What Is Tumor Necrosis Factor?

In 1975, EA Carswell et al. Discovered that after injection of BCG-injected mice with bacterial lipopolysaccharide, a substance that can cause hemorrhagic necrosis of various tumors appeared in the serum, and named it tumor necrosis factor (TNF). It was discovered in the 1980s that it played an important role in wasting disease, also known as cachexia. TNF is mainly produced by activated macrophages, NK cells and T lymphocytes. In 1985, Shalaby named TNF produced by macrophages as TNF- and lymphotoxin (LT) produced by T lymphocytes as TNF-. Although TNF- and TNF- are only about 30% homologous, they share a common receptor. The biological activity of TNF accounts for 70% to 95% of the total TNF activity, so TNF is often referred to as TNF-. The cloning of the TNF gene opened the era of clinical trials in 1984. It was the first cytokine to be used in tumor biotherapy, but because of its lack of targeting and severe side effects, it is currently only used for local treatment.

Zhang Qingyun	(Chief Physician)	Department of Laboratory Medicine, Peking University Cancer Hospital
Zhang Meng	(Doctor)	Department of Laboratory Medicine, Peking University Cancer Hospital
Wang Bing	(master's degree)	Department of Laboratory Medicine, Peking University Cancer Hospital

In 1975, EA Carswell et al. Discovered that after injection of BCG-injected mice with bacterial lipopolysaccharide, a substance that can cause hemorrhagic necrosis of various tumors appeared in the serum, and named it tumor necrosis factor (TNF). It was discovered in the 1980s that it played an important role in wasting disease, also known as cachexia. TNF is mainly produced by activated macrophages, NK cells and T lymphocytes. In 1985, Shalaby named TNF produced by macrophages as TNF- and lymphotoxin (LT) produced by T lymphocytes as TNF-. Although TNF- and TNF- are only about 30% homologous, they share a common receptor. The biological activity of TNF accounts for 70% to 95% of the total TNF activity. Therefore, TNF is often referred to as TNF-. The cloning of the TNF gene opened the era of clinical trials in 1984. It was the first cytokine to be used in tumor biotherapy, but because of its lack of targeting and severe side effects, it is currently only used for local treatment.

Western Medicine Name: Tumor necrosis factor

Affiliated Department: Internal Medicine-
Contagious: Non-contagious

Tumor necrosis factor alias

Cachectin

Macrophage cytotoxin

Necrosin

Cytotoxin

Tumor necrosis factor-

Hemorrhagic factor

Macrophage cytotoxic factor

Differentiation-inducing factor

Tumor necrosis factor source

Macrophages

Natural killer cells

T-lymphoblastoid Cells

B-lymphoblastoid Cells

Mast cells

Fibroblasts

Smooth muscle cells

Breast tumor cells

Ovarian tumour cells

Astrocytes

L-929 cells

Kupffer's cells

Epidermal cells

Granulosa cells

TNF Characteristics of tumor necrosis factor TNF gene

The human TNF- gene was successfully cloned in 1985, located at 6p21.4, about 3.6 kbp in length, and has 4 exons and 3 introns. It is tightly linked to the major histocompatibility complex (MHC) gene. The MHC3 gene region between HLA-B and HLA-C2 loci consists of TNFA and TNFB, which encode TNF and TNF, respectively. Single nucleotide polymorphisms at positions 238 and 308 in the promoter region are believed to regulate the transcription level of TNF and are related to the susceptibility to chronic diseases such as chronic hepatitis B, autoimmune diseases, insulin resistance, and tumors. The TNF gene encodes about 1.7 kbp of mRNA, and in its 3 untranslated region, there is a conserved TATTTTAT sequence (AU-rich element, ARE) that many cytokines have.

Phorbol ester is an inducer of TNF, which can induce the transcription of TNF through a short sequence near the TATAA box of the promoter region.

TNF Characteristics of tumor necrosis factor TNF protein

1 Human TNF- precursor is composed of 233 amino acids (26 kDa), which contains a signal peptide consisting of 76 amino acid residues. Under the action of the TNF converting enzyme TACE, the signal peptide is excised to form a mature 157 amino acid residue TNF- (17 kDa). Because there are no methionine residues, there is no glycosylation site, in which the two cysteines at positions 69 and 101 form an intramolecular disulfide bond. Human TNF- has 79% amino acid composition homology with mouse TNF-, and the biological effect of TNF- does not seem to have obvious species specificity. Recently, it has been reported that 155 amino acid human TNF- with less 2 amino acids (Val, Arg) at the N-terminus is expressed by genetic engineering technology, which has better biological activity and antitumor effect. In addition, there are genetic engineering methods that delete 7 amino acid residues at the amino terminal of the TNF- molecule, and then change 8Pro, 9Ser, and 10Asp to 8Arg, 9Lys, and 10Arg, or change 157Leu to 157Phe at the same time. TNF- has a 1000-fold increase in killing L929 cells in vitro compared to natural TNF, and the tumor hemorrhagic and necrotic effects in vivo are also significantly increased. The natural forms of TNF- and exerting biological effects are homotrimers.

