Cytokines: Families and Functions

Overview of Cytokines: Families and Functions

What are Cytokines?

Cytokines are small secreted proteins released by cells that are a major communication mechanism used by the immune system. They also have specific effects on the interactions between cells. Cytokines can either have pro or anti-inflammatory effects on the host depending on the family of the cytokine.

Cytokines are classified according to their cell of origin or their mechanism of action and are produced throughout the body. Cytokine is an umbrella term and these secreted molecules are given other names based on their effector function, cell of secretion or target of action. For example, cytokines that are produced by lymphocytes and are involved in the interactions between those cells are called lymphokines or interleukins (ILs). Additionally, cytokines produced by macrophages and monocytes are called monokines.

Cytokines can be produced by a variety of different cell types including immune cells such as macrophages, B cells and T cells. T cells that produce cytokines include T helper cells (CD4+) which include Th1, Th2, Th17 and regulatory T cells (Treg). Additionally, cytotoxic T cells also produce a variety of cytokines. Other cells involved in the production of pro inflammatory cytokines and anti inflammatory cytokines include mast cells, endothelial cells, fibroblasts and various stromal cells (connective tissue cells of any organ). Certain cytokines can be produced by more than one cell type.

Cytokines interact with cell surface receptors in order to be activated and carry out their specific functions. The are particularly important in the immune system as they regulate the balance between cell-based and humoural responses. They also modulate the maturation, growth, and activity of certain cell populations. Cytokines also have the ability to enhance or inhibit the action of other cytokines. They can do this by increasing or dampening down specific signals that stimulate the production, activation and migration of other cytokines to a site of infection.

Figure 1: Diagram conveying what cell types that produce what cytokine. (Source)

Cytokine Families

Various cells of the body produce a large family of cytokines and this includes the cytokine superfamily which is composed of interleukins, colony-stimulating factors (CSFs), interferons, chemokines and the transforming growth factors (TGF) and tumour necrosis factor (TNF) families. Within each of the families, the different cytokines within the group have similar structures but can have diverse cytokine functions.

Families of cytokines share similar sequence structures and share similