IL 6 production may be cell type specific. The synthesis and release of IL 6 was stimulated by TGFP,, under conditions in which TNFa and IL 1 production were inhibited. The finding that IL 1 and TNFa are regulated independently of IL 6 in PBM is consistent with the observed differences between these cytokines. IL 6 is functionally dissimilar to IL 1 and TNFcr, because it is unable to induce collagenase and collagen degradation, and it does not induce bone resorption or prostaglandin production in systems in which both IL 1 and TNFa are effective (C. Richards, Strangeways Laboratory, Cambridge, personal communication, 1989). IL 6 is also unable to induce IL 1 or TNFa production (D. Chantry, unpublished results) or the production of a low molecular weight neutrophil chemotactic factor, which is highly inducible by IL 1 and TNF.16 It is conceivable that IL 6 may potentially mediate some of the effects of TGFJ3, in immune response. TGFP,, in contrast to its inhibitory effects on IgG production, enhances the production of the IgA subclass of antibodies” and it is likely IL 6 may play a role in this response. Like TGFP,, IL 6 has recently been shown to inhibit IL 1 and TNF production in some systems.18 IL 6 and TGFP, have also been shown to cooperate in the inhibition of endothelial cell proliferation.” The possibility that IL 6 may mediate some of the effects of TGF@, is therefore consistent with IL 6 having some antiinflammatory properties, such as the ability to induce proteinase inhibitors by the liver.20 In contrast to the concordant effects of IL 6 and TGF/3,, the latter has been shown to inhibit thymocyte and IL 2-driven T lymphocyte proliferation,2,6 while IL 6 is a potent thymocyte and T cell mitogen.21322 It therefore seems that TGFP, may have the capacity to selectively modulate IL 6 biological activities. To support this theory, we have recently shown that TGF/3, can inhibit thymocyte proliferation induced by IL 6.23Taken together, our data indicate that TGFP, induces IL 6, and we suggest that this mechanism may be involved in mediating some of the biological properties of TGF/3,. In view of the ability of TGFP, to induce IL 6 production, it is an oversimplification to consider the effects of TGF/3, on the immune system, as being solely immunosuppressive.
MATERIALS AND METHODS Materials Peripheral blood mononuclear cells, (PBM) were isolated from plateletpheresis residues from healthy donors (provided by the North London Blood Transfusion Service), on FicollHypaque gradients (Lymphoprep). Cells were maintained and stimulated in complete medium (CM), which consisted of
Transforming growth factor p, induces IL 6 / 215 RPM1 1640, supplemented with 10% (vol/vol) fetal calf serum and 2 mM glutamine, (Gibco). Phytohemagglutinin (PHA) was purchased from Difco laboratories, Salmonella typhimurium lipopolysaccharide (LPS), polyinosinic-cytidilic acid (poly 1:C) and phorbol 12-myristate, 13-acetate (PMA) were purchased from Sigma. Recombinant human TNF (5 x lo7 U/mg; Endotoxin content c; 0.25 EU/mg), recombinant human IFNy (2 x 10’ U/mg; Endotoxin content c: 0.1 EU/mg) and recombinant human TGFP, (endotoxin content < 0.125 EU/mg) were provided by Genentech Inc. Recombinant human IL 6 (specific activity 5 x 1 O6u/mL in a CESS cell assay) was provided by Genetics Institute. The cDNA probe for IL 6 was a Taq I-Ban II fragment from pBSF-2. 38.1,24 which was kindly provided by Prof. T. Kishimoto (Osaka University, Japan).
IL 6 Bioassay IL 6 bioactivity was analyzed by measuring growth of the B9 hybridoma cell line (kindly provided by L.A. Aarden, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service and Laboratory for Experimental and Clinical Immunology, Amsterdam), which is dependent on the presence of IL 6.25 5000 B9 cells were cultured together with conditioned supernatants or known amounts of human recombinant IL 6 for 72 hr in a total volume of 200 ~1. Proliferation was determined by a 4 hr pulse with 1 &i/well of ‘H methyl-thymidine (specific activity 85 Curies/mmol, Amersham International). Cells were harvested using a semiautomatic cell harvester (LKB/Pharmacia 1205 Betaplate), and the amount of ‘H methyl-thymidine incorporated into DNA/ culture was assessed by liquid scintillation counting. Levels of IL 6 are expressed either as counts per minute (CPM) or units/ml, determined by comparison to a standard curve (dilutions of a known amount of recombinant IL 6; one unit of IL 6 is defined as the amount of IL 6 that induces half maximal proliferation of the B9 cells).
