June 1996, Volume 46, Issue 6

Review Articles

Cachectin/Tumor Necrosis Factor

Abdus Salam Khan Gandapur  ( Department of Basic Medical Sciences, Gomal University, Dera Ismail Khan. )
Salman Malik  ( Department of Biology, Quaid-e-Azam University, Islamabad. )

Cachectin/tamor necrosis factor (a 17-KDapolypeptide honnone produced mainly by the cells of monocyte/macro­phage lineage), has been implicated in the pathogenesis of both local and systemic inflammatory reactions. It is aprimary mediator in the pathogenesis of infection, injury and inflam­mation and in the beneficial processes of host defence and tissue homeostasis. Depending on its concentration, duration of cell exposure and presence of other mediators in the cellular environment, the net biological effects of this peptide regula­toty factor may be ultimately beneficial or injurious to the host. Thus, acute systemic release of cachectin/TFNF causes septic shock and tissue injury. Persisting cachectin/TNF production provokes Cachexia and these sequelae are syner­gistically influenced by other mediators like interleukin-I (IL-i) and interferon-v (LFN-v). When lesser amounts are released in tissues, the beneficial effects may predominate to mediate enhanced host defence, against pathogens and to coordinate normal tissue remodelling. In this article we shall review the history, chemistry, synthesis, mechanism of action, beneficial and injurious effects, as well as diagnostic and therapeutic potentials of the molecule.
History
Cachectinwas first isolated during a reseaich directed at the identification of basic mechanisms of cachexia in chronic disease. When rabbits are infected with tiypanosoma brucei, they lose upto 50% of their body weight. The rabbits develop striking lipaemia during the final stage of the disease1,2. The hyperlipemia (hypertriglyceridemia) is due to complete sup­pression of the enzyme lipoprotein lipase (LPL); hence preventing the uptake of exogenous triglycerides by fat cells and causing paradoxical lipaemia frequently associated with infection3,4 orneoplastic disease5-7. Subsequent obseivations suggested that a serum factor produced by macmphages in response to invasive stimuli resulted in LPL suppression and contributed to the development of cachexia8.
Beutler and coworkers first isolated cachectin from the supematant of murine macrophages activated by endo­toxin/lipopoly sacchride9. Later, when the gene encoding cachectin was identified and cloned10 it was found to be identical to tumor necrosis factor - (TNFa), a molecule that had been studied for its role as macrophage mediator of haemorrhagic necrosis of certain transplantable tumors11. The availability of recombinant human cachectiniTNF (rh-TNF) 12 and the realization that single mediator participates in the development of both cell cytotoxicity and cachexia prompted investigation into the biologic properties of this newly identified cytokine.
Biochemistry and Biosynthesis
The gene for human TNF is located on the short arm of chromosome 6 and encodes, a prohonnone of 233 amino acids, that is processed to a 157 residue mature protein (MW17KD) by cleavage of a 76 residue single peptide.
Mature cachectin/FNF polypeptide shares a 28% amino acid sequence homology with another cytokine, lymphotoxin 9TNFB), with which it shams some biological properties and these molecules may compete for a common receptor. Lymphotoxin is encoded by a separate gene and is also located on the short ann of chromosome 6 in man possibly indicating ancestral tandem duplication. The biological function of cachectin/TNF also overlaps with that of another 17/KD cytokine IL-i, but the molecules am structurally different and do not compete for a common receptor and several another inflammatory mediators contain 33 nucleotide receptor. The mRNA transcripts of these 3-untranslated sequence is com­posed of repeated and overlapping copies of the consensus octamer UUAUUUAU10. This sequence apparently shortens mRNA life, thereby, reducing the chances for incidental or inappropriate ?roduction of large quantities of these potent polypeptides13
Synthesis
Cachectin/TNF is synthesized by various activated phagocytic and non-phagocytic cells, including macrophages, monocytes, lymphocytes, natural killer cells, astmcytes and microglial cells of the brain and kupifer cells of the liver. A pivotal role of TNF in inflammation is suggested by a wide variety of infectious or inflammatory stimuli capable of triggering cachectin/TNF biosynthesis e.g., bacterial endo­toxin, lipopolysaccharide (LPS), enterotoxin, toxic shock syndrome, toxin-I mycobacterial card factor, viruses, c5a, fungal or parasitic antigens, IL-I and in an autocrine manner cachectinfrNF itself. In response to Iipopolysaccharide (LPS), both transcription and translation of the cachectiniTNF precursor are increased and large amounts of the mature protein are released within minutes. Dexamethasone inhibits cachectin/TNF biosynthesis, but this effect is not observed if the steroid is given after the cells have been exposed to LPS. By contrast interferon (IFNv) exerts a permissive influence that enhances cachectinlr/TNF biosynthesis. The up or down regulation of cachectin/TNF biosynthesis by INFv or Dex­amethasone, respectively, probably contributes to the pro-in­flammatory oranti-inflammatozy effect of these mediators14. Two different forms of cachectinffNF are produced; the 17-KD secreted form and a 26KD membrane associated form15.
