cell factor

Small molecule protein

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This entry is made by Compilation and application of scientific encyclopedia entries of "Science Popularization China" to examine.

from Immune cell (such as single core macrophage 、 T cell 、 B cell , NK cells, etc.) and some non immune cells（ endothelial cells 、 Epidermal cell 、 Fibroblast It is a kind of small molecule protein with wide biological activity that is synthesized and secreted by stimulation. Cytokines generally regulate cell growth, differentiation and effect by binding to corresponding receptors, and regulate immune response. Cytokine (CK) is Immunogen , mitogen or other stimulators induce a variety of cells to produce low molecular weight soluble proteins, which can regulate innate immunity and adaptive immunity, hematopoiesis, cell growth APSC pluripotent cell And repair of damaged tissues. Cytokines can be divided into interleukin, interferon, tumor necrosis factor superfamily Colony stimulating factor 、 Chemotactic factor , growth factors, etc.

Chinese name: cell factor
Foreign name: cytokines,CK

Essence: Small molecule protein
Features: Multi efficiency, overlap, synergy, etc

catalog

one brief introduction
two Restructuring requirements and status
three classification
four recipient
▪ concept

brief introduction

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Cytokine (CK) is composed of Histiocyte Small molecular polypeptides or glycoproteins (mainly immune cells) synthesized and secreted. Cytokines can mediate the interaction between cells and have a variety of biological functions, such as regulating cell growth, differentiation and maturation, function maintenance, regulating immune response, participating in inflammatory reaction, wound healing and tumor growth and decline. At first, people did not know the essence of cytokines, so they named them according to their biological activities. As a result, the same cytokine has multiple names. Later, people realized that cytokines are mainly synthesized by white blood cells and mainly mediate the interaction between white blood cells. Therefore, these factors were uniformly named as interleukin (IL), and they were named with Arabic numerals in the order of discovery, such as IL-l, IL-2, IL-3, etc. Since the discovery of interferon by Lssac in 1957, more than 200 cytokines have been discovered so far. All interleukins, interferons, tumor necrosis factors, hematopoietic factors, growth factors, chemokines, etc. are collectively referred to as cytokines. Modern genetic engineering and Cell engineering technology The rapid development of cytokines has provided technical conditions for discovering more cytokines and studying their structure and function. The research achievements of cytokines have provided a scientific basis for clinical prevention, diagnosis and treatment of diseases. In particular, the use of cytokines to treat tumors, infections, hematopoietic disorders, autoimmune diseases, etc., has a very broad application prospect^[1] 。

Cytokines（ cytokine，CK） It is immunogen Mitogen Or other stimulators to induce a variety of cells to produce low molecular weight soluble protein It has multiple functions such as regulating innate immunity and adaptive immunity, blood cell generation, cell growth, and repair of damaged tissues. Cytokines can be divided into interleukin, interferon, tumor necrosis factor superfamily, colony stimulating factor, chemotactic factor, growth factor, etc. Many cytokines play a role in the body through paracrine, autocrine or endocrine, and have multiple physiological characteristics, such as pleiotropy, overlap, antagonism, synergy, etc., forming a very complex cytokine regulation network and participating in many important physiological functions of the human body.

Action mode of cytokines

Restructuring requirements and status

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Requirements for recombinant cytokines:

1. Authenticity: The recombinant protein product has been subjected to N-terminal amino acid sequence analysis consistently; If necessary, SDS-PAGE, RP-HPLC and mass spectrometry (MS) shall be used to detect its authenticity.

2. Purity: SDS-PAGE and RP-HPLC methods shall be used to analyze the product purity.

three biological activity : Carry out corresponding in vitro or in vivo activity tests.

4. Protein content: detected by UV spectrum analysis and SDS-PAGE electrophoresis; If necessary, use HPLC quantitative standard protein solution.

five endotoxin : Kinetic LAL method was used to detect endotoxin.

6. Microorganism: The recombinant protein is filtered and sterilized before bottling.

Status of recombinant cytokines:

Mainstream in the market Recombinant protein Products, mostly excluding Carrier protein Or other additives (such as BSA, HAS, sucrose, etc, Make the protein components that may be attached to the pipe cover or pipe wall gather at the bottom of the freeze-drying chamber (it is normal to see white precipitates at this time).

classification

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（1） Classification according to the types of cells producing cytokines

cell factor

1. Lymphokine, named after, is mainly produced by lymphocytes, including T lymphocytes B lymphocytes and NK cells. Important lymphatic factors include IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-14, IFN- γ、 TNF- β、 GM-CSF and Neuroleukin Etc.

2. Monokine mainly consists of monocyte or macrophage Generated, such as IL-1, IL-6, IL-8, TNF- α、 G-CSF, M-CSF, etc.

3. Not lymphocyte The cytokines produced by non monocyte macrophages are mainly composed of stromal cells in bone marrow and thymus vascular endothelial cell , fibroblasts and other cells, such as EPO, IL-7, IL-11, SCF, endothelial cell derived IL-8 and IFN- β Etc.