Northern
Blotting
Total cellular RNA was prepared by guanidine isothiocyanate lysis and cesium chloride gradient ultracentrifugation, electrophorezed through 1% agarose gels containing 6% formaldehyde, with the addition of 10 ng/mL ethidium bromide to the gel running buffer, as previously described.8 Following electrophoresis for 18 to 20 hr at 2OV, the gels were photographed under ultraviolet illumination and RNA transferred onto nitrocellulose by capillary blotting. Following transfer, the gel was inspected again under ultraviolet light to insure efficiency of the transfer. Filters were prehybridized in a solution containing 50% formamide, 5 x SSC (1 x SSC = 150 mM NaCl, 15 mM sodium citrate, pH 7.0), 7.5 xDenhardts solution (1 x Denhardts = 0.05% ficoll 400, 0.05% polyvinylpyrollidone, 0.05% bovine serum albumin), 50 mM phosphate buffer (pH 6.6), 0.5 mg/mL denatured salmon sperm DNA at 42OC for 4 to 16 hr. cDNA inserts were resolved on low melting point agarose gels, and labeled by random priming to high specific activity (7 to I2 x IO9 cpm/pg DNA) using [a-32P] dCTP (Amersham), then hybridized to filters overnight at 42OC. Filters were washed twice in 2 xSSC, 0.1% Sodium Dodecyl Sulphate (SDS) at room temperature, and
twice in 0.2 xSSC, 0.1% SDS at 50°C and exposed to Fuji X ray film at -70°C with intensifying screens.
Measurement
of TNFcY and IL-l/3
IL lb levels in tissue culture supernatants were determined using the Cistron IL 10 specific ELISA (Laboratory Impex Ltd.) Human TNFa was detected using ELISA reagents kindly provided by Dr. A.B. Chen (Genentech), the characteristics of this ELISA have been described in detail elsewhere.26
Acknowledgments We thank Dr. C. Richards for discussion of unpublished results and Dr. E. Abney for helpful suggestions, Dr. H.M. Shepard (Genentech) for recombinant human TGFP,, TNFa and IFNy, Dr. Steven Clark (Genetics Institute) for recombinant human IL 6, Dr. L. Aarden for B9 cells and anti-IL 6, Dr. T. Kishimoto for the IL 6 cDNA and Dr. Contreras and her colleagues at the National Blood Transfusion Laboratory, Edgeware for buffy coat residues. REFERENCES 1. Sporn MB, Roberts AB (1988) Peptide growth factors are multifunctional. Nature 332:217-220. 2. Kehrl JH, Wakefield LM, Roberts AB, Jakowlew S, AlvarezMon M, Derynck R, Sporn MB, Fauci AS (1986) Production of transforming growth factor fl by human T lymphocytes and its potential role in the regulation of T cell growth. J Exp Med 163:1037-1049. 3. Espevik T, Figari IS, Ranges GE, Palladino MA Jr (1988) Transforming growth factor 01 and recombinant human tumour necrosis factor reciprocally regulate the generation of lymphokine activated killer cell activity. J Immunol 140:23 12-23 16. 4. Ranges GE, Figari IS, Espevik T, Palladino MA Jr (1987) Inhibition of cytotoxic T cell development by transforming growth factor /3 and reversal by recombinant tumor necrosis factor LY. J Exp Med 166:991-998. 5. Rook AH, Kehrl JH, Wakefield LM, Roberts AB, Sporn MB, Burlinaton DB. Lane HC. Fauci AS (1986) Effects of transforming grow&factor beta on the’functions of‘ natural killer cells: Depressed cytolytic activity and blunting of interferon responsiveness. J Immunol 136:3916-3920. 6. Kehrl JH, Roberts AB, Wakefield LM, Jakowlew S, Sporn MB, Fauci AS (1986) Transforming growth factor fl is an important immunomodulatory protein for human B lymphocytes. J Immunol 137:3855-3860. 7. Tsunawaki tion of macrophages 334:260-262.
