TUMOUR NECROSIS FACTOR AS AN AUTOCRINE TUMOUR GROWTH FACTOR FOR CHRONIC B-CELL MALIGNANCIES

969 of the N-linked oligosaccharides of serum IgG in these diseases are not known, but recent results indicate a modulation of p-galactosyltransferase activity in B

specific 0-galactosyltransferase which lymphocytes: transfers UDP-Gal to an asialo-agalacto IgG has been reported to be present in B lymphocytes.13,14 The affinity of this enzyme for UDP-Gal in the B lymphocytes of patients with rheumatoid arthritis is lower than in B lymphocytes from a control group, and the specific activity of the galactosyltransferase towards asialo-agalacto IgG was found

2. Ansell BM. Juvenile chronic arthritis. Curr Orthop 1986; 1: 81-89. 3. Parekh RB, Dwek RA, Rademacher TW. Rheumatoid arthritis as 4.

a

to

be reduced

to

50-60% of control levels in adult

rheumatoid patients. Further analysis of the mechanism underlying the galactosylation defect may yield important insights into the pathogenesis of both disorders. We thank Daryl Fernandes and David Ashford for help with the statistical analysis and Mrs P. M. Rudd for technical assistance. R. B. P., R. A. D., and T. W. R are members of the Oxford Glycobiology Unit, which is supported by the Monsanto Company.

Correspondence should be addressed to T. W. R., Department of Biochemistry, University of Oxford, South Parks Road, Oxford OXl 3QU. REFERENCES 1. Morrow

WJW, Isenberg DA. Autoimmune rheumatic Scientific, 1987: 161-67.

disease. Oxford: Blackwell

TUMOUR NECROSIS FACTOR AS AN AUTOCRINE TUMOUR GROWTH FACTOR FOR CHRONIC B-CELL MALIGNANCIES F. T. CORDINGLEY A. BIANCHI A. V. HOFFBRAND J. E. REITTIE H. E. HESLOP A. VYAKARNAM A. MEAGER M. TURNER M. K. BRENNER

Department of Haematology, Royal Free Hospital, London; Department of Immunology, Sunley Research Institute, Charing Cross Hospital, London, and National Institute of Biological Standards and Control, South Mimms, Herts

Recombinant tumour necrosis factor (TNF) promotes survival and induces proliferation in the tumour cells from two malignancies of B lymphocytes—hairy-cell leukaemia and B-chronic lymphocytic leukaemia. Culture with TNF also induces TNF mRNA and protein, so the cytokine may act as an autocrine tumour growth factor. These growth promoting effects are antagonised by alpha but not by gamma interferon.

Summary

Introduction TUMOUR necrosis factor (TNF) may induce lysis of malignant cells and cell-lines and regression of some animal tumours;l.2 this effect has led to investigation of the therapeutic value of TNF in clinical oncology. More recently, however, TNF has been shown to act as a growth

factor for normal fibroblasts,3 T cells,4 and B cells. We now show that TNF can also act as a tumour growth factor and promote the survival and proliferation of tumour cells in two types of B-cell malignancy-hairy-cell leukaemia (HCL) and B-chronic lymphocytic leukaemia (B-CLL). Patients and Methods - PanMH.—Ten patients, three with HCL and seven with B-CLL, were studied on one to four occasions. Diagnosis of HCL was made on the basis of circulating cell morphology, phenotype

5. 6.

7.

8.

9.

a

glycosylation

disorder. Br J Rheumatol (in press). Parekh RB, Dwek RA, Sutton BJ, et al Association of rheumatoid arthritis and primary osteoarthritis with changes m the glycosylation pattern of total serum IgG. Nature 1985; 316: 452-57. Ropes MW, Bennett GA, Cobb S, Jacobs R, Jessar RA. 1958 revision of diagnostic criteria for rheumatoid arthritis. Bull Rheum Dis 1958; 9: 175-76. Isenberg DA, Martin P, Hajirousou V, Todd-Pokropek A, Goldstone AH, Snaith ML. Haematological re-assessment of rheumatoid arthritis. Br J Rheumatol 1986; 25: 125-27. Ashford D, Dwek RA, Welply JK, et al. The &bgr;1 →2-D-xylose and &agr;1 →3-L-fucose substituted N-linked oligosaccharides from Erythrina cristagalli lectin. Eur J Biochem 1987; 166: 311-20. Parekh RB, Isenberg DA, Roitt IM, Dwek RA, Rademacher TW. Age-specific galactosylation of N-linked oligosaccharides of human serum IgG. J Exp Med (in