（2） Classification according to the main functions of cytokines

1. Interleukin (IL) was named in 1979. Cytokines produced by lymphocytes, monocytes or other non mononuclear cells play an important role in the regulation of cell interaction, immune regulation, hematopoiesis and inflammation. The cDNA gene cloning and expression of all named interleukins have been successful, and more than 30 kinds (IL-1-IL-38) have been reported.

two Colony Stimulating factor (CSF) stimulates according to different cytokines Hematopoietic stem cells Or hematopoietic cells at different stages of differentiation Semi solid medium Different cell colonies were formed in, which were named G (granulocyte) - CSF, M (macrophage) - CSF, GM (granulocyte, macrophage) - CSF, Multi (multiple) - CSF (IL-3), SCF, EPO, etc. Different CSFs can stimulate different Developmental stage The proliferation and differentiation of hematopoietic stem cells and progenitor cells can also promote the function of mature cells.

three interferon (interferon, IFN) Cytokines discovered in 1957 were originally found that cells infected by one virus can produce a substance that can interfere with the infection and replication of another virus, hence the name. According to the source and structure of interferon production, it can be divided into IFN- α、 IFN- β And IFN- γ， They are produced by white blood cells, fibroblasts and activated T cells, respectively. The biological activities of different IFNs are basically the same, with antiviral, anti-tumor and immunomodulatory effects.

cell factor

4. Tumor necrosis factor (TNF) is named after the discovery that this substance can cause tumor tissue necrosis. It can be divided into TNF according to its source and structure- α And TNF- β The former is produced by monocyte macrophages, and the latter is produced by activated T cells, also known as Lymphotoxin （lymphotoxin, LT）。 The basic biological activities of the two types of TNF are similar, except that they have Killing tumor cells In addition, immune regulation and participation in fever and inflammation occur. High dose TNF- α Can cause cachexia, so TNF- α also called Cachexia （cachectin）。

five Transforming growth factor- β Transforming growth factor- β family, TGF- β Family) is produced by a variety of cells, mainly including TGF- β 1、TGF- β 2、TGF- β 3、TGF β one β 2 and Bone morphogenetic protein (BMP), etc.

6. Growth factor (GF), such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), fibroblast growth factor (FGF) Hepatocyte growth factor （HGF）、 insulin IGF-1, IGF - Ⅱ Leukemia inhibitory factor (LIF), nerve growth factor (NGF), tumor suppressor M (OSM), platelet derived endothelial growth factor (PDECGF) Transforming growth factor- α （TGF- α）、 Vascular endothelial growth factor (VEGF), etc.

7. Chemokine family includes four subfamilies: (1) C-X-C/ α Subspecies, major chemotaxis Neutrophils The main members are IL-8, melanoma cell growth stimulating activity (GRO/MGSA) Platelet factor -4 (PF-4), platelet basic protein, CTAP - Ⅲ and β- Thromboglobulin, inflammatory protein 10 (IP-10), ENA-78; （2）C-C/ β Subgroup, mainly chemotactic monocytes, whose members include macrophage inflammatory protein 1 α （MIP-1 α）、 MIP-1 β、 RANTES、 Monocyte chemotactic protein -1 (MCP-1/MCAF), MCP-2, MCP-3, and I-309. (3) Type C subfamily is represented by lymphocyte chemotactic protein. (4) CX3C subfamily, Fractalkine is a CX3C chemokine, which has chemotaxis on monocyte macrophages, T cells and NK cells.

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concept

Cytokines are polypeptide molecules that are produced by a variety of cells and have a broad role in regulating cell function. Cytokines not only act on immune system And hematopoietic system, and also widely acts on nerves endocrine system It plays an important role in regulating the interaction between cells, cell proliferation, differentiation and effect function. Cytokines play a wide variety of biological functions by binding to receptors on the surface of target cell membrane and transmitting signals to the cell interior. Therefore, understand Cytokine receptor Its structure and function are essential for further study of the biological function of cytokines. With the in-depth study of cytokine receptors, it is found that the cytokine receptors are different Subunit There is a shared chain phenomenon, which provides a basis for clarifying the similarity and difference of biological activities of many cytokines at the receptor level. Most cytokine receptors exist in soluble form. Mastering the production rule of soluble cytokine receptors and their physiological and pathological significance will certainly expand people's understanding of the role of cytokine networks. Detection of cytokines and their receptors has become an important aspect of basic and clinical immunology research.

classification

1、 Structure and classification of cytokine receptors

According to the homology and structure of the cDNA sequence of the cytokine receptor and the amino acid sequence of the extracellular domain of the receptor, the cytokine receptor can be divided into four types: Immunoglobulin superfamily （IGSF）、 haemopoietic cytokine receptor superfamily Nerve growth factor receptor superfamily and chemokine receptor. In addition, the structure of some cytokine receptors has not been fully understood, such as IL-10R, IL-12R, etc; Although the structure of some cytokine receptors has been clarified, they have not been classified, such as IL-2R α Chain (CD25). （1） Immunoglobulin superfamily

All members of this family have one or more immunoglobulin (Ig) - like structure. It is known that IL-1RtI (CD121a), IL-1RtII (CD121b), IL-6R of cytokine receptors belonging to IGSF members α Chain (CD126), gp130 (CDw130), G-CSFR, M-CSFR (CD115), SCFR (CD117) and PDGFR, and can be divided into several different structure type , different IGSF structure types recipient Their signal transduction pathways are also different.

(1) M-CSFR, SCFR and PDGFR: 5 Ig like products are found in the extracellular domain Domain There is one V-like structure near the cell membrane area, and the other four are C2 like structures. The receptor is usually in the form of dimer with the corresponding Homodimer Ligand binding. Receptor cytoplasmic region It contains protein tyrosine kinase (PTK) structure.

(2) IL-1RtI and IL-1RtII: The extracellular region contains three C2 like structures, and the receptor cytoplasmic region serine /Threonine Phosphorylation It may be related to receptor mediated signal transduction.

（3）IL-6R α Chain, gp130 and G-CSFR: there is a C2 like region at the N end of the extracellular region, and there is one on the side near the membrane red blood cell poietin Receptor superfamily In addition, there are 2-4 fibronectin domains in the extracellular domain. Tyrosine phosphorylation in gp130 cytoplasm is related to signal transduction. such structure type The amino acid sequence and molecular structure of IL-6, OSM, LIF and G-CSF ligands are also very similar.

(II) haemopoietic cytokine receptor superfamily

Hematopoietic cytokine receptor superfamily cytokine receptor family (Cytokine receptor family), which can be divided into erythropoietin receptor superfamily (ERS) and interferon receptor family.