S, Sporn M, Ding A, Nathan by transforming growth
8. Chantry D, Turner M, Modulation of cytokine production beta. J Immunol 142:4295-4300.
C (1988) Deactivafactor beta. Nature
Abney E, Feldmann M by transforming growth
(1989) factor
9. Espevik T, Figari IS, Shalaby MR, Lackdies GA, Lewis GD, Shepard HM, Palladino MA Jr (1987) Inhibition of cytokine production by Cyclosporin A and transforming growth factor beta. J Exp Med 166:571-576. 10. Wahl SM, Hunt DA, Wakefield LM, McCartney-Francis N, Wahl LM, Roberts AB, Sporn MB (1987) Transforming growth factor type p induces monocyte chemotaxis and growth factor production. Proc Nat1 Acad Sci USA 84:5788-5792. Francis
11. Wahl SM, Hunt DA, Wong HL, Dougherty N, Wahl LM, Ellingsworth L, Schmidt JA,
S, McCartneyHall G, Roberts
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AB, Sporn MB (1988) Transforming growth factor-p is a potent immunosuppressive agent that inhibits IL-l-dependent lymphocyte proliferation. J Immunol 140:3026-3032. 12. Chantry D, Turner M, Feldmann M (1989) Interleukin 7 (pre B cell growth factor/Lymphopoietin 1) stimulates the growth of murine thymocytes: Regulation by transforming growth factor /3. Eur J Immunol 19:783-786. 13. Hirano T, Kishimoto T (1990) Interleukin 6. In Sporn MB, Roberts A (eds). Handbook of experimental pharmacology “Peptide growth factors and their receptors.” Springer-Verlag, Berlin. 14. Lee G, Namen AE, Gillis S, Ellingsworth LR, Kincade PW (1989) Normal B cell precursors responsive to recombinant murine IL-7 and inhibition of IL-7 activity by transforming growth factor 0. J Immunol 142:3875-3883. 15. Shalaby MR, Wagge A, Espevik T (1989) Cytokine regulation of interleukin 6 production by human endothelial cells. Cell Immunol 121:372-382. 16. Strieter RM, Kunkel SL, Showell HJ, Marks RM (1988) Monokine induced gene expression of a human endothelial cell derived neutrophil chemotactic factor. Biochem Biophys Res Comm 156: 13401345. 17. Coffman RL, Lebman DA, Shrader B (1989) Transforming growth factor@ specifically enhances IgA production by lipopolysaccharide-stimulated murine B lymphocytes. J Exp Med 170: 1039- 1044. 18. Aderka D, Le J, Vilcek J (1989) IL-6 inhibits lipopolysaccharide-induced tumor necrosis factor production in cultured human monocytes, U937 cells, and in mice. J Immunol 143:3517-3523. 19. May LT, Torcia G, Cozzolino F, Ray A, Tatter SB, Santhanam U, Sehgal PB, Stern D (1989) Interleukin-6 gene expression in human endothelial cells: RNA start sites, multiple IL-6
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proteins and inhibition of proliferation. Biochem Biophys Res Comm 159:991-998. 20. Gauldie J, Richards C, Harnish D, Lansdorp P, Baumann H (1987) Interferon @2/B-cell stimulatory factor type 2 shares identity with monocyte derived hepatocyte stimulating factor and regulates the major acute phase protein response in liver cells. Proc Nat1 Acad Sci USA 84:7251-7255. 21. Helle M, Boeije L, Aarden LA (1989) IL-6 is an intermediate in IL-l induced thymocyte proliferation. J Immunol 142:43354338. 22. Uyttenhove C, Coulie PG, Van Snick J (1988) T cell growth and differentiation induced by interleukin-HPl/IL-6, the murine hybridoma/plamsacytoma growth factor. J Exp Med 167:1417-1427. 23. Turner M, Chantry DH, Lane A, Feldmann M (1990) Modulation of cytokine production and function by transforming growth factor-p. In, Molecular and Cellular Biology of cytokines. Oppenheim JJ, Powanda MC, Kluger MJ, Dinarello CA (eds) Alan Liss, New York (in press) 24. Hirano T, Yasukawa K, Harada H, Taga T, Watanabe Y, Matsuda T, Kashiwamura S, Nakajima K, Koyama K, Iwamatsu A, Tsunasawa S, Sakiyama F, Matsui H, Takahara Y, Taniguchi T, Kishimoto T (1986) Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin. Nature 324:73-76. 25. Aarden LA, De Groot ER, Schaap OL, Lansdorp PM (1987) Production of hybridoma growth factor by human monocytes. Eur J Immunol 17:1411-1416. 26. Prince WS, Harder KJ, Saks S, Reed BR, Chen AB, Jones AJS (1986) ELISA for quantification of tumour necrosis factor-alpha in serum. J Pharm Biomed Analysis 5:793-798.