press). Godfrey K. Simple linear regression

in medical research. N Engl J Med 1985; 313: 1629-36. 10. Miller ML, Glass DN. The major histocompatibility complex antigens in rheumatoid arthritis and juvenile arthritis. Bull Rheum Dis 1981; 31: 21-24. 11. Torrigiani G, Ansell BM, Chown EEA, Roitt IM. Raised IgG antiglobulin factors in Still’s disease. Ann Rheum Dis 1969; 28: 424-27. 12. Hay FC, Nineham LJ, Fletcher MR, Roitt IM. An improved method for the detection of IgG and IgM rheumatoid factors in rheumatoid diseases. In: Jayson MIV, ed. Still’s disease-juvenile chronic polyarthritis. London: Academic Press, 1976:

135-44. 13. Furukawa K, Matsuta

K, Takeuchi F, et al. Alteration of a galactosyltransferase in B cells of rheumatoid arthritis patients. From the IXth international symposium on glycoconjugates. Tourcoing (France): A. Lerouge, 1987: E56. 14. Axford JS, Mackenzie L, Lydyard PML, Hay FC, Isenberg DA, Roitt IM. Reduced B-cell galactosyltransferase activity in rheumatoid arthritis Lancet 1987; ii: 1486-88.

(with B-cell markers and a monoclonal antibody, FMC7, that detects a hairy-cell-associated marker), cytochemistry (positivity for tartrate-resistant acid phosphatase), and trephine or splenic histology, B-CLL was diagnosed by means of cell morphology, number, and phenotype (CD5 and CD20 positive). All patients were selected for high circulating HCL or B-CLL cell numbers. Two of the three HCL patients had undergone splenectomy 5 and 6 years previously. No patient was receiving therapy at the time of

study. Recombinant cytokines.-Recombinant (r) cytokines, expressed in Escherichia coli, were the gift of Knoll-AG (BASF) (rTNF), Biogen (interferon -IFN and Kirby Warwick (rIFN-ot). Specific activity of rTNF was 6-3 x 106 u/mg protein, of rIFN-y 3-3 x 106 u/mg protein, and of rIFN-a 2-5 x 106 u/mg protein. Cell preparation and culture.-Peripheral blood mononuclear cells were prepared by ’Ficoll’ sedimentation and then depleted of monocytes by adherence and of T cells by double-E rosetting. Cells used in this study contained < 0-5% T cells or monocytes; > 95 % of the mononuclear cell population from HCL patients and > 98 % from CLL patients were neoplastic cells. Viability counts were carried out daily in 96-well microculture plates (Nunc). 180 )1 of cell suspension at 106/ml in RPMI 1640 medium and 10% fetal calf serum selected for low mitogenicity were added to each well followed by 10 III of either cytokines or RPMI 1640. At intervals during incubation at 37°C in 5 % CO2, viability counts were carried out with 0 25% ’Nigrosine’. All estimations were carried out in triplicate. Proliferation was estimated by pulsing plates prepared as above with 1 pCi 3I-I-thymidine for 6 h at 37°C and then harvesting DNA and measuring thymidine incorporation in a beta-scintillation counter.