It has been suggested that the membrane-bound form may act primarily as a mediator of local (paracnne) tissue effects and is processed to the 17-KD circulating form. High affinity membrane receptois for TNF are present in a variety of tissues (most notably liver, kidney, spleen, lung, muscle, endothelium and intestine) and mediate maximal cellular responses even with low receptor occupancy. Two different TNF receptors (P55 and P75) have recently been identified and sequenced and their genes cloned16,17.
Mechanism of action and metabolic effects of cachectin
After binding to its receptor, TNF induces transcrip­tional changes that lead to a reduction of the MRNA, for several key lipogenic enzymes and LPL, without altering the biosynthesis of normal house keeping genes. The metabolic effects of these cellular responses is lipolysis, with net losses. of free fatty acids and a depletion of the triglyceride storage pool. Muscle cells also become catabolic when incubated with TNF, they are rapidly depleted of glycogen stores, release. lactate and develop a defect in the ability to maintain a normal resting transmembrane potential18. In contrast to the mono­cyte and adipocyte effects, hepatocytes display relatively anabolic responses to TNF and manifest increased rates of lipogenesis and glucagon-mediated amino acid uptake accel­erated biosynthesis of acute phase proteins and decreased albuminbiosynthesis19.
Taken together, the data suggests that the net effects of TNF induces mobilization of peripheral tissue energy stores from adipose and muscle, that can then be used by the liver in order to meet the increased energy and synthetic demands associated with infection or cancer. However, as with other cases immunologically mediated tissue injuiy or death (e.g., anaphylaxis), the inappropriate or prolonged production of TNF is capable of mediating progressive catabolism and injurious wasting that may ultimately kill the host.
Septic shock
After intravenous administration of endotoxin/LPS in man Cachectin/TNF achieve peak levels within two hours, coinciding with the onset of fever, rigors, myalgia, headache and nausea20.
Larger quantities of LPS stimulate much higher serum cachectin/TNF concentrations, that trigger lethal shock awl tissue injury. Administration of human recombinant cachectinf/TNF, that is virtually endotoxin free, produces metabolic effects and lethal tissue injury that is almost identical to septic/toxic shock syndrome21.
It has recently become clear that many of the systemic effects of TNF are mediated by secondary factors (Table)

but the importance of cachectin/TNF in lethal/toxic and septic shock has been established by studies showing a protective effect of anticachectin/TNF antibodies in vivo. In primates disassociated from the presence of bacteria or LPS. Septic shock syndrome seems to be the result of immunological over-responsiveness (similarto anaphylactic shock), whenthe death of the host may be caused by an excessive immune response to an invasive stimulus.