Turner*,
David Chantry,
Marc Feldmann
Previous studies have indicated that the cytokine transforming growth factor & (TGFP,) has immunosuppressive properties and can inhibit the production of tumor necrosis factor (TNF) and Interleukin 1 (IL 1) by human peripheral blood mononuclear cells. In this study, we have examined the effects of TGF/3r on the production of Interleukin 6 (IL 6) by human peripheral blood mononuclear cells. Treatment with only TGFP, leads to the induction of IL 6, and this was both dose- and time-dependent. The effect of TGF& was evident at the level of IL 6 mRNA, suggesting TGFP,-induced de novo synthesis of IL 6. Induction of IL 6 by TGFP, was specific, as other cytokines made by mononuclear cells (TNF and IL 1) were not induced by TGFP,. Furthermore, when a panel of stimuli were compared for their ability to induce IL 1, TNF and IL 6 in the presence or absence of TGFP,, IL 6 levels were augmented in the presence of TGF&, while the induction of IL 1 and TNF was inhibited significantly. These results indicate that TGFP, has complex effects on the production of cytokines by peripheral blood mononuclear cells and that TGFP, is not inhibitory for all cytokine production. The ability of TGF/3, to induce IL 6 suggests that IL 6 may mediate some of the effects of TGFBr. o 1990 by W.B. Saunders Company.
Transforming growth factor pi (TGFP,), a member of a superfamily of growth and differentiation factors, has recently been shown to mediate a number of immunosuppressive effects.’ TGFP,-inhibited IL 2-driven lymphocyte proliferation,’ lymphokine activated killer (LAK) cell generation3 cytotoxic T lymphocyte (CTL) generation in mixed lymphocyte culture4 and natural killer (NK) activity.5 TGFP, has also been shown to inhibit B cell proliferation and IgG production.6 In addition to its effects on lymphoid cells, TGFP, has been shown to block H,O, production and other macrophage functions.7 Many of the effects of TGFP, on immune-celleffector-function may be secondary to the inhibition of cytokine production or responsiveness, which is essential for the generation of these activities. It has been shown that TGFP, blocks tumor necrosis factor (TNF) production by peripheral blood mononuclear cells (PBM), in
From
the Charing Cross Sunley Research Centre, Hammersmith, London W6 SLW UK *To whom reprint requests should be addressed. o 1990 by W.B. Saunders Company. 1043-4666/90/0202-0001$05.00/0 KEY
WORDS:
CYTOKINE,
TGF
P/IL-6/Human
Vol. 2, No.
3 (May),
PBM
1990: pp 211-216
Lurgan
Avenue,
response to lipopolysaccharide (LPS).**’ In addition phytohemagglutinin (PHA)-induced IFNr production by PBM was blocked by the pretreatment of human lymphocytes with TGFP,.’ The role of TGFP, in regulation of IL 1 production is less clear. TGF/3, has been shown to induce IL 1 mRNA,sV’o~ll but our study did not lead to the induction of detectable IL 1 protein.8 In contrast, other work has shown a biological activity consistent with the presence of IL 1 in murine thymocyte and fibroblast proliferation assays.lO~ll The ability of TGFP, to mediate inhibitory effects on immune cells depends on the nature of the inducing stimulus and the activation state of the responding cells. For example, IL l-driven thymocyte proliferation can be completely blocked by TGFP,, but thymocyte proliferation that is stimulated by IL 7 is considerably more resistant to TGFP, inhibition.12 Induction of IL lp and TNF by bacterial LPS is blocked by TGFP,, while induction of these cytokines by phorbol ester (PMA) is unaffected by TGFP,. This suggests different pathways of cytokine induction are differentially susceptible to the effects of TGF&.* We demonstrate, in this study the ability of TGFP, to induce production and secretion of interleukin 6 (IL 6) by peripheral blood mononuclear cells under circumstances where it can inhibit the production of IL l/3 and TNFcE. 211
212
/
Turner,
Chantry,
Feldmann
CYTOKINE,
RESULTS TGFP, has been shown to inhibit the release of a number of cytokines including TNFLu, IL 1, and IFNy.*” It was therefore of interest to examine the effects of TGFP, on the production of IL 6, a cytokine that is highly inducible by IL 1 and TNF.13 PBM were separated on Lymphoprep and cultured for 16 hr with various doses of TGFP,. The resulting supernatants were assayed for IL 6 using the IL 6-dependent cell line B9. TGFP, induced a dose-dependent increase in IL 6 activity in the conditioned medium (Fig. la). The kinetics of the induction of IL 6 activity by TGFP, were established by incubating PBM for up to 96 hr in the presence of 10 ng/mL TGFP,. TGFP, elicited an increase in IL 6 production as early as 4 hr, and IL 6 levels continued to accumulate in a time-dependent manner (Fig. lb). Next we determined the effect of TGFP, on IL 6-induced B9 proliferation, because the conditioned medium used for the IL 6 assay contained significant quantities of bioactive recombinant TGFP, remaining from its addition’ and it can interfere with some bioassays for cytokines.’ TGFP, alone had no stimulatory effect on the proliferation of B9 cells. Furthermore, TGFP, did not inhibit or enhance the proliferative response of B9 cells to exogenously added recombinant human IL 6 (Fig. 2a). Having shown that TGF/3, did not influence the IL 6 bioassay, we verified that the identity of the B9 stimulatory activity present in TGFP, conditioned medium was