TNF receptor assay.-r TNF was iodinated with 1251 (Amersham Radiochemicals) by the iodogen method,6 producing a specific activity of > 1000 fCi/ug of protein. TNF receptor assays were carried out by the competitive displacement method7 and cellbound rTNF-125I was separated from unbound TNF by centrifugation through phthalate oil.a All estimations were carried out in triplicate and analysed with the LIGAND computer

program.9 Enzyme-linked immunosorbent assay (ELISA).-Microtitre plates (Micro Elisa, Dynatech, Billingshurst) coated with rabbit anti-human IgM (DAKO, Weybridge) or goat anti-human IgG chain-specific antiserum (Sigma) were incubated with supernatants from HCL or B-CLL cultures for 2 h; alkaline-phosphataseconjugated goat anti-human antibodies specific for IgM or IgG, or for anti-human kappa or lambda light chains (Sigma), were then

970

concentrations of

Fig 3-Thymidine incorporation ofB-CLL cells from individuals at different days of culture with increasing concentrations of rTNF.

Mean (SD) viable counts from quadruplicate wells are shown from a single experiment, representative of the eight undertaken (see legend to fig 2). rTNF concentration; nil; S 100 u/ml ; 0 500 u/ml; S 1000 u Jml; 1 5000 u/ml.

Key for rTNF concentration as for fig 2. Paired t testing of data from all experiments showed a significant effect for rTNF 100 (p < 0-02) and 500 u/ml (p < 0-005) on days 7 and 9 of culture (n = 10).

added for 2 h. For visualisation, the substrate p-nitrophenyl phosphate (Sigma phosphatase substrate tablets) was used, and optical density of individual wells was determined with a ’Multiscan MC’ (Titertek). Assay sensitivity was 10-40 ng immunoglobulin/ ml. Results are presented for day 15 assays. Slot-blot analysis of TNF mRNA.-Total cellular RNA was extracted from 5 x 10’ purified HCL or B-CLL cells" after culture for 0-48 h with and without rTNF 500 u/ml and samples were slotted on to nitrocellulose filters. Under stringent conditions, the filters were hybridised with rTNF cDNAlI labelled with 32P by random oligopriming and then exposed to X-ray film at - 70°C.

in these cells, with a lag period of up to 6 days of action (fig 2). Again the response was dose dependent and declined at the highest dose level. The TNF stimulation index (thymidine incorporation with 500 u rTNF/thymidine incorporation in control cultures) on the day of peak uptake ranged from 37 to 201 (mean =9 32, n 8). Once DNA synthesis had declined, hairy cells from two of the patients could be restimulated with rTNF and have been maintained in continuous culture for > 5 months, with a doubling time of 7-12 days. The effects of rTNF are not due to stimulation of residual T cells, normal B cells, or monocytes in the nominally purified HCL population, since immunophenotyping of rTNF-stimulated cells showed that they lacked T cell and monocyte markers (CD5 and VIM D2) but that 90-99% were strongly positive for tartrate-resistant acid phosphatase, the B cell marker RFB4, and for the hairy-cellassociated marker FMC7. Even after 3 months of culture, the rTNF-stimulated HC lymphocytes were negative for Epstein Barr virus nuclear antigen by immunofluorescence. TNF receptors were also present on B-CLL cells from six of the seven patients studied. In the remaining patient,

Fig I-Viability

of HCL cells with

increasing

rTNF.

Results Scatchard analysis of the binding of rTNF to fresh HCL cells showed the cells had 200-830 TNF receptors, with a mean Kd of 1-76x10-" mol/I (SEM=0-83, n=7). Examination of cell cultures showed that rTNF maintained rather than diminshed cell integrity and induced areas of focal proliferation. This action was reflected in an rTNFdose-dependent maintenance of viable HCL cell numbers (fig 1) (mean viable cell count on day 9 with rTNF 500 u=0-92 x 105 Irnl vs 0-23 x lO5/m1 in control cultures, n = 8, p < 0-002). Measurement of thymidine incorporation confirmed that rTNF was a strong inducer of DNA

synthesis

before

onset

=

Fig 2-Mean thymidine incorporation of the HCL cells shown in fig 1 at different days of culture with increasing concentrations of rTNF.

rTNF concentrations nil; 9t 100 u/ml; 0 500 u/ml; 1 5000 u/ml. Paired t testing of data from all experiments showed a significant effect of rTNF at 100 u/ml (p - 0 027) and 500 u/ml (p = 0-005) on day 9/10 of culture

(n = 8).

