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years, only comprehensive clinical trials will reveal the true medical significance of this family of cytokines. While emphasis has been placed thus far upon assessing therapeutic applications of individual ILs, it is likely that their use in combination with additional cytokines may display greater therapeutic advantage.
TUMOUR NECROSIS FACTORS (TNFs)
The tumour necrosis factor (TNF) family of cytokines essentially consists of two related regulatory factors: TNF-a (cachectin) and TNF-b (lymphotoxin). Although both molecules bind the same receptor and induce very similar biological activities, they display limited sequence homology. The human TNF-a and -b genes are located adjacent to each other on chromosome 6, being separated by only 1100 base pairs. Both contain three introns and their expression appears to be coordinately regulated. TNF-a, sometimes referred to simply as TNF, has been studied in significantly greater detail than lymphotoxin.
TNF-a is also known as cachectin, macrophage cytotoxic factor, macrophage cytotoxin and necrosin. As some of these names suggest, activated macrophages appear to represent the most significant cellular source of TNF-a, but it is also synthesized by many other cell types (Table 5.9). Producer cells do not store TNF-a, but synthesize it de novo following activation.
Human TNF-a is initially synthesized as a 233 amino acid polypeptide, which is anchored in the plasma membrane by a single membrane-spanning sequence. This TNF pro-peptide, which itself displays biological activity, is usually proteolytically processed by a specific extracellular metalloprotease. Proteolytic cleavage occurs between residues 76 (Ala) and 77 (Val), yielding the mature (soluble) 157 amino acid TNF-a polypeptide. Mature human TNF-a appears to be devoid of a carbohydrate component and contains a single disulphide bond.
Monomeric TNF is biologically inactive — the active form is a homotrimer in which the three monomers associate non-covalently about a three-fold axis of symmetry, forming a compact bell-shaped structure. X-ray crystallographic studies reveal that each monomer is elongated and characterized by a large content of anti-parallel b-pleated sheet, which closely resembles subunit proteins of many viral caspids (Figure 5.10).
A number of stimuli are known to act as inducers of TNF production (Table 5.10). Bacterial lipopolysaccharide represents the most important inducer, and TNF mediates the pathophysiological effects of this molecule. TNF biosynthesis is regulated by both transcriptional and post-transcriptional mechanisms. Macrophages appear to constitutively express TNF-a mRNA,
Table 5.9. The major cellular sources of human TNF-a. As is clearly evident, TNF-a synthesis is not restricted to cells of the immune system, but is undertaken by a wide variety of different cells in different anatomical locations, including the brain
Macrophages B and T lymphocytes
NK cells Polymorphonuclear leukocytes
Hepatic Kupffer cells Langerhans cells
Glomerular mesangial cells Brain microglial cells
Fibroblasts Various transformed cell lines
CYTOKINES: INTERLEUKINS AND TUMOUR NECROSIS FACTOR 247
Figure 5.10. 3-D structure of TNF-a. Photo from Eck & Sprang (1989), by courtesy of the Protein Data
Table 5.10. Major physiological inducers of TNF-a production
Lipopolysaccharide Bacterial enterotoxin
Mycobacteria Various viruses
Antibody-antigen complexes Various cytokines (e.g. IL-1)
Inflammatory mediators TNF-a (autocrine activity)
which is translated only upon their activation. After activation, the rate of gene expression may increase only three-fold, although cellular TNF-a mRNA levels may increase 100-fold and secretion of soluble TNF-a may increase 10000-fold.
Biological activities of TNF-a
The availability of large quantities of recombinant TNF-a facilitates rigorous investigation of the effects of this cytokine on cells in vitro, as well as its systemic in vivo effects. Like most cytokines, TNF-a exhibits pleiotropic effects on various cell types. The major biological responses induced by this cytokine include:
• activation of certain elements of both non-specific and specific immunity, in particular in response to Gram-negative bacteria;
• induction/regulation of inflammation;
• selective cytotoxic activity against a range of tumour cells;
• mediation of various pathological conditions, including septic shock, cachexia and anorexia.