Cachexia
Initial experiments with partly purified material from munne macrophages indicated that persistent exposure to cachectinf/TNF may induce cachexia, these observations were confirmed with recombinant cachectint/TNF25. Recent work­ers have used antibodies against TNF in animal models of tumorassociated cachexiato identify the role of endogenously produced TNF. The antibody treated animals were partially protected against the development of anorexia and wasting, as evidenced by improved food intake and reduced losses of whole body protein and lipid26. Interestingly, the antibody’ treatment did not protect against the development of anaemia or acute phase protein biosynthesis, suggesting either that the antibodies did not completely neutralize the cachexia induc­ing effects of TNF produced locally in tissues or that other factors directly mediated these effects. Stovroff and others (in animal studies) demonstrated that endogenous production of TNF during tumor invasion contributed to the development of malnutrition and wasting27.
Studies in patients and healthy volunteers given rh-TNF offer additional evidence of the role of TNF in cachexia and of the involvementofa complex cascade of secondary mediators.
Following intravenous administration of TNF, whole body energy expenditure protein turnover and lipogenesis are enhanced28 and amino-acid release from extremity skeletal muscle is increased29. In addition to these catabolic responses, higher doses of TNF are extremely toxic and have been complicated by the development of hypotension, capillary leak syndrome, fever, anorexia and inflammation at the site of injection30. Recently, it has been found that the systemic effects of TNF are mediated by sccondaiy factors. For example, TNF stimulates pituocytes biosynthesis of ACTh that in turn stimulates cortisol secretion. The role of ACTH in the regulation of increased protein breakdown is well docu­mented31. In addition, TNF stimulates interleukin 1 (IL-i) biosynthesis from endothclial cells and macrophages, which in turn synergistically increases biological responses to TNF32.Thus the available evidence indicates that the net catabolic effect of TNF in vivo are the result of the complex interplay bthveen its direct catabolic effects and the influence of other factors that are induced during illness.Tracey and Cerami demonstrated that the whole body nutritional re­sponses to TNF are dependent on the site of tissue production, regardless of the predominate blood level32.
Malignancy
Identification of cachectin/TNF as the agent promoting tumor lysis in the serum of endotoxaemic mice initially stimulated interest in its development, as a potential antineo­plastic agent. It is cytotoxic to some human tumor cell lines and causes haemorrhagic necrosis of the centres of some implantable tumors in vivo. However, animal studies indicate that the therapeutic efficacy of cachectin/TNF is unimpressive and limited by severe toxicity. Moreover, various human epithelial cell lines are resistant to and actually synthesize the cytokine33.
Clinical trials of recombinant human CachectinTrNF are underway. Phase-I studies confirmed many of the observa­tions initially made in animals; profound systemic and organ specific toxicity has been observed, fever, rigors and myalgias develop frequently and dose limiting aftereffects include hypotension, increased fluid requirements and hepatotoxic­ity30 It remains to be established whether the toxicity of cachectin can be decreased without reducing any intrinsic antineoplastic efficacy. Trials of cachectin given in combina­tion with other cytokines are also in progress, but the evidence available suggest that the effects of cachectin/TNF in cancer are not specifically tumouricidal, but resemble the effects of bacterial endotoxin.
Parasitic diseases
In 1986, Scuderi and his co-workers for the first time measured the serum levels of tumor necrosis factor (TNFa) in healthy people and patients with neoplastic or infectious disease. Only patients with Kala-azar (visceral leishmaniasis) and malaria were found to have strikingly increased frequency of raised TNF levels. They found that 7.9% of samples from both healthy subjects and patients with neoplastic disease contained measurable TNF. The discovery of elevated TNF levels in the sera of patients with parasitic diseases suggests that this cytokine may play a part in host defence against parasitic infections34.
Malaria
Raised TNF levels were detected in patients with malaria in 1986 34. Since then, interest has focussed on the possible relation of TNF to severe and cerebral malaria. There is now considerable evidence linking TNF with various aspects of malarial pathology34. Recently more detailed studies have shown that serum TNF levels correlate with disease severity in malaria35. The authors also found very highly significant difference (P<0.00i) in TNF levels in severe malaria as compared to mild malaria (unpublished observations).