2 The human TNF- molecule is composed of 205 amino acid residues and a signal peptide containing 34 amino acid residues. The mature TNF- molecule is 171 amino acid residues and has a molecular weight of 25 kDa.

3 Human TNF- and TNF- have 56% DNA homology and 36% homology at the amino acid level. X-ray crystal diffraction technology was used to prove that TNF is a dense trimer composed of three identical monomer subunits. The monomer subunits are wedge-shaped, and the -sheet structure is formed by -sheet folding.

TNF Tumor necrosis factor TNF receptor

1.1 Typing of TNF-R

TNFR can be divided into two types

Type I TNF-R (also known as TNFR1, CD120a, p55), 439 amino acid residues, 55kDa, its corresponding mRNA is 4.5Kbp, can be expressed on all types of cells, and plays a major role in cytolytic activity.

Type II TNFR (also known as TNFR2, CD120b, p75) has 426 amino acid residues and 75kDa, and its corresponding mRNA is 3Kbp, which is only expressed on immune and endothelial cells, which is related to signal transmission and T cell proliferation.

1.2 Structural and functional characteristics of TNFR

Both types of TNFR are glycoproteins, including the extracellular, transmembrane and intracellular regions. The extracellular region is 28% homologous, but there is no homology in the cytoplasmic region, which may be related to mediation. Different signal transduction pathways are involved. Several studies have confirmed that tumor necrosis factor exerts its biological activity mainly through interaction with TNF-R1. TNF protein combined with the extracellular domain of TNF-R1 induces TNF-R1 aggregation and releases death domain silencers (SODD), and then TRAD combines with the death domain in TNF-R1 to recruit more adapter proteins, such as RIP, TRAF -2 and FADD etc. These adapter proteins recruit other important proteins involved in signal transduction to play a role.

At present, little is known about the structure and function of TNF-R2, but it lacks a death domain and therefore cannot promote the process of apoptosis. However, TNF-R2 can interfere with programmed cell death (PCD) by activating the NF-B and JNK pathways or inhibiting TRAF-2.

1.3 Distribution of TNFR

TNFR exists on the surface of a variety of normal and tumor cells. Generally, the number of receptors per cell is 500 ~ 5000 / cell. For example, ME-800 tumor cell line TNFR is about 2000 / cell, and Kd is 2 × 10-10M. The number and affinity of TNFR on different cell surfaces do not seem to be parallel to the cell's sensitivity to TNF-.

1.4 Soluble TNFR

TNF-binding protein (TNF-BP) is a soluble form of TNFR. There are sTNFR (TNF-BP) and sTNFR (TNF-BP). It is generally believed that sTNFR has the function of limiting TNF activity or stabilizing TNF, and has an important regulatory role in the cytokine network. Seckiner found in 1988 that TNF inhibitors were present in the urine of fever patients with a molecular weight of 33 kDa. Olsson also found TNF-BP in the blood and urine of patients with chronic renal insufficiency in 1989. TNF-BP can specifically bind to TNF, inhibit TNF activity, such as inhibiting its cytotoxic activity and inducing the production of IL-1, can promote the growth of Meth A virus subcutaneously inoculated, which may be one of the mechanisms of tumor escape host anti-tumor. TNF-BP in normal human serum is 1-2 ng / ml, which can also be found in normal pregnancy urine. Inflammation, endotoxemia, meningococcal infection, SLE, HIV infection, renal insufficiency, and tumors can be elevated. Soluble TNFR can effectively reduce the pathological changes of adjuvant arthritis and septic shock.

TNF Tumor necrosis factor TNF receptor superfamily

Researchers have now confirmed the existence of 29 different receptors in the tumor necrosis factor protein homofamily superfamily (Figure 1). These receptors can be divided into three main groups:

The first type of receptor contains a death domain (DD) in the cytoplasmic tail. Receptors containing death domains can be recruited by binding ligands to corresponding death domain-containing receptors, such as Fas-related death domain protein (FADD / MORT1) and TNF receptor-related death domain protein (TRADD ), Which together form the so-called death-inducing signal complex (DISC). These molecules cause caspase activation and induce apoptosis, but can also recruit family members of the TNF receptor-related factor (TRAF).

The second type of receptor contains one or more TRAF interaction motifs (TIM) in the cytoplasmic tail region. Activation of such receptors can directly recruit members of the TRAF family and eventually activate key molecules of multiple signal transduction pathways, such as mitogen-activated protein kinase (MAPK) (such as c-Jun N-terminal kinase JNK), P38 (P38MAPK), Extracellular signal-regulated kinase (ERK), nuclear factor kappa-B inhibitor kinase (IKK), and phosphatidylinositol 3-kinase (PI3K).

The third type of receptor does not contain functional intracellular signaling domains or motifs. Although these "bait" receptors are not involved in intracellular signaling, they can compete with the other two groups of receptors to bind to the corresponding ligands. ^[1]

TNF Biological activity of tumor necrosis factor TNF

The biological effects of TNF- and TNF- are very similar, which may be related to the similarity of molecular structure and the identity of the receptor. But there are also differences in some biological effects.

1.1 Kill or inhibit tumor cells

TNF can kill certain tumor cells in vivo and in vitro, or inhibit proliferation. Tumor cell lines have very different sensitivities to TNF-, and TNF- even stimulates a small number of tumor cells. Treating tumor cells (such as mouse fibroblast cell line L929) with actinomycin D, mitomycin C, and actinomycin can significantly increase the tumor cell killing activity of TNF-. The tumor response to TNF- in vivo is also very different, and it is not parallel to the sensitivity of TNF- to cell lines in vitro. The same cell line may have sensitive and resistant strains such as L929-S and L929-R. In addition, the expression of endogenous TNF in target cells may make the cells resistant to the cytotoxic effects of exogenous TNF, so the sensitivity of the cells to exogenous TNF can be changed by inducing or inhibiting the expression of endogenous TNF. Macrophage-bound TNF may be involved in killing target cells.

The mechanism of TNF killing tumors is not very clear. Compared with complement or perforin killer cells, TNF killer cells have no perforation, and the killing process is relatively slow. TNF killing tumor tissue cells may be related to the following mechanisms.

1.1.1 Direct killing or inhibiting effect. After binding to the corresponding receptor, TNF moves into the cell and is taken up by the target cell's lysosome, resulting in a decrease in lysosomal stability, leakage of various enzymes, and cell lysis. It is also believed that TNF activates phospholipase A2, releases superoxide and causes DNA breakage, and phospholipase A2 inhibitors can reduce the anti-disease effect of TNF. TNF can alter the glucose metabolism of target cells, reduce the intracellular pH, and cause cell death.

1.1.2 Promote the killing of tumor cells by T cells and other killer cells through the regulatory effect of TNF on the body's immune function.

1.1.3 TNF acts on vascular endothelial cells, damages endothelial cells or causes vascular dysfunction, causes vascular injury and thrombosis, causes local blood flow blockage in tumor tissues, and causes bleeding and hypoxia and necrosis.

1.2 Improve the phagocytic ability of neutrophils, increase the production of peroxide anions, enhance ADCC function, stimulate cells to degranulate and secrete myeloperoxidase. TNF cultured with endothelial cells in advance can increase the expression of MHC class I antigen, ICAM-1, the secretion of IL-1, GM-CSF and IL-8, and promote the adhesion of neutrophils to endothelial cells, thereby stimulating the body Local inflammatory response, this induction of TNF- is stronger than TNF-. TNF stimulates monocytes and macrophages to secrete IL-1 and regulates the expression of MHC class II antigens.

1.3 Anti-infection: Such as inhibiting the growth of Plasmodium, inhibiting virus replication (such as adenovirus type II, herpes virus type II), inhibiting viral protein synthesis, virus particle production and infectivity, and killing virus-infected cells. The antiviral mechanism of TNF is not very clear.

1.4 TNF is an endogenous pyrogen that causes fever and induces protein synthesis in the acute phase of liver cells. The fever caused by TNF may be caused by directly stimulating the hypothalamus to question the regulatory center and stimulate the release of IL-1 by macrophages. It can also stimulate other cells to produce IL-6 through IL-1 and TNF-.