Vol. 2, No. 3 (May
indeed IL 6. This was done by specific neutralization with goat antiserum that was raised against recombinant human IL 6, while normal goat serum, used at the same dilutions, was ineffectual (Fig. 2b). To characterize the mechanism of IL 6 induction by TGFP,, we extracted RNA from PBM treated at various times with TGFP, and performed northern blots with an IL g-specific probe. TGF/3, transiently induced IL 6 mRNA (Fig. 3). Levels of ribosomal RNA, which served as a control, were unchanged by treatment with TGFP,, and verified equal loading on each lane of the gel. Conditioned medium, collected from these cells, showed a continued time-dependent accumulation of IL 6 bioactivity (data not shown). We have previously shown that treatment of PBM with TGF/3, causes an transient increase in steady state levels of mRNA for IL la, IL l/3 and TNFa,’ and, as shown here, of IL 6. However no release of immunoreactive or bioactive IL 1 or TNFa was demonstrable, even using sensitive bioassays combined with anti-TGF& antibodies.8 Therefore, it was of interest to determine simultaneously the effects of TGF@, on the production of all three cytokines. We tested a panel of stimuli which have been shown to be potent inducers of cytokine production, both in the presence and absence of TGFP, (Fig. 4). PBM, cultured with medium alone, produced little IL l/3, TNFol or IL 6 protein, and there was no enhancement of IL I/? or TNFa production when TGFP, was included at 10 ng/mL. In contrast, IL 6 levels in the same supernatants were significantly elevated
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1990: 211-216)
of IL-6.
(A) PBM at 1 x lo6 cells/ml in RPM1 1640 containing 10% FCS were cultured for with varying doses of TGFB, and conditioned medium was collected after 20 hr and assayed for ability to support B9 cell proliferation as described in Materials and Methods. (B) Kinetics of IL 6 induction by TGFP,. Freshly separated PBM were cultured for varying lengths of time at 1 x lo6 cells/ml in RPM1 1640 containing 10% FCS and 10 ng/mL TGFP,. Conditioned media was collected and assayed for IL 6.
Transforming
I
1
0
Figure
2.
2 4 IL-6 (U/ml) Effect
of TGFj3,
on the IL-6
8
I : 100
growth
I
1:1000
.
factor
I
1:10,000
p, induces
-
I
1:100,000
IL 6 / 213
.
I
00
Dilution of antisera assay.
(A) Serial dilutions of recombinant human IL-6 were added to B9 cells in the presence or absence of 0.1 ng/mL or 10 ng/mL TGFP,. Proliferation was determined by tritiated thymidine uptake, the results are expressed as mean counts per minute i standard error of the mean. (B) The IL 6 bioactivity is immunologically identical to IL 6. Medium that had been conditioned for 16 hr by treating PBM at 1 x 106/mL with 10 ng/mL TGFP, was incubated with varying dilutions of goat anti IL-6 serum or normal goat serum (NGS) and assayed for ability to stimulate B9 cell proliferation as described in Materials and Methods
above background. Combinations of stimuli which included the phorbol ester, PMA were potent inducers of all three cytokines and were not susceptible to inhibition by TGFP,, as shown in our previous findings.’ IFNy
IL -6L3
DISCUSSION
18S* time
3 8 16 (hours)
of IL-6
by TGFB,.
0 Figure
3.
PBM were FCS and harvested. denaturing under UV nitrocellulose
Northern
alone, or IFNy with TGFP, caused small but significant elevations in the production of all three cytokines. With the other stimuli tested, which included PHA, LPS, TNF, LPS + IFNy, TNF + IFNy and poly I:C, the production of IL l/3 and TNFa was inhibited in the presence of TGFP,. In contrast, there was a marginal increase in the amount of IL 6 produced, however, this effect was not synergistic (Fig. 4).
analysis
1 induction
cultured at 1 x lo6 cells/ml in RPM1 1640 containing 10% 10 ng/mL TGFP, and at the indicated times RNA was Twenty pg of total cellular RNA was separated on a agarose gel and the 18s ribosomal RNA photographed illumination (bottom panel). The RNA was transferred to a filter and hybridized with an IL 6 probe (top panel).