1. The extracellular area of all ERSERS members Erythropoietin The extracellular domain of (EPO) receptor has high homology in amino acid sequence and molecular structure, so it is named.

(1) ERS members: members belonging to ERS include EPOR Thrombopoietin R、IL-2 β Chain (CD122), IL-2R γ Chain, IL-3R α Chain (CD123), IL-3R β、 IIL-4R（CDw124）、IL-5R α Chain, IL-5 βα Chain, IL-5R β Chain, IL-6R α Chain (CD126), gp130 (CDw123), IL-7R, IL-9R, IL-11R, IL-1240kDa Subunit 、G-CSFR、GM-CSFR α Chain, GM=CSFR β Chain, LIFR, CNTFR, etc. In addition, some hormones such as growth hormone Receptor (GRGR) and Prolactin Receptors (PRLR) also belong to ERS.

(2) The structural characteristics of ERS: the erythropoietin receptor superfamily members have a 210 Amino acid residue The main characteristics of the homologous region are as follows: ① There are four high and conservative homologous regions near the N end Cysteine Residues Cysl, Cys2, Cys3, Cys4 and a conservative palladium amino acid form two disulfide bonds between Cys1 and Cys2, and between Cys3 and Cys4. ② The homologous region is close to the cell membrane, and there is a tryptophan serine X at the 18-22 amino acid base outside the cell membrane- Tryptophan -Serine Motif The so-called Trp Ser Xaa Trp Ser is the WSXWS motif, and its biological function is still unknown. IL-3 α Chain, IL-3R β Chain, GM-CSFR β The chain and LIFR office have two ERS Domain , where GM-CSFR β The first ERS structure of the chain has a WSXWS like motif, namely proline - serine -Lysine tryptophan serine (PSKWS) motif. In 1994, Hilton et al. synthesized the corresponding oligonucleotide of WSXWS motif as a probe CDNA Library IL-11 Receptor of Middle Clone Mice α Chain cDNA was successful. IL-6R α The chain and gp130 and G-CSFrN end have an IGSF structure. Only Cys1 and Cys3 are located near the N-terminal side of IL-7R. Compared with other members, IL-7R lacks Cys2, Cys4 and tryptophan residues. IL-1240kDa Subunit It has the homologous structure of ERS, but is not membrane bound, and forms heterodimer with another 35kDa subunit of IL-12 through disulfide bond. The GM CSFrN terminal in ERS can be regarded as composed of two type Ⅲ fibronectin, each type Ⅲ fibronectin Domain Anti parallel by 7 strands β fold The strand forms a barrel structure, and the groove between the two barrel structures is the conservative region outside the membrane of the ligand, which has obvious evolution Homology The degree of homology is similar to that among IGSF members. The EPoR like family has higher homology with other family members and may play a dominant role in evolution. The cytoplasmic regions of ERS vary in length from 54 Amino acid residue To 568 amino acid residues, except IL-2R β In addition to certain homology in the cytoplasmic region between the chain and EPOR, other members have no obvious homology in the cytoplasmic region. The cytoplasmic regions of ERS members do not have PTK structure, and their signal transduction pathways and mechanisms are different. IL-2R β The Local Duality Region of Chain Cytoplasmic Region and Tyrosine kinase The serine rich region is related to the non kinase dependent pathway. IL-2R γ The chain cytoplasmic region has SH2 structure and participates in signal transmission. PTK and PKC in cytoplasm may participate in IL-4R mediated signal transduction. Serine rich region and tyrosine rich region of gp130 cytoplasmic region Phosphorylation It is related to gp130 mediated signal transduction. In addition, Tyrosine phosphorylation And IL-7R, GM-CSFR β Chain, IL-3R β Chain, IL-5R β Chain mediated signal transduction.

2. IFN is a member of the interferon receptor family- α/β R、IFN- γ R and tissue factor (TF) Cell membrane receptor ）Its structure is similar to that of erythropoietin receptor family, but there are only two Conservatism Cys, there are 7 amino acids between two Cys. There are also two conservative Cys near the membrane, and there are 20-22 amino acids between the two Cys. IFN- α/β R consists of two Domain Consists of.

（3） Nerve growth factor Receptor superfamily

1. The members of the NGFR superfamily belong to this family. In addition to the nerve growth factor receptor NGFR, there are TNF-R Ⅰ (CD120a), TNF-R Ⅱ (CD120b), CD40, CD27, T-cell cDNA-41BB coding product, rat T-cell antigen OX40 and human myeloid cell surface activation antigen Fas (CD95).

2.NGFR Superfamily The structural characteristics of the NGFR superfamily members include 3-6, about 40 amino acid The Cys rich region, such as NGFR, TNF-R Ⅰ and TNF-R Ⅱ, consists of four Domain CD95 has three domains and CD30 has six domains. The first region at the N-end of all members contains six conservative Cys and one Tyr, Gly and Thr residue respectively, and other regions also contain 4-6 Cys. The cytoplasmic region between TNF-R Ⅰ, CD95 and CD40 molecules is about 40-50% Homology 。

（4） Chemokine receptor

Since the successful cloning of IL-8 gene in 1988 Chemotactic factor (chemokine). So far, there are at least 19 members of chemokine family. The receptors of some chemokines have been basically clarified, and they all belong to G protein coupling GTP binding protein coupled receptor Transmembrane area , also known as 7 membrane penetration areas Receptor superfamily （sevenpredicatedtransmembranedomainreceptorsuperfamily,STRsuperfamily）。 G protein coupled receptor (or STR) covers a wide range, except chemokine receptors, such as some amino acids acetylcholine , monoamine receptors, and classical chemotactic agents (C5a, fMLP, PAF) receptors are all G-protein coupled receptors/STRs.

1. Type and structure of chemokine receptor

(1) Types of chemokine receptors: the discovered chemokine receptors include IL-8RA, IL-8RB and MIP-1 α/ RANTESR, NCP-1R and red blood cell chemokine receptor (RBCCKR). Someone classified IL-8RA, IL-8RB and RBCCKR (Duffy antigen) that can bind to IL-8 as IL-8 Receptor family 。

(2) Structure of chemokine receptors: All chemokine receptors belong to G protein coupled receptors/STRs, with N terminal outside the cell membrane and C terminal in the cytoplasm. 7 Transmembrane area (transmembranedomain, TMD) is α screw At TMD Ⅱ, Ⅳ, Ⅴ, Ⅵ and Ⅶ α The lung amino acid in the spiral is kinked, and there are hydrophilic amino acid The three "one nots" are called e1-e3 (e: extracellularly connecting loops) and il-i3 (iintracellularly connecting loops) for short. Two conservative Cys form a disulfide bond between e1 and e2, and some receptors also form a disulfide bond between the extracellular N-terminal and e3, such as IL-8Ra30Cys and 277ys Disulfide bond 。 In the STR superfamily, chemokine receptors and classic chemokine receptors have the following characteristics: (1) Their length is the shortest in the STR superfamily, about 350 amino acids, which is mainly due to the short N-terminal and C-terminal, and the i3 ring only contains 16-22 amino acids; (2) At amino acid level, the homology is more than 20%; (3) I3 is rich in Basic amino acid , with positive electricity; (4) N-terminal imitation acid, with negative is electricity; (5) The cytoplasmic region contains multiple serine and threonine , may be Phosphorylation Locus; (6) MRNAs are mostly expressed in leukocytes.

2. The IL-8 receptor family, the IL-8R family, is the general name of different chemokine receptors that can bind to IL-8, including IL-8RA, IL-8RB and RBCCKR.

(1) IL-8RA: IL-8RAcDNA, 1991 Gene cloning The success is that Holmes and others from neutrophil cDNA Expression library The human IL-8RA gene is located on chromosome 2q35, closely linked and highly homologous to the IL-8RB gene, which may be copied from the same ancestor gene. From cDNA, IL-8RA is calculated to be 350 amino acid Composition, with 5 N connections Glycosylation Locus. The naked peptide has a molecular weight of 40kDa, 55-69kDa after glycosylation, and is similar to IL-8RB in amino acid level Homology 77%. IL-8RA only binds to ligand IL-8 (basic, PI8.0-8.5), which is related to the structure of IL-8RA Acid amino acid It is the location of binding with IL-8. Gly275 and Arg280 in Asp11 and e3 at the N-terminal are crucial for binding with the ligand. Because a disulfide bond is formed between Cys30 and Cys277, Asp11, Glu275 and Arg280 are very close in space and jointly participate in binding with the ligand. There are a wide range of cells expressing IL-8RA gene, such as neutrophils monocyte PGA activated T cell , monocyte like cell line, melanoma cell line, synovial fibroblasts, HL60 cell line and premedullary like cell line THP-1, etc.

(2) IL-8RB: IL-8RBcDNA was first cloned from HL60 cells, and the deduced amino acid residue was 335, with a potential N connection Glycosylation Locus. IL-8RB can be compared with IL-8, GRO in CXC subfamily α、 GRO β、 GRO γ And NAP-2. Human IL-8RB is mainly expressed in Myeloid cell , such as neutrophils, HL60, THP-1 and AML193 cells.

(3) RBCCKR: This receptor binding ligand has a wide specificity, also known as multi specific receptor, which can bind IL-8, NAP-2, GRO in CXC subfamily α And CC subfamily. Human RBCCKRcDNA was successfully cloned in 1993, and the gene was located in 1q21-q25, mature Receptor molecule 338 amino acid Composition, molecular weight of 39kDa, and IL-8RB and MIP-1 α/ RANTESR has 27% and 23% homology respectively. The extracellular domain is 66 amino acids and contains two potential N junctions Glycosylation Point, acidic. 24 cytoplasmic regions at the C-end Amino acid residue RBC-CKR does not seem to be regulated by G protein and may be an uncoupled receptor of G protein. RBCCKR is a human red blood cell Duffy antigen (gpD), which is also a micro day plasmodium Plasmodium vivax receptor. Duffy blood type Negative individuals do not express Duffy antigen/RBCCKR despite the existence of the cause of the blood type. RBCCKR acts as a clear receptor to clear chemokines in the blood. The binding affinity Kd of this receptor to ligand is 5nM, and the level of IL-8 in normal serum is at pM level. In adult respiratory distress syndrome (ARDS) and sepsis, the level of serum IL-8 can rise to 8 nM, and the excessive level of IL-8 can be combined with RBCCKR and cleared. Chemokines such as IL-8 lose their effect on target cells when they bind to red blood cells. The significance of this scavenging effect of red blood cells also lies in maintaining an appropriate concentration of chemokines, and ensuring that neutrophils and other sensitive cells move from the blood to inflammatory sites with high concentration of chemokines. RBCCKR is not only expressed on red blood cells, but also expressed in kidney and brain. The gene table is also found in spleen , lungs and thymus Etc.

3. The third one in the signal transduction IL-8RA and IL-8RB of the receptor Transmembrane area The second intracellular loop (i2) of (TMD Ⅲ) has a highly conserved DRYLAIVHA sequence, which is closely related to receptor signal transduction. DRY is necessary for the receptor to effectively couple G protein. If this sequence is changed by mutation, although it does not affect the binding of receptor and ligand, it almost completely loses the biological activity stimulated by ligand. IL-8R combines with ligand to decompose heterotrisomy G protein bound to receptor into α Subunit and βγ Subunit, α Subunit activation Phospholipase C (phospholipase CPLC), leading to three Phosphoinositol (IP3) and Diacylglycerol (DAG) increased, which induced the activation of Ca 2 and PKC released from Ca library in cytoplasm, respectively. In addition, phosphorylation of serine and threonine residues at IL-8RA and IL-8RBC terminals may be related to signal transduction.