Several hypotheses have been proposed to explain the development of coma in cerebral malaria. The most important one is called cytokines hypothesis. It is based on the observation that in African children with cerebral malaria, plasma concentrations of TNF, interleukin-1 and other cytok­ines correlate with disease severity, asjudgedby parasitaemia, hypoglycemia, case fetality and the incidence of neurological sequelae. Cytokines, particularly tumor necrosis factor re­leased by macmphages under the influence of a malaria toxin Glycosyl phosphatidyl inositol (GPI) released at schizont rupture could be involved in enhancing cytoadherence by increasing the expression of endotheial receptors) such as intercellular adhesion molecule-i (ICAM-1), CD36 and can induce fever, hypoglycaemia, coagulopathy, dysexythropoi­esis and leukocytosis37.
Saissy and his co-workers compared the clinical and biologic aspects of adult severe falciparurn malaria, with those found in Children in West Africa (in malaria endemic area) and confirmed the prognostic significance of serum levels of TNF, IL-6 and IL- 2SR in severe malaria38. Kumaratilake and co-workers39 synthesized tumor necrosis factor, agonist peptide TNF (70-80) and demonstrated that it enhanced the human PMN-mediated killing of plasmodium falciparum in vitro and reduced the plasmodium chabaudi parasitaemia in mice. They concluded that the host protective effects of TNF can be retained while the toxic effects are eliminated using a selected, characterized subunit of the cytokine.
Treatment of malaria with neutralizing antibodies to TNF seems a distinct possibility, but., the role of antibodies, directed against other cytokines, can be of immense clinical importance.

References

1. Guy, MW. Serum and tissue fluid lipids in rabbits experimentally infected with trypanosoma brucci. Trans. R. Soc. Trop. Med.Hyg. 1975;69:429 abstract.
2. Kawakami, M. and Cerami, A. Studies on aidotoxin induced decrease in lipoprotein lipase activity J. Exp. Med., 1981,154:631-39.
3. Beutler, B., Mahoney, J., Trang, L.N. et al. Purification of Cachectin, A lipoprotein lipase suppressing hormone secreted by endotoxin induced RAW 264. 7 cells. J. Exp. Med. 1985;161 ;984-995.
4. Caput, D., Beutler, B., Hartog, K. et al. Identification of a common Nucleotide soquence in the 3*.untranslated region of MRNA molecules specifying inflammatory mediators. Pro. Nat. Acad. Sci. USA. 1986;83:1670-74.
5. Carswell, E.A. Old, Li, Kassel, R.L. et al, An endotoxin induced factors that causes necrosis of tumors. Proc. Nat. Acad. Sci. USA, 1975;72:3666-3670.
6. Traccy, K.J., Beutler, B., Lowry, S.F. ci at. Shock and tissue injwy induced by recombinant human Cachectin. Science, 1986;234:470-74,
7. Snaw, G., Kansas, RA. and Conserved, AU. Sequence from the 3’. untranslated region of GM-CSF MRNA mediates selective MRNA degradation. Cell, 1986;46:659-667.
8. Beutler, B.T., Kacenko, V., Milsak, I. et at, Effect of gamma interferon on Cachectin expression by mononuclear phagocytes. Reversal of Ipsd phenotype J.Exp.Med., 1986;164;1791-96.
9. Kriegler, M., Perez, C., Defay, K. et at. Novel form of TNF/Cachectin is a cell surface cytotoxic transmembrne protein ramifications of the complex physiol­ogyonTNF. Cell, 1988;53:45-53.
10. Schall, T.J., Lewis, M., Koller, K.J. Molecular cloning and expression of a receptor. Tumor necrosis factor. Cell, 1990;61 :361-70.
11. Cosman, 0. and Goodwin, R.G. A receptor for tumor necrosis factor dcfmes an unusual family of cellular and viral proteins. Science, 1990;248:1019-1023.
12. Tracey, K. I., Lowsy, S.F.. Beutler, B. et at. Tumor necrosis factor mediates changes of Skeletal muscle transmembranc potential J. Exp. Med., 1986;164;1368-73.
