Immunology 7bday, vol. 5, No. 6, 1984 identify SCs, their precursors, and the possible interaction of the SC line with cells of the B-cell lineage in birds and m a m m a l s . O u r concept predicts that in-vivo experiments in m a m m a l s would identify SCs in various lymphomyeloid tissues, leading to conclusions similar to those we have d r a w n from experiments in birds 22. Future experiments in the chicken should identify the precursor SC and provide evidence of an interaction of SCs with cells of the B lineage. [:['1 Acknowledgements This work was presented, in part, as the Halpin Memorial Lecture, held at the University of Wisconsin, Madison, on 21 April 1983. This is Journal Article No. 5631 from the MississippiAgriculture and Forestry Experimental Station. We wish to thank Paige Chamblee for secretarial assistance.

References 1 Gliek, B. (1977) Int. Rev. Cytol. 48, 345 2 Golub, E. S. (1981) The Cellular Basis of the Immune Response, Sinauer Associates, Massachusetts

165 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Baba, T. and Kita, M. (1977) Immunology 32, 271 Glick,8. (1960) PoultrySd. 39, 1097 Brand, A., Gilmour, D. G. and Gotdstein, G. (1976) Science193, 319 Subba-Rao, D.S.V. andGlick, B.(1977)inAvianImmunology(Benedict, A. A., ed.), pp. 87-98, Plenum Press, New York Stinson,R. S., Mashaly, M. and Glick, B. (1979) Immunology36, 769 Hemmingson,E. J. and Linna, T. J. (1972) Int. Arch. Allergy 42,693 Olah, I. and Glick, B. (1978) Experlentia42, 693 Olah, I. and Glick, B. (1979) PoultrySci. 58, 195 Olah, I. and Glick, B. (1982)Am. J. Anat. 165, 445 Kineade,P. and Cooper, M. D. (1971),f Immunol. 106, 371 Grossi, C. E., Lydyard, P. and Cooper, M. D. (1977)J. hnmunol 19, 149 Glick,B. (1983) inAvlan Biology(King,J. R., Farner, D. S. and Parkes, K. C., eds), AcademicPress, New York Fitzimmons, R. C., Garrod, E. M. F. and Garnett, I. (1973) Cell. Immunol. 9, 377 Jankovic,B. D., Isakovie,K., Markovic,B. M. and Rajcevic,M. (1977) Immunology 32, 689 Granfors, K., Martin, C., Lassila, O. etal. (1982) Clin. Immunol. Immunopathol. 23, 459 Hoshi, H. (1972) TohokuJ. Exp. Med. 106, 785 Sugimura,M. and Hashimoto, V. (1980)J. Anat. 131,441 Olah, I. and Glick, B.J. LeukocyteBiol. (in press) Olah, I. and Glick, B. (1983)Anat. Roe. 205, 287 Szakal,A. K., Holmes, K. L., and Tow,J. G. (1983)J. Immunol. I31, 1714

What is tumour necrosis factor really for? J. H. L. Playfair, J. Taverne and N. Matthews A m o n g the approximately 60 substances secreted by macrophages, one n a m e stands out by its purposeful~ sound - t u m o u r necrosis factor ( T N F ) . This is the n a m e originally given to the material in some endotoxininduced sera which, on injection, causes certain transplanted tumours in mice to necrose and occasionally to regress completely 1. T u m o u r necrosis serum ( T N S ) is usually prepared by injecting mice or rabbits first with B C G or Corynebacterium parvum and then some weeks later with endotoxin, and bleeding them 1 ½ -2 hours later. T h e same serum at high dilution will also kill m a n y lines of t u m o u r cells in vitro. Because of the relative insensitivity of the in-vivo assay, progress in characterization of the anti-tumour effect has largely been m a d e on the basis of in-vitro cytotoxicity assays. These have shown the cytotoxic factor to be a product of macrophages, especially activated macrophages 2 4. T h e cytotoxin from rabbit serum is a protein with a molecular weight in the range of 39 000-55 000 on gel filtration, with an isoelectric point in the range of p H 5.1-5.25,6. T h e mouse cytotoxin has an isoelectric point of p H 4.8 7, but on gel filtration multiple forms have been described - a low molecular weight component of M, 50 000-60 0002`s and higher molecular weight forms o f M r 100 000-225 0008. T h e component of mouse T N S that is active in vivo and causes necrosis is restricted to a fraction of approximate molecular weight 1500008,9 . Thus, although it was originally thought that the same factor in T N S was responsible for both in-vivo and in-vitro effects, this is now questionable and it is probably safer to refer to these factors simply as cytotoxins. Department of Immunology, Middlesex Hospital Medical School, London W1P 9PG, U K and Department of Medical Microbiology, Welsh National School of Medicine, Cardiff CF4 4XN, UK.