TGFP, is a member of a family of growth and differentiation factors, which have among their properties a number of immunosuppressive effects.2T7Because of the multiple effects of TGFP, in the cytokine network, it is conceivable that these immunosuppressive effects are manifested through inhibiting production of stimulatory cytokines, or through interfering with their biological functions. TGFP, has been shown to block the production of TNFa, IL 1 and IFNy,*” and cause inhibition of IL 2 and IFNa receptor expression.235 It was surprising to find that TGFP, was able to induce the production of IL 6. These observations extend further the number of cytokines that have been shown to induce IL 6, including platelet-derived growth factor (PDGF), IL 1, TNFa, lymphotoxin (also known as TNFP), granulocyte macrophage colony stimulating factor (GMCSF) (D. Chantry unpublished data) and IL 7.13,14A recent report has demonstrated the ability of TGFP, to inhibit IL l/3 and TNFa-induced IL 6 production in
214
/
Chantry,
Turner,
LPSllFNy TNFlIFMy Poly kc Untreated
CYTOKINE,
Feldmann
r.,..A] 0
2000
1000
3000
4000
5000
IL-1 (pg/ml f sem) PMA PHA PHAlPYA 1% LPS TNF LPSIIFNy TNFIIFNy
B
Poly IC Untreated
2000
1000
0
3000
IL-6 (U/ml f sem)
PMA PHA PHAIPMA IFNV LPS LPSllFNy Poly kc Untreated I.
0
I.
I.
2000
1000
8..
3000
I
4000
5000
lNF (pglml f sem) Figure
4.
Influence
of TGF/31
on IL 6, IL l/3 and TNFa
production.
PBM were cultured at 1 x lo6 cells/ml in RPM1 1640 containing 10% FCS in the presence (closed bars) or absence (open bars) of 10 ng/mL TGF/3, and were further stimulated with the following agents: PMA 50 ng/mL, PHA 1 pg/mL, IFNr 1000 U/mL, LPS 10 rg/mL, TNFa 2500 U/mL, poly 1:C 100 wg/mL in the indicated combinations. Conditioned medium was collected and assayed for IL 6 using B9 cells, and IL l@ and TNFol using ELISA as described in Materials and Methods. Statistically significant values determined using the Students t test are denoted by asterisks: *P < 0.05, **P -c 0.01, ***p < 0.005.
Vol. 2, No. 3 (May
1990: 211-216)
cultures of primary human endothelial cells,15 suggesting that the stimulatory effects of TGFP, on IL 6 production may be cell type specific. The synthesis and release of IL 6 was stimulated by TGFP,, under conditions in which TNFa and IL 1 production were inhibited. The finding that IL 1 and TNFa are regulated independently of IL 6 in PBM is consistent with the observed differences between these cytokines. IL 6 is functionally dissimilar to IL 1 and TNFcr, because it is unable to induce collagenase and collagen degradation, and it does not induce bone resorption or prostaglandin production in systems in which both IL 1 and TNFa are effective (C. Richards, Strangeways Laboratory, Cambridge, personal communication, 1989). IL 6 is also unable to induce IL 1 or TNFa production (D. Chantry, unpublished results) or the production of a low molecular weight neutrophil chemotactic factor, which is highly inducible by IL 1 and TNF.16 It is conceivable that IL 6 may potentially mediate some of the effects of TGFJ3, in immune response. TGFP,, in contrast to its inhibitory effects on IgG production, enhances the production of the IgA subclass of antibodies” and it is likely IL 6 may play a role in this response. Like TGFP,, IL 6 has recently been shown to inhibit IL 1 and TNF production in some systems.18 IL 6 and TGFP, have also been shown to cooperate in the inhibition of endothelial cell proliferation.” The possibility that IL 6 may mediate some of the effects of TGF@, is therefore consistent with IL 6 having some antiinflammatory properties, such as the ability to induce proteinase inhibitors by the liver.20 In contrast to the concordant effects of IL 6 and TGF/3,, the latter has been shown to inhibit thymocyte and IL 2-driven T lymphocyte proliferation,2,6 while IL 6 is a potent thymocyte and T cell mitogen.21322 It therefore seems that TGFP, may have the capacity to selectively modulate IL 6 biological activities. To support this theory, we have recently shown that TGF/3, can inhibit thymocyte proliferation induced by IL 6.23Taken together, our data indicate that TGFP, induces IL 6, and we suggest that this mechanism may be involved in mediating some of the biological properties of TGF/3,. In view of the ability of TGFP, to induce IL 6 production, it is an oversimplification to consider the effects of TGF/3, on the immune system, as being solely immunosuppressive.