13. Fein-Gold, K.R. and Grunfeld, C. Tumor necrosis factor. alpha stimulates hepatic lipogenesis in the rat in Vivo J. Chin. Invest., 1987;80: 184-190.
14. Hesse, D.G. and Tracey, K.i. Cytokine appearance in human endotoxaemia and primate bacteremia, Surg. Gynecol. Obstet., (abstract) 1988;166:147-153.
15. Tracey, K.J., Beutler, B.. Lowry, S.F. et al. Shock and tissue injury induced by recombinant human Cachectin. Science, 1986;234:470-74.
16. Traccy, K.J. TNF and other Cytokines in the metabolism of septic shock and Cachexia. Clin. Nut. 1992;11:1-11.
17. Tracey, k.J., Vlassara, H. and Cerami, A. Cachectin tumor necrosis factor Lancet, 1989;1:1122-1126.
18. Tracey, KJ., Fong, Y. and Hesse, DO. Anticachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteramia. Nature, 1987;330:662-64.
19. Traccy, K.J.. Wei, H.. Manoguc, KR. et at. Cachectin/Tumor necrosis factor induces, Cachexia, anaemia and inflammation 3. Exp. Med., 1988; 167:1211-27.
20. Sherry, BA., Gelin, J. Fongy, A. er at. Anticachectin/Tumor necrosis factor antibodies attenuate development of Cachexia in tumor models FASEBJ, 1989;3:1956-62,
21. Stovroff,M.C,,Fraker,D.L.,Travis, W.D. etal, Altered macrophageactivity and tumor necrosis factor. J. Surg. Res., 1989;46:4.62-69.
22. Stames, HF.. Warren, R. S.. Jee-Vanandam, M. et at. Tumor necrosis factor and the acute metabolic response to tissue injury in man. J. Chin Invest., 1988;83:1321-1325.
23. Warren, R.S., Starnes, HF., Gabritove, J.L. et al. The acute metabolic effects of tumor necrosis factor administration in humans. Arch. Surg., 1987;122:1396-1400.
24. Spnggs, D.R., Sherman, M.L., Frei, E. et al. Clinical studies with tumor necrosis factor. Bock G. Marsh, J. cds. Tumor necrosis factor and related cytotokines (Ciba Foundation symposium 1310. Chichcstcr, England, wiley, 1988. pp. 206-227.
25. Michic, HR., Manogue, K.R., Spriggs, D.R. et at. Detection of circulating tumor necrosis factor after endotoxin administration N. EngI. J. Med., 1988;318:1481-1486.
26. Traccy, K.J. and Cerami, A. Tumor necrosis factor in the malnutrition (Cachexia) of infection and cancer. Ann. 1. Trop. Med. Hyg., 1992;47:2-7.
27. Spnggs, D.K.. Sherman, M.L. Sariban, E. et at. Tumor necrosis factor expression in human epithelial cell lines. J. Clin. Invest., 1988;81:455-60.
28. Vander-Meer, J.WM., Endres, S., Lonneman, 0. et at. Concentrations of immunorcactive tumor necrosis factor alpha produced by human mononuclear cells invitro. J. Leukoc. Bio., 1988;43:216-223.
29. Grau, G.E., Taylor,T.E., Molyneaux, M. et at. Tumor necrosis factor and disease severity in children with falciparum malaria. New Engl. J. Med., 1989;320:1586-91.
30. Gilles, H.M. and Warrel, D.A. In Bruce-Chwatts Ed Essential Malariology, London, Edward Arnold. 1993, pp. 52-53.
31. Saissy, J.M. and Vit-ris, M. Prognostic values of cytokines in severe adult and childhood malaria in seasonal endemic area in Africa. (Abstract) Press med., 1994;23: 1426.
32. Kumaratilake, L.M., Rathje,,D.A., Mack, P. et at. A synthetic tumor necrosis factor alpha agonist peptide enhances human polymorphonuclear leukocyte mediated killing of plasmodium falciparum in vitro and suppcresses, Plasmo­ dium chabaudi infection in mice. J. Chin. Invest. 1995;95:2315-2323.

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