4. Chemokine receptors and viruses. It is found that some open reading frame products infected with human or primate viruses have high levels of chemokine receptors Homology This may be related to the pathogenicity of the virus and some biological characteristics of the virus.

（1） Human cytomegalovirus (human cytomegalovirus HCMV): It is a kind of infectious person epithelial cells , medullary and Lymphoid cell Of β Herpes virus（ β Herpesvirus） molecular structure STR can be simulated on, wherein US28 product and human MIP-1 α/ RANTESR has about 30% homology and 56% homology with the N-terminal of the receptor. US28 products can be associated with chemokines β MIP-1 in subfamily α、 MIP-1 β、 MCP-1 is combined with RANTES, but not α Chemokines in subfamilies.

(2) Saimiri herpes virus (HerpesvirussaimiriHVS): It is a kind of virus that infects T-lymphocytes of primates γ Herpes virus（ γ Herpesvirus）。 HVS open reading frame ECRF3 product has nearly 30% Homology And 44% homology with IL-8RbN. ECRF3 product and IL-8, GRO α And NAP-2 to some extent.

HCMV-US28 and HVS-ECRF3 probes cannot hybridize with human genomic DNA, suggesting that Herpes virus Not only from host Obtained chemokine receptor in vivo Gene copy , and has been modified. Similar phenomenon can be seen in anthropophilia B lymphocytes Of γ Herpes virus EB virus (EBV), EBV open reading frame BCRF1 is the IL-10 gene obtained from the host, and the BCRF1 product is also called viral IL-10 (vIL-10), which can simulate the anti-inflammatory and anti proliferative effects of mammalian IL-10.

Sharing chain

2、 Shared chain in cytokine receptor

Most cytokine receptors are composed of two or more Subunit Composed of heterodimers or POLYMER , usually including a Specific ligand combination α Chain and a participating signal β Chain. α The chain forms a low affinity receptor, β In general, chains alone cannot bind to cytokines, but they are involved in the formation of high affinity receptors and signal transduction. Application of ligand competitive binding test, functional similarity analysis and Molecular cloning technology It was found that different cytokine receptors share the same chain in cytokine receptors.

（1） Types of cytokine receptor shared chains

Among many cytokines, some of them have very similar effects, such as IL-3, IL-5 and GM-CSF hemopoietic system Promote the proliferation of hematopoietic stem cells or directional stem cells. IL-6, IL-11, LIF, and OSM can all act on Hepatocyte 、 Megakaryocyte 、 Plasmacytoma And play a similar biological role. IL-2, IL-4, IL-7, IL-9 and IL-13 can stimulate the proliferation of T cells or B cells. The similarity of the above cytokine functions has been partly explained at the receptor level, which is largely determined by the shared chain of cytokine receptors. It is known that the cytokine sharing chain mainly includes gp310 and GM-CSFR β Chain and IL-2R γ Chain.

1. Gp130/LIFR is IL-6R, monoclonal antibody MT18 in myeloma Cell line A 130kDa glycoprotein named gp130 was obtained from the coprecipitation of U266. Hibi was successfully cloned in 1990, gp130, which belongs to hematopoietic factor Receptor family 。 IL-6 and IL-11 can stimulate the proliferation of IL-6 trusted mouse plasma cell tumor line T1165, and shorten bone marrow under the effect of IL-3 and GM-CSF Pluripotent stem cell Go phase of IL-3-dependent human and mouse megakaryocytes Colony To promote the formation of Specificity Antibody reaction can induce the production of acute phase protein of hepatocytes. Anti gp130 can block the proliferation of TF1 cells induced by IL-6 and IL-11 cytokines respectively, while anti IL-5R can only block the proliferation of TF1 cells induced by IL-6, indicating that IL-6 and IL-11 receptors share a signal transduction chain. There are two kinds of OSM receptors: low affinity and high affinity. The low affinity receptor, gp130, forms a high affinity receptor with LIFR. Unlike in IL-6R and IL-11R, gp130 only forms a low affinity receptor in OSMR and cannot transmit cytokine signals alone. The high affinity LIF receptor consists of LIFR and gp130. OSM and LIF can compete to bind the high affinity LIF receptor, but not the low affinity LIF receptor.

（4）IL-11R α Chain (mouse) and IL-6R α Chain and CNTFR α The homology of chain amino acids was 24% and 22%, respectively.

2. KH97/AIC2B is shared by IL-3R, IL-5R and GM-CSFR. In terms of hematopoiesis, both IL-3 and GM-CSF can promote Immature cell , mixed cells and granulocyte macrophage Colony To activate monocytes and promote Eosinophils Colony formation. IL-5 in addition to promoting B cell differentiation In addition to secreting antibodies, it can also stimulate eosinophil differentiation. Use GM-CSFR β Chain and IL-3, IL-5, GM-CSFR respectively α The chain co transfection test proved that β The chains in mice and humans are AIC2B and KH97, respectively, with 56% homology.

3. IL-2 receptor γ Chain division IL-2R contains γ Out of the chain, IL-2R is also shared in IL-4R, IL-7R, IL-9R and IL-13R complexes γ Chain（ γ c）。 The corresponding ligands of these receptors are a group of growth factors that mainly act on T cells. With IL-2 γ X interlock with chain abnormality as the main feature is serious Immunodeficiency syndrome The patient shows abnormal development of T cells, and the lack or number of T cells is significantly reduced, indicating that IL-2 γ Chain in T cell It plays a vital role in the development of. IL-4 and IL-7 all play a role in the development of T cells, and they share a signal transduction chain, IL-2R γ Chain T cell proliferation The signal of. In the IL-2 receptor system, α The chain constitutes a low affinity receptor, and the medium affinity receptor is composed of β、γ The high affinity receptor consists of α、β、γ It is composed of three chains, among which, γ Chain equivalent to other cytokine receptors β Chain, participating in signal transmission, and αβ The chain is equivalent to α Chain, which mainly plays the role of recognizing and binding ligands.