1.5 Promote the differentiation of myeloid leukemia cells into macrophages, such as promoting the differentiation of myeloid leukemia cells ML-1, monocyte leukemia cells U937, and promyelocytic leukemia cells HL60, the mechanism is unclear. TGF- can inhibit a variety of biological activities of TNF-, but it is not consistent with the induction of myeloid leukemia cells by TNF-, and even has a synergistic effect.

1.6 Promote cell proliferation and differentiation: TNF promotes the expression of T cell MHC class I antigens, enhances the proliferation of IL-2 dependent thymocytes and T cells, promotes the poor differentiation of lymphokines such as IL-2, CSF and IFN-, and enhances mitogen or foreign Antigens stimulate B cell proliferation and Ig secretion. TNF- has growth factor-like effects on certain tumor cells, and cooperates with the proliferation-promoting effects of EGF, PDGF and insulin, and promotes the expression of EGF receptors. TNF can also promote the expression of proto-oncogenes closely related to cell proliferation, such as c-myc and c-fos, causing the cell cycle to change from G0 to G1. Recently, TNF- (LT) is an autocrine growth factor of EB virus-transformed lymphoblasts. Anti-LT antibodies, sTNFR and TNF- have been able to proliferate EB virus-transformed lymphocytes.

IL-1, IFN- and GM-CSF have obvious enhancement effects on the biological effects of TNF, which may be related to increasing the expression of TNF receptors in cells. An anti-TNF- monoclonal antibody has been reported to mimic certain biological effects of TNF-, a phenomenon that has not been seen in other factors. ^[2]

TNF Tumor necrosis factor TNF and clinical

1.1 Application in tumor treatment

TNF has demonstrated antitumor or antitumor activity and immunomodulatory activity in human, mouse tumor cell lines or primary cultured human cancer cells, as well as in tumor-bearing nude mice. The application of TNF in the treatment of tumors is mostly in the clinical trial stage. It can also be used in combination with IL-2 to treat tumors. At present, systemic drugs are not as effective as local drugs. The latter, such as intra-focal injection, have high local concentrations and mild side effects. . In recent years, TNF gene therapy has been used for clinical verification of tumors such as melanoma. In addition, intra-pleural administration of TNF can significantly reduce or completely disappear cancer cells in the pleural fluid of patients with metastatic gastric cancer and breast cancer.

In 2003, domestically, the world's first mutant human recombinant tumor necrosis factor (nrhTNF) was approved for production.

1.2 Septic shock

At present, it is believed that diffuse intravascular coagulation and toxic shock caused by gram-negative bacilli or meningococci are caused by bacterial endotoxins that stimulate the body to produce excessive TNF-, which causes fever, severe damage to the heart and adrenals, respiratory failure, and even causes respiratory failure. Death, and its TNF level is positively correlated with mortality. The pathogenesis may be that TNF stimulates endothelial cells, leading to inflammation, tissue damage and coagulation. TNF is also an important factor in acute liver necrosis. Viral fulminant liver failure induces TNF and IL-1 activity in peripheral blood cells, and is related to the severity of the disease. The mechanism of TNF-mediated endotoxin shock is not clear at present. It is believed that TNF can promote the production of IL-1 and leukotriene by phagocytes and endothelial cells, leading to DIC and endotoxin shock. TNF antibodies (antiserum or monoclonal antibodies) effectively prevent lethal endotoxin shock in mice, rabbits and baboons.

1.3 cachexia

TNF-, also known as cachexia, can induce cachexia in the body.

1.4 Relationship between TNF and pathogens

TNF also has an anti-viral effect similar to IFN, preventing the synthesis of early proteins of the virus, thereby inhibiting the replication of the virus, and synergizing the anti-viral effect with IFN- and IFN-. On the other hand, TNF induces the expression of the HIV-1 gene in T cells. TNF activates or induces NF-b in HIV-infected CD4 + cells. NF-B binds to the enhancer site of HIV's long terminal repeat (LTR) and activates HIV genes, which may be related to the pathogenesis of AIDS. The production of TNF- in monocytes of AIDS patients increased, and the level of TNF- in serum increased. In addition, TNF also shows antibacterial and antimalarial effects.

1.5 Relationship to Rheumatoid Arthritis

TNF can be detected in synovial fluid of patients with rheumatoid arthritis, which is thought to be related to the onset of arthritis. Many anti-inflammatory drugs can reduce TNF production. There are currently TNF antagonists on the market, such as etanercept, which can be used for the treatment of active rheumatoid joints, active ankylosing spondylitis and so on. ^[3-4]