Fig 4-Inhibition ofrTNF-induced thymidine incorporation in HCL and B-CLL cells by rIFN-a but not rIFN-y. The effects were analysed on the day of peak rTNF-induced proliferation and mean thymidine incorporation (SEM) (n 7) is shown. All cytokines were added at a concentration of 500 u/ml at initiation of culture. =

971

Fig 5-Slot-blot analysis of TNF mRNA of stimulation experiment on an HCL patient.

one

representative

Left track 5 wg RNA; right track 2 5 ug RNA. Fresh cells showed only a highly attenuated signal with the labelled rTNF probe. By 48 h of culture in medium alone, specific message is detectable, but signal strength is greatly increased when cells are cultured in the presence of rTNF. After 96 h, lysates of cells cultured in RPMI + fetal calf serum alone contained < 0.1 u rTNF/10’ cells, whereas cells cultured with rTNF contained 16 u/107 cells.

receptors were not detected until day 6 of culture. Scatchard analysis of fresh B-CLL cells showed 190-3300 receptors/ cell with a Kd not significantly different from HCL cells (mean 3-76 x 10-9 mol/l, SEM 1 °49, n 6). Cells from four patients were cultured with rTNF; all responded with increased cell survival over controls (mean viable cell =

count on

day 10 with rTNF 500 u

=

0-85

x

106/ml compared

with 0,05 106/ml in control cultures) and with increased DNA synthesis (fig 3). Peak thymidine incorporation was seen between days 7 and 13 and the stimulation index on the day of maximum uptake was 2-7-74-2 (mean 21 °1, n = 10). Again the effects were dose dependent. Unlike HCL lymphocytes, B-CLL cells did not respond to restimulation with rTNF. TNF-dependent DNA synthesis in cells from all but one of the HCL and B-CLL patients could be antagonised by addition of rIFN-cx at the beginning of culture (fig 4): thymidine incorporation was significantly less than that in rTNF-treated cultures (analysis of variance, p < 0-0 1) and did not differ significantly from control values. In contrast, thymidine incorporation in cultures treated with both rIFN-y and TNF did not differ significantly from that in cultures treated with TNF alone. The antagonistic effects of rIFN-fx could be detected at a concentration of 10 u/ml and <

were

x

maximum

at

500

u/ml.

Although rTNF induced DNA synthesis in HCL and B-CLL cells, it did not induce terminal differentiation, and no monoclonal immunoglobulin was detected in culture supernatants by ELISA assay on day 15. TNF does not function as a constitutively produced autocrine growth factor for these cells. No TNF is detected in the supernatant of cultured HCL/B-CLL cells either by cytotoxic bioassay with L929 cells12 or by a two-site monoclonal antibody capture radioir=unoassay.13 However, whilst fresh cells contain barely detectable TNF mRNA, co-culture with TNF protein greatly enhances TNF message levels and induces synthesis of TNF protein (illustrated for HCL in fig 5), indicating that a positive feedback loop can be generated by exogenous TNF. Discussion

The capacity of TNF to inhibit tumour cell growth in vitro has led to an extensive search for tumours likely to be susceptible to this agent. We have now shown that rTNF is also a tumour growth factor, maintaining HCL and B-CLL