MATERIALS AND METHODS Materials Peripheral blood mononuclear cells, (PBM) were isolated from plateletpheresis residues from healthy donors (provided by the North London Blood Transfusion Service), on FicollHypaque gradients (Lymphoprep). Cells were maintained and stimulated in complete medium (CM), which consisted of
Transforming growth factor p, induces IL 6 / 215 RPM1 1640, supplemented with 10% (vol/vol) fetal calf serum and 2 mM glutamine, (Gibco). Phytohemagglutinin (PHA) was purchased from Difco laboratories, Salmonella typhimurium lipopolysaccharide (LPS), polyinosinic-cytidilic acid (poly 1:C) and phorbol 12-myristate, 13-acetate (PMA) were purchased from Sigma. Recombinant human TNF (5 x lo7 U/mg; Endotoxin content c; 0.25 EU/mg), recombinant human IFNy (2 x 10’ U/mg; Endotoxin content c: 0.1 EU/mg) and recombinant human TGFP, (endotoxin content < 0.125 EU/mg) were provided by Genentech Inc. Recombinant human IL 6 (specific activity 5 x 1 O6u/mL in a CESS cell assay) was provided by Genetics Institute. The cDNA probe for IL 6 was a Taq I-Ban II fragment from pBSF-2. 38.1,24 which was kindly provided by Prof. T. Kishimoto (Osaka University, Japan).
IL 6 Bioassay IL 6 bioactivity was analyzed by measuring growth of the B9 hybridoma cell line (kindly provided by L.A. Aarden, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service and Laboratory for Experimental and Clinical Immunology, Amsterdam), which is dependent on the presence of IL 6.25 5000 B9 cells were cultured together with conditioned supernatants or known amounts of human recombinant IL 6 for 72 hr in a total volume of 200 ~1. Proliferation was determined by a 4 hr pulse with 1 &i/well of ‘H methyl-thymidine (specific activity 85 Curies/mmol, Amersham International). Cells were harvested using a semiautomatic cell harvester (LKB/Pharmacia 1205 Betaplate), and the amount of ‘H methyl-thymidine incorporated into DNA/ culture was assessed by liquid scintillation counting. Levels of IL 6 are expressed either as counts per minute (CPM) or units/ml, determined by comparison to a standard curve (dilutions of a known amount of recombinant IL 6; one unit of IL 6 is defined as the amount of IL 6 that induces half maximal proliferation of the B9 cells).
Northern
Blotting
Total cellular RNA was prepared by guanidine isothiocyanate lysis and cesium chloride gradient ultracentrifugation, electrophorezed through 1% agarose gels containing 6% formaldehyde, with the addition of 10 ng/mL ethidium bromide to the gel running buffer, as previously described.8 Following electrophoresis for 18 to 20 hr at 2OV, the gels were photographed under ultraviolet illumination and RNA transferred onto nitrocellulose by capillary blotting. Following transfer, the gel was inspected again under ultraviolet light to insure efficiency of the transfer. Filters were prehybridized in a solution containing 50% formamide, 5 x SSC (1 x SSC = 150 mM NaCl, 15 mM sodium citrate, pH 7.0), 7.5 xDenhardts solution (1 x Denhardts = 0.05% ficoll 400, 0.05% polyvinylpyrollidone, 0.05% bovine serum albumin), 50 mM phosphate buffer (pH 6.6), 0.5 mg/mL denatured salmon sperm DNA at 42OC for 4 to 16 hr. cDNA inserts were resolved on low melting point agarose gels, and labeled by random priming to high specific activity (7 to I2 x IO9 cpm/pg DNA) using [a-32P] dCTP (Amersham), then hybridized to filters overnight at 42OC. Filters were washed twice in 2 xSSC, 0.1% Sodium Dodecyl Sulphate (SDS) at room temperature, and
twice in 0.2 xSSC, 0.1% SDS at 50°C and exposed to Fuji X ray film at -70°C with intensifying screens.
