TNF-alpha

See also TNF generally

See also TNF-alpha regulation

What does TNF-alpha do?

TNFα is a proinflammatory cytokine. Although it is important for immune response against pathogens, it can cause many pathogenic responses such as septic shock, cachexia and rheumatoid arthritis when overproduced. Inhibition of TNF-alpha is thus a therapeutic option.

Originally, TNFα was identified as a macrophage produce mediating cytotoxicity against certain cell types, especially transformed cell lines. It also activates neutrophils and T cells at a low concentration. At high concentrations they activate an IL-1 & Il-6 cascade and result in cachexi (wasting). TNF-alpha enhances the phagocytic and microbicidal activities of macrophages and stimulates the production of other proinflammatory cytokines including IL-1 and IL-6. Mice lacking TNF-alpha or TNFRs are acutely susceptible to intracellular infections.

Tumor necrosis factor-α (TNF) is a cytokine that induces programmed cell death, apoptosis, in a number of cell types and is also employed by cytotoxic T cells to eliminate virus infected cells. The stimulation of apoptosis by TNF in many cell types in vitro often requires inhibition of protein synthesis.

TNFalpha induces the expression of IFNy and IL-12 through activation of the NF-kB pathway. TNF induces transient phosphorylation of the p65 subunit of NF-kB leading to its association with the p50 subunit and translocation to the nuclues, where the heterodimer transactivates target genes by assocaition with the p300 transcriptional co-activator.

Organization of the TNF-alpha Gene, protein and its receptors

TNFα coding regions consist of 4 exons arranged over about 3 kb of DNA. More than 80% of the sequence contributing to mature TNF is encoded in the fourth exon, while exons I and II contain almost entirely leader peptide sequence. The TNF amino acid sequence is conserved between species. The region corresponding to residues 114 to 130 is most highly conserved, suggesting that a functionally important structure is encoded there.

TNF is initially synthesized as a 26-kDa membrane associated protein which is cytotoxic only when associated with membrane components as microsomes. This protein is cleaved to form the secreted 17-kDA TNF protein by metalloproteinase TNFα converting enzyme.

TNF can use 2 receptors called TNFRI (p55) and TNFRII (p75).

Activation of TNF-alpha

Induction of TNF can occur through a variety of stimuli, including LPS, TPA, cytokines, calcium flux, and oxygen free radical mechanisms. A common pathway for these diverse agents remains unknown. The activation of monocytes by LPS may involve a number of signal transduction pathways, including phospholipase C, phospholipase A2, protein kinase C, calcium flux and cyclic nucleotides.

TNFα is also induced by dsRNA (as by viral infection). This requires protein kinase R (PKR) activation (interesting induction of IL-1β induction follows a PKR-independent pathway.

TNF induces its own expression at both the RNA and protein levels in HL-60 cells.

TNF and IL-1 are frequently induced by the same stimuli and appear to have regulatory mechanisms that are similar but not identical.

What signalling pathways does TNF-alpha Activate itself?

Activation of TNFR members like TNFα results in constitutive activation of nuclear factor-kB (NF-kB). A functional NF-kB molecule is a heterodimer composed of members of the Rel family of proteins, which includes RelA (p65), RelB, c-Rel, p50 and 52. The major form of NF-kB that is rapidly induced after sitmulation is the RelA/p50 complex and this dimer is what is commonly referred to as NF-kB. NF-kB is maintained in an inactive form in the cytoplasm by the inhibitor IkB, which binds to NF-kB and masks its nuclear localization signal. There are several IkB protiens that are regulated differently and have different affinities for individual NF-kB complexes. IkB-alpha is the best characterized. Following activation of NF-kB through TNFR family members, TRAF molecules (adaptor proteins) trigger the activation of the IkB kinase complex (IKK) which then phosphorylates 2 serine resides in the N-terminus of IkB, allowing for the ubiquitinization and degradation of IkB and release of NF-kB. NF-kB is then able to translocated tot he nucleus where it stimualtes the transcription of a wide variety of genes, including cytokines, cell adhesion moelcules, and acue pahse response proteins, which are involved in proliferation and survival as well as the inflammatory response.

 Cells which produce TNFα        See also cells which produces cytokines

TNFα is produced and secreted by granuloctyes, macrophages (primary producer), fibroblasts, and epithelial cells as well as lymphocytes.

Murine peptone-induced peritoneal macrophages show an optimal response to LPS at a concenration of 10 ng/ml and an inhibition of TNFα at higher concentrations.

HL-60 cells have been exposed to VitD3 for 4 d, cells harvested and TNFα mRNA measured by Northern blots. There is a 7.5 fold accumulation of TNFα mRNA in VitD3-stimulated cells compared with unstimulated cells.