cell survival in vitro and inducing proliferation without terminal differentiation. These results imply that administration of rTNF to patients with chronic B-cell malignancies would have adverse effects. They also suggest that endogenous paracrine/autocrine TNF production14 may be important for growth and survival of these B-cell malignancies because levels of TNF mRNA and protein are greatly increased when cells are cultured with rTNF itself. Endogenous TNF production may be especially important for the growth of HCL cells since rIFN-K blocks rTNFinduced proliferation in vitro (fig 3) and also decreases HCL cell numbers in vivo. Moreover, rIFN-y, which does not affect rTNF-HCL interaction, lacks therapeutic efficacy. 15 However, this evidence for the role of endogenous TNF is indirect since rIFN-Qt may antagonise other cytokines with growth-promoting activity for HC lymphocytes.16 It is less likely that endogenous TNF or other growth factors are important for B-CLL survival and growth because the antagonistic effects of rIFN-Ot on TNF-induced proliferation in vitro are not matched by comparable clinical effects.17 Whatever the importance of endogenous TNF production for tumour cell growth, it will be interesting to determine whether rTNF can act as a growth factor for other malignancies derived from normal cells that proliferate in response to the cytokine.3-5 Correspondence should be addressed to: M. K. B., Department of Haematology, Royal Free Hospital, Pond Street, Hampstead, London NW3 2QG. We thank the Wellcome Trust for fmancial support, Dr C. M. Rooney for EBV immunofluorescence, and Mrs Megan Evans for preparation of the typescript. F. T. C. is a recipient of an SAW Medical Research Fellowship (University of Western Australia). REFERENCES 1. Carswell EA, Old LJ, Kassel RL, Green S, Fiore N, Williamson B. An endotoxin-induced serum factor that causes necrosis of tumors. Proc Natl Acad Sci USA 1975; 72: 3666-71. 2. Williamson BD, Carswell EA, Rubm BY. Human TNF produced by human B cell lines: synergistic cytotoxic interaction with human IF. Proc Natl Acad Sci 1983; 80: 5397-401. 3. Vilcek J, Palombella VJ, Henriksen-De Stefano, et al. Fibroblast growth enhancing activity of TNF and its relationship to other polypeptide growth factors J Exp Med 1986; 163: 632-44. 4. Zucali JR, Elfenbein GJ, Barth KC, Dinarello CA. Effects of human IL-1 and human TNF on human T lymphocyte colony formation. J Clin Invest 1987; 80: 772-77. 5. Kehrl JH, Miller A, Fauci AS. Effect of TNF&agr; on mitogen activated human B cells. J Exp Med 1987; 166: 786-91. 6. Fraker PJ, Speck JC Jr. Protein and cell membrane estimations with a sparingly soluble chloramide 1,3,4,6,-tetrachloro-3&agr;,6&agr; diphenylglycouril. Biochem Biophys Res Comm 1978; 80: 849-57. 7. Cuatrecasas P, Hollenberg MD. Membrane receptors and hormone action. Adv Protein Chem 1976; 30: 251-451. 8. Finbloom DS, Hoover DL, Wahl LM. The characteristics of binding of human recombinant IFNy to its receptor on human monocytes and human monocyte-like cell lines. J Immunol 1985; 135: 300-05. 9. Munson PS. LIGAND: A computerised analysis of ligand binding data. Meth Enzymol 1983; 92: 543-76. 10. Tumer M, Londei M, Feldmann M. Human T cells from autoimmune and normal individuals can produce tumour necrosis factor. Eur J Immunol 1987; 17: 1807-14. 11. Pennica D, Nedwin GE, Hayflick JS, et al. Human tumour necrosis factor: precursor structure, expression and homology to lymphotoxin. Nature 1984; 312: 724-29. 12. Flick DA, Gifford GE. Comparison of in vitro cell cytotoxic assays for tumour necrosis factor. J Immunol Meth 1984; 68: 167-72. 13. Meager A, Parti S, Leury H, et al. A two site immunoradiometric assay of human lymphotoxin with monoclonal antibodies and its applications. J Immunol Meth 1987; 104: 31-42. 14. Philip R, Epstein LB. Tumour necrosis factor as an immunomodulator and mediator of monocyte cytotoxicity induced by itelf, &ggr;-interferon and interleukin-1. Nature 1986; 323: 86-89. 15. Balkwill FR, Smyth JF Interferons in cancer therapy: a reappraisal. Lancet 1987; ii: 317-19. 16. Paganelli KA, Evans SS, Han T, Ozer H. B cell growth factor-induced proliferation of hairy cell lymphocytes and inhibition by type I interferon in vitro. Blood 1986; 67: 937-42. 17. Foon KA, Bottino GG, Abrams PG, et al. Phase II trial of recombinant leucocyte A interferon in panents with advanced chronic lymphocytic leukemia. Am J Med

1985; 78: 216-20.