Independently of these developments, a n u m b e r of infectious organisms have also been shown to be susceptible to killing by secreted macrophage products; these include arginase metabolites (Herpes simplex, Schistosomules) and oxygen metabolites (Staphylococci, Leishmania, Trypanosoma cruzi, Toxoplasma) I°. T o date, however, only one parasite has been convincingly shown to be killed by T N S , namely the malaria parasite Plasmodium, and in this brief article we propose that herein m a y lie the real biological importance of these cytotoxins. Anti-malarial activity of T N S was first demonstrated by Clark et al ~ who showed that two intraperitoneal injections of mouse T N S would reduce parasitaemia and prevent mortality from the normally lethal P. vinckei infection in mice. At about the same time we showed a cytotoxic effect of T N S against P.yoelii and P. berghei in vitro 12'13, and subsequently the h u m a n parasite P. falciparum has been found to be equally susceptible (A. O. Wozencraft, H. M. Doekrell, J . Taverne, J. H. L. Playfair and G. A. T. Targett, unpublished observations). Since macrophages in malaria are highly activated, such that an injection of endotoxin causes the release of T N S cytotoxin into the serum H'12, it seemed a reasonable hypothesis that the cytotoxin might be released spontaneously during the infection and might contribute to parasite killing. Fitting in rather well with this idea was our finding that injections of T N S in vivo were very m u c h more effective against a non-lethal parasite (P.yoeli 0 than against the invariably lethal P. berghei 13; resistance to the cytotoxin in vivo might be one reason for the inability of mice to control the lethal infection. However, when tested in vitro, both these parasites were found to be equally susceptible to killing by T N S , which suggests that the effect of T N S in vivo is not a simple, direct one. O n e unsolved question arising from this hypothesis is: what triggers eytotoxin release in the absence of © 1984,ElsevierSciencePublishersB.V. Amsterdam 0167 4919/84/$02.00

166 endotoxin? Small amounts of endotoxin from the gut might in fact enter the circulation in malaria if liver function is disturbed, or the parasite might release a n endotoxin-like substance la. A n alternative idea is that T cells (via lymphokines) or antibody. (via Fc receptors) might be able to trigger activated macrophages to release these cytotoxins, as has been shown for other cytotoxic factors ~4. Another problem is that T N S contains a large variety of molecules with different biological activities. However, we are fairly convinced that the factors in T N S which kill t u m o u r cells in vitro and which kill malaria parasites in vitro are one and the same because: (1) they co-purify on both gel filtration and ion-exchange chromatography; a n d (2) parasitized red cells can competitively inhibit killing of t u m o u r cells in vitro by T N S , and vice versa (J. Taverne, N. Matthews, P. Depledge and J. H. L. Playfair, u n p u b lished observations). With certain exceptions, it is not possible to absorb tumour-killing activity from T N S with susceptible t u m o u r cell lines and we conclude that the cytotoxin probably reacts with a surface molecule on both t u m o u r cells a n d parasitized red cells with relatively low affinity. There is a close correlation a m o n g t u m o u r cell lines between susceptibility to the cytotoxin a n d to natural killer (NK) cells, and in view of the recent suggestion that NK-cell-mediated killing operates via the target cell receptor for transferrin 15, it is a tempting speculation that the cytotoxin does likewise. Perhaps surprisingly, malaria parasites have been shown to be highly dependent on a supply of iron exogenous to the red celP 6 a n d red cells, particularly the younger ones which m a n y species of malaria parasite preferentially inhabit, are rich in transferrin receptors ~5. It remains, of course, to be demonstrated that the eytotoxin actually does operate in this way d u r i n g malaria, b u t the same can be said of its role in cancer. Interestingly, we have been u n a b l e to kill a variety of bacteria or fungi in vitro with T N S (M. L. Neale and N. Matthews, unpublished observations), although T N S has been found to kill a small proportion (about 10%) of normal lymph-node T and B lymphocytes in mice ~7. This subpopulation has not been characterized but it m a y be significant that the transferrin receptor is thought to be crucial for proliferation of a n u m b e r of types of cell including lymphocytes, and here again the cytotoxin a n d N K cells might share a c o m m o n role in the regulation of proliferation. O f the two, the cytotoxin seems a less likely candidate for everyday regulation since it appears to require extreme macrophage activation for its production. So we m a y reasonably ask the question: if the cytotoxin is a natural product and not a laboratory artefact, what is