（2） Shared chain and cytokine receptor signal transduction

Cytokine signal transduction first requires the ligand to bind to the receptor and induce the formation of the dimer (or trimer) of the receptor, so that the cytoplasmic part of the dimer (or trimer) interacts, resulting in different signal transduction pathways. In the IL-2R system β、γ Chain dimerization is necessary for signal transduction, lacking β IL-2R in the cytosolic region of the strand could not transduce the signal generated by IL-2 stimulation. The stimulation and signal transduction of most cytokines to cells are related to the activation of tyrosine kinases and tyrosine phosphorylation of intracellular proteins. The binding of cytokines to receptors can cause tyrosine phosphorylation of receptor components. 60 near the membrane end of the ERS cytoplasmic region Amino acid residue It is highly conserved. This homologous sequence plays a key role in the signal transduction of IL-6, G-CSF, EPO, and IL-7, suggesting that these receptors may use similar intracellular signal transduction mechanisms.

1. Gp130 mediates signal transduction in the signal transduction common chain gp130 of IL-6R, IL-11R, OSMR, LIFR, CNTFR, and its cytoplasmic region contains about 277 amino acid residues serine Rich region, nucleotide binding region and four GTP binding mode regions. The serine rich region also exists in G-CSFR and IL-2R β、 IL-4R, EPOR and other ERS members have obvious homology. One fragment is conservative in all ERS members, and the other fragment exists in G-CSFR, EPOR, KH97. In these two short fragments, no matter which mutation occurs, gp130 will not be able to produce tyrosine Phosphorylation , lose the energy of signal transduction. The LIFR/gp130 heterodimer can not phosphorylate tyrosine with tyrosine phosphogp130 and lose the function of signal transduction. LIFR/gp130 heterodimer is also related to tyrosine phosphorylation. Although most members of the hematopoietic factor receptor family do not have tyrosine kinases Domain , but they are associated with the tyrosine kinase type Growth factor receptor Similarly, growth factors cause Receptor tyrosine kinase The formation and activation of dimer, and hematopoietic factors may induce its receptors Dimer It forms and causes the activation of related tyrosine kinases. It was found that tyrosine phosphorylation of protein with molecular weight of 97/95 kDa was detected in TF1 cells stimulated by IL-6 and IL-11, which is important in the signal transduction of anti gp130. In different Cell line 3T3-L1、 B-cell hybridoma The protein with different molecular weight is found to be tyrosine phosphorylated in myeloid leukemia lines, suggesting that there are cell specific tyrosine kinases and their respective specific substrate This may be one of the reasons for the different biological functions of IL-6, IL-11, LIF, CNTF, and OSM sharing gp130 in different cells. JAK2 is a non receptor type Tyrosine kinase , can be activated by EPO, IL-3, G-CSF, IL-6 and other cytokines, JAK2 may be these different cytokine receptors signal transduction pathway This JAK2 kinase associated with the receptor may catalyze different substrates due to different receptor structures, which leads to JAK2 mediating many different biological functions. In addition, gp130 also undergoes its own tyrosine phosphorylation after stimulation by IL-6, IL-11, CNTF and LIF.

2. KH97/AIC2B mediated signal transduction There are also two regions necessary for generating different signals in the cytoplasmic region of the signal transduction chain KH97/AIC2B of IL-3, IL-5, GM-CSF: one is the region of about 60 amino acids near the membrane end upstream of Glu517, which is necessary for inducing c-myc and pim-1; The other region is the cytoplasm region of about 140 amino acids from Leu623 to Ser763, which is Ras, Raf, MAP (mitogen activated protein kinase ）It is necessary for the induction of c-fos and c-jun. hGM-CSFR α、β The chain has no catalytic region of any known enzyme and is co transfected with hGM-CSF α、β The increase of C-Myc and pim-1 levels in Ba/F3 cells was associated with the increase of C-Myc and pim-1 levels. Transfection of GM-CSF in mouse lymphocyte line α、β Post chain can induce tyrosine phosphorylation of several proteins in the cell and cause proliferation reaction, α、β Chain co transfection of mouse NIH3T3 cells expressing GM-CSFR high affinity receptors can cause β The chain cytoplasmic region and another protein containing 40-45kDa protein in the capsule are rapidly phosphorylated by tyrosine.

solubility

3、 Soluble cytokine receptor

In natural state, cytokine receptor (CK-R) mainly exists in two forms: membrane bound cytokine receptor (mCK-R) and soluble cytokine receptor (sCK-R) in serum and other body fluids. The complex biological activity of cytokines is mainly mediated by the binding of mCK-R with the base, while sCK-R has unique biological significance. The relationship between the changes of sCK-R level and some diseases has been paid more and more attention by scholars. Some recombinant sCK-R (rsCK-R) genetic engineering products have entered clinical validation, and the basic research on the generation mechanism, structural characteristics and basic immunological functions of sCK-R has also made considerable progress. （1） Generation mechanism and action characteristics of sCK-R

HUT102B2, MT-2 and other cells infected by human T-cell leukemia virus type I (HTLV-I), myeloid leukemia Cell (HL-60, KG1) and some human B Cell line (Raji) In addition to expressing a variety of mCK-R, sCK-R can also be generated in different ways, such as HUT102B2 cell culture High levels of sCK-2R and sIL-6R can also be detected in Fengqingzhong. Human PMC also produces a large number of sCK-2R and sCK-6R after PHA stimulation in vitro.