Measurement
of TNFcY and IL-l/3
IL lb levels in tissue culture supernatants were determined using the Cistron IL 10 specific ELISA (Laboratory Impex Ltd.) Human TNFa was detected using ELISA reagents kindly provided by Dr. A.B. Chen (Genentech), the characteristics of this ELISA have been described in detail elsewhere.26
Acknowledgments We thank Dr. C. Richards for discussion of unpublished results and Dr. E. Abney for helpful suggestions, Dr. H.M. Shepard (Genentech) for recombinant human TGFP,, TNFa and IFNy, Dr. Steven Clark (Genetics Institute) for recombinant human IL 6, Dr. L. Aarden for B9 cells and anti-IL 6, Dr. T. Kishimoto for the IL 6 cDNA and Dr. Contreras and her colleagues at the National Blood Transfusion Laboratory, Edgeware for buffy coat residues. REFERENCES 1. Sporn MB, Roberts AB (1988) Peptide growth factors are multifunctional. Nature 332:217-220. 2. Kehrl JH, Wakefield LM, Roberts AB, Jakowlew S, AlvarezMon M, Derynck R, Sporn MB, Fauci AS (1986) Production of transforming growth factor fl by human T lymphocytes and its potential role in the regulation of T cell growth. J Exp Med 163:1037-1049. 3. Espevik T, Figari IS, Ranges GE, Palladino MA Jr (1988) Transforming growth factor 01 and recombinant human tumour necrosis factor reciprocally regulate the generation of lymphokine activated killer cell activity. J Immunol 140:23 12-23 16. 4. Ranges GE, Figari IS, Espevik T, Palladino MA Jr (1987) Inhibition of cytotoxic T cell development by transforming growth factor /3 and reversal by recombinant tumor necrosis factor LY. J Exp Med 166:991-998. 5. Rook AH, Kehrl JH, Wakefield LM, Roberts AB, Sporn MB, Burlinaton DB. Lane HC. Fauci AS (1986) Effects of transforming grow&factor beta on the’functions of‘ natural killer cells: Depressed cytolytic activity and blunting of interferon responsiveness. J Immunol 136:3916-3920. 6. Kehrl JH, Roberts AB, Wakefield LM, Jakowlew S, Sporn MB, Fauci AS (1986) Transforming growth factor fl is an important immunomodulatory protein for human B lymphocytes. J Immunol 137:3855-3860. 7. Tsunawaki tion of macrophages 334:260-262.
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C (1988) Deactivafactor beta. Nature
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9. Espevik T, Figari IS, Shalaby MR, Lackdies GA, Lewis GD, Shepard HM, Palladino MA Jr (1987) Inhibition of cytokine production by Cyclosporin A and transforming growth factor beta. J Exp Med 166:571-576. 10. Wahl SM, Hunt DA, Wakefield LM, McCartney-Francis N, Wahl LM, Roberts AB, Sporn MB (1987) Transforming growth factor type p induces monocyte chemotaxis and growth factor production. Proc Nat1 Acad Sci USA 84:5788-5792. Francis
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AB, Sporn MB (1988) Transforming growth factor-p is a potent immunosuppressive agent that inhibits IL-l-dependent lymphocyte proliferation. J Immunol 140:3026-3032. 12. Chantry D, Turner M, Feldmann M (1989) Interleukin 7 (pre B cell growth factor/Lymphopoietin 1) stimulates the growth of murine thymocytes: Regulation by transforming growth factor /3. Eur J Immunol 19:783-786. 13. Hirano T, Kishimoto T (1990) Interleukin 6. In Sporn MB, Roberts A (eds). Handbook of experimental pharmacology “Peptide growth factors and their receptors.” Springer-Verlag, Berlin. 14. Lee G, Namen AE, Gillis S, Ellingsworth LR, Kincade PW (1989) Normal B cell precursors responsive to recombinant murine IL-7 and inhibition of IL-7 activity by transforming growth factor 0. J Immunol 142:3875-3883. 15. Shalaby MR, Wagge A, Espevik T (1989) Cytokine regulation of interleukin 6 production by human endothelial cells. Cell Immunol 121:372-382. 16. Strieter RM, Kunkel SL, Showell HJ, Marks RM (1988) Monokine induced gene expression of a human endothelial cell derived neutrophil chemotactic factor. Biochem Biophys Res Comm 156: 13401345. 17. Coffman RL, Lebman DA, Shrader B (1989) Transforming growth factor@ specifically enhances IgA production by lipopolysaccharide-stimulated murine B lymphocytes. J Exp Med 170: 1039- 1044. 18. Aderka D, Le J, Vilcek J (1989) IL-6 inhibits lipopolysaccharide-induced tumor necrosis factor production in cultured human monocytes, U937 cells, and in mice. J Immunol 143:3517-3523. 19. May LT, Torcia G, Cozzolino F, Ray A, Tatter SB, Santhanam U, Sehgal PB, Stern D (1989) Interleukin-6 gene expression in human endothelial cells: RNA start sites, multiple IL-6
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