Immunology Today, vol. 5, No. 6, 1984

its function? The answer is inevitably teleological, but teleology is usually profitable and always enjoyable. To put it briefly, malaria exerts a powerful selective effect on the evolution of susceptible species and tumours do not. Freedom of parts of the h u m a n race from malaria is probably a very recent luxury, and early m a n surely had mosquitoes and malaria parasites as his constant companions. Even today, there are at least a million deaths per year from malaria in Africa alone, a n d these are almost exclusively in childhood, i.e. before the age of reproduction. At a rough estimate, African children up to the age of 15 are about 30 times more likely to die of malaria than of any form of cancer 18. Moreover, death from malaria can be extremely acute (within days of parasites appearing in the blood) a n d while antibody undoubtedly plays a n important protective role in ' i m m u n e ' adults and, via the placenta a n d milk, in neonates, it is unlikely that a primary antibody response alone would be quick enough to save the life of a 1-yearold child with acute P. falciparum malaria. A n y rapidly produced substance that had a significant slowing effect on the acute growth rate of the blood-stage infection would therefore be intensely selected for. If the in-vitro experiment s bear any relation to events in vivo t h e same factor m a y turn out to cause t u m o u r necrosis and recovery from malaria, in which case ' p l a s m o d i u m necrosis factor' m a y be a more logical n a m e for it. [~1 References 1 Carswell,E, A., Old, L. J., Kassel, R. L. et al. (1975)Proc. NatlAcad. &i. USA72, 3666 2 Mannel,D. M., Moore, R. N. and Mergenhagen,S. E. (1980) Infect. Immun. 30, 523 3 Matthews, N. (1978)Br. J. Cancer38, 310 4 Matthews,N. (1981)Immunology 44, 135 5 Ruff, M. R. and Gifford,G. E. (1980)J. Immunol. 125, 1671 6 Matthews, N., Ryley, H. C. and Neale, M. L. (1980)Br. J. Cancer42, 416 7 Mannel,D. M., Mehzer, M. S. and Mergenhagen,S. E. (1980)Infect. Immun. 28, 204 8 Kull, F. C., Jr and Cuatrecasas, P. (1981)J. Immunol. 126, 1279 9 Green, S., Dobrjansky,A., Carswell, E. A. et al. (1976) Proc. Natl Acad. Sci. USA 73, 381 10 Adams,D. O. and Nathan, C. F. (1983)Immunol. Today 4, 166 11 Clark,I. A,, Virelizier,J.-L., Carswell,E. A. and Wood, P. R. (1981) Infect. Immun. 32, 1058 12 Taverne, J., Dockren, H. M. and Playfair, J. H. L. (1981) Infect. Immun. 33, 83 13 Taverne,J., Depledge, P. and Playfair,J. H. L. (1982) Infect. Immun. 37, 927 14 Adams,D. (1982)Immunol. Today 3, 285 15 Newman, R., Schneider, C., Sutherland, R. et al. (1982) Trends Biochem. ScL 7, 397

16 Pollack,S. (1983)Br. J. Haematol. 53, 181 17 Playfair,J. H. L., De Souza,J. B. and Taverne,J. (1982) Clin. Ex]). ImmunoL, 47, 753 18 Edington, G. M. and Gilles, H. M. (1976) Pathology in the Tropics, Edward Arnold, London