1. The production of sCK-R mostly comes from the shedding of membrane receptor, so the membrane receptor cation Cytolysis It is a method to obtain a large number of sCK-R. Most of the amino acid sequences of sCK-R are homologous to the extracellular domain of mCK-R, only lacking transmembrane and cytoplasmic domains, but still corresponding to ligand happen Specificity Combined with. except Membrane receptor In addition to the form of sCK-R produced by cleavage and abscission of SCK-R, another mechanism of producing sCK-R is through different splicing of receptor mRNA to produce Secretory type MRNA, which is directly secreted out of cells after translation, has been confirmed that cells can contain the same CK-R in different forms of cDNA. sIL-4R、sIL-5R α Chain, sIL-6R α Chain, sIL-7R and sG CSFR can be generated in this form.

2. The biological effect of sCK-R Most of the affinity of sCK-R to the corresponding cytokines is lower than that of mCK-R, which may be related to the single chain structure of sCK-R or the lack of some structural regions. Some sCK-Rs, such as sIL-4R, have the same affinity with natural mIL-4R and corresponding ligands, and even low-dose sIL-4R can specifically inhibit IL-4 induced cell proliferation Reaction. SCK-R exerts its unique immunological function in many ways.

(1) As a cytokine Transporter And transport cytokines to the relevant production sites of the body, resulting in local high concentration areas of cytokines to give full play to the biological effects of cytokines.

(2) The membrane receptor is normal Metabolic pathway It is conducive to the recovery of activated cells to normal levels.

(3) Competitively bind the corresponding ligand of mCK-R to inhibit the biological effect mediated by mCK-R.

（2） SCK-R and clinical

1. Detection of sCK-R level in clinical application Detection of certain sCK-R levels assists in early clinical diagnosis of certain diseases, understands the development and prognosis of the disease course, and can evaluate the immune function and prognosis of patients Clinical treatment It also has some guiding significance.

(1) Detection of sIL-2R: Scholars at home and abroad have conducted a large number of studies on sIL-2R, and found that its level changes in serum and other body recognition and clinical diseases such as organ transplant Rejection reaction Viral infection , malignant tumor, trauma and Autoimmune disease The condition and course of disease are closely related.

(2) Detection of other sCK-R: a 50kDasIL-6R molecule, called IL-6R-SUP, was found in the urine, which can promote the growth of mouse plasma cell tumor T1165 induced by low-dose IL-6. Multiple myeloma The level of sIL-6R in patients' plasma was significantly increased. The serum sTNFR level in patients with rheumatic disease was increased abnormally, and a high level of sTNF-R could also be detected in the synovial fluid after the closure of the cavity, and the level in the active phase was significantly higher than that in the inactive phase. Only TNF-R Ⅱ type sTNFR can be detected in the urine of normal women, while TNF-rI and TNF-R Ⅱ type sTNFR can be detected in the urine of pregnant women. With the increase of fetal age, the level of sTNFR gradually increases, and then decreases after delivery, which may be a protective mechanism to protect the fetus from TNF. High levels of sTNFR can also be detected in liver infectious ascites and cancerous ascites. In addition, it was found that the increase of sTNFR level was closely related to the decline of renal function.

2. Prospects of clinical application of sCK-R Most of sCK-R and cytokines combine to cut off the relationship between cytokines and Membrane receptor To inhibit the biological activity of cytokines, the application of sCK-R to alleviate or prevent the pathological damage caused by inflammatory cytokines provides a new therapeutic approach. Animal experiment results showed that local injection of sIL-1R could inhibit IL-1-mediated inflammatory reaction. SIL-1R can reduce mouse allograft heart Rejection of transplantation, experimental arthritis and allergic encephalitis in rats. SIL-1R can significantly inhibit the proliferation of bone marrow cells from patients with acute myeloid leukemia in vitro. The application of IL-1R genetic engineering products began to enter clinical validation for the treatment of arthritis, diabetes and prevention and treatment of organ transplantation rejection. In vivo injection of sIL-4R into animals can prolong the survival of homograft, inhibit GVHR and reduce type I hypersensitivity. The application of sTNFR can alleviate the pathological damage mediated by TNF in autoimmune diseases, and can alleviate septic shock.

principle

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In order to maintain the physiological balance of the body, resist the invasion of pathogenic microorganisms, and prevent the occurrence of tumors, many cells in the body, especially Immune cell Synthesis and secretion of many kinds of micro polypeptide factors. They transmit information between cells, regulate the physiological process of cells, improve the immunity of the body, and may also cause fever, inflammation, shock and other pathological processes under abnormal conditions. Hundreds of such factors have been found, collectively referred to as cytokines, including those produced by lymphocytes Lymphokine , produced by monocytes Mononuclear factor , various growth factor Etc. Many cytokines are named according to their functions, such as Interleukin （IL）、 interferon （IFN）、 Colony stimulating factor （CSF）、 tumor necrosis factor (TNF), erythropoietin (EPO), etc.

effect

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Cytokines play a biological role by binding to the corresponding cytokine receptors on the cell surface. Cytokines and their receptors aggregate to initiate complex interactions between intracellular molecules and eventually cause cells Gene transcription Changes.

cell factor

participate in immune response It regulates innate immunity and adaptive immune response; Stimulating hematopoietic function; Stimulate cell activation, proliferation and differentiation; Induce or Suppressor cell Toxic effect, inducing apoptosis. Action mode of cytokines: 1 autocrine effect. 2. Paracrine effect. 3. Endocrine function.

Characteristics of cytokines: Pleiotropy , overlap Synergy , antagonism and duality.

Cytokine research has very important theoretical and practical significance. It helps to clarify the immune regulation mechanism at the molecular level, and helps to prevent, diagnose and treat diseases, especially by using Genetic engineering technology The produced recombinant cytokines have been used in the treatment of tumors, infections, inflammation, hematopoietic dysfunction, etc., and have received good effects, with a very broad application prospect.

Cytokines are gradually used as adjuvants in vaccine development, such as interleukin-2, IL-12, etc.

characteristic

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Many cytokines have the following common characteristics.

(1) The vast majority of cytokines are glycoproteins with a mass of less than 25kDa, and those with low quality, such as IL-8, are only 8kDa. Most cytokines exist in monomer form, and a few cytokines such as IL-5, IL-12, M-CSF and TGF- β They play a biological role in the form of two bodies. Most genes encoding cytokines are single copy genes (IFN- α Except), and composed of 4-5 exons and 3-4 exons Intron form.

(2) It mainly regulates the immune response of the body Hematopoietic function It is related to inflammatory reaction.

(3) Usually paracrine or autocrine (autocarine) acts on nearby cells or cytokine producing cells themselves. Under physiological conditions, most cytokines only work locally.

(4) High efficiency effect, generally at pM (10-12M) level, has obvious biological effect.

(5) Exists in cell surface Corresponding height of Affinity The number of receptors is small, ranging from 10 to 10000 per cell. The research progress of cytokine receptor is quite rapid gene DNA sequence and receptor extracellular domain amino acid sequence Homology And structure, which can be divided into four types: Immunoglobulin superfamily Hematopoietic factors Receptor superfamily Nerve growth factor receptor superfamily and chemokine receptor.

(6) A variety of cells produce one IL, which can be produced by many different cells under different conditions, such as IL-1 except monocytes, macrophages or Macrophage system In addition to production, B cells, NK cells, fibroblasts endothelial cells 、 Epidermal cell They can synthesize and secrete IL-1 under certain conditions.

(7) Multiple regulatory actions. Different regulatory actions of cytokines are related to their own concentrations, the types of target cells and other types of cytokines that exist at the same time. Sometimes the animal species are different, and the biological effects of the same cytokines can be quite different. For example, human IL-5 mainly acts on eosinophils, while mouse IL-5 can also act on B cells.

(8) Overlapping regulation, such as IL-2, IL-4, IL-9 and IL-12, can maintain and promote the proliferation of T lymphocytes.

(9) It functions in the form of network, and the network function of cytokines is mainly through the following three ways: (1) One cytokine induces or inhibits the production of another cytokine, such as IL-1 and TGF- β Promote or inhibit the production of IL-2 in T cells respectively; (2) Regulate the expression of the same cytokine receptor, for example, high dose of IL-2 can induce NK cells to express high affinity IL-2 receptor; (3) Induces or inhibits the expression of other cytokine receptors, such as TGF- β It can reduce the number of IL-2 receptors in T cells, while IL-6 and IFN- γ It can promote the expression of IL-2 receptor in T cells.

(10) Together with hormones, neuropeptides and neurotransmitters, they form the intercellular signal molecular system.

（11） Self limited secretion 。

structure

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From the perspective of molecular structure, cytokines are small polypeptides, most of which are composed of about 100 amino acids. Cytokines can exert their biological effects only by specifically binding to cytokine receptors on the surface of target cells. These effects include promoting the proliferation and differentiation of target cells, enhancing anti infection and tumor killing effects, promoting or inhibiting the synthesis of other cytokines, promoting inflammatory processes, and affecting cell metabolism. These effects of cytokines have the characteristics of network, that is, each cytokine can act on a variety of cells; Each cell can be regulated by various cytokines; Different cytokines interact with each other, which forms a complex immune regulatory network of cytokines. People's understanding of this network is far from clear.

R&D history

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In recent years, recombinant cytokines, as a new type of biological response regulator, have made remarkable achievements in clinical application. For example, the earliest interferon used in clinic α In treatment leukemia And virus infection. China's interferon a1 passed the new drug review in 1991 and has been widely used. Cytokine drugs that have been approved for production internationally also include EPO and interferon γ、 GM-CSF, G-CSF, IL-2, etc. Because cytokines are human body's own components, they can treat diseases by regulating the physiological process of the body and improving immunity. They can play a role in low doses, so it is a new biological therapy with significant curative effect and small side effects, and will soon achieve rapid development.

The research of cytokines started from the research of interferon in the 1950s and the research of colony stimulating factors in the 1960s. Due to the rapid development of genetic engineering technology, the research of cytokines has made breakthrough progress. In the 1980s and 1990s, a large number of cytokines were cloned. The chemical essence of cytokines is polypeptide. From the perspective of information transmission, cytokines are an important class of biological factors First Messenger Molecules are intracellular gene expression The product of.

In natural state, cytokine receptor (CK-R) mainly exists in two forms: membrane bound cytokine receptor (mCK-R) and soluble cytokine receptor (sCK-R) in serum and other body fluids. The complex biological activity of cytokines is mainly mediated by their binding with the corresponding mCK-R, while sCK-R has unique biological significance. The relationship between the changes of sCK-R level and some diseases has been paid more and more attention by scholars. Some recombinant sCK-R (rsCK-R) genetic engineering products have entered clinical validation, and basic research on the generation mechanism, structural characteristics and immunological functions of sCK-R has also made considerable progress.

Studies have shown that various cytokines in vivo do not exist in isolation, but have complex interactions. They form a complex network of cytokine interactions through the mutual regulation of synthesis and secretion, the mutual regulation of receptor expression, and the mutual influence of biological effects.

Biological function

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Cytokines have other biological functions, such as promoting apoptosis and promoting wound tissue repair. Some cytokines in the tumor necrosis factor superfamily can directly kill or induce apoptosis of target cells, such as TNF-a and LT- α It can kill target cells or tumor cells infected by the virus. Transforming growth factor B (TGF-B) can stimulate fibroblasts and Osteoblast The epidermal growth factor can promote the proliferation of epithelial cells, fibroblasts and endothelial cells, and promote the healing of skin wounds^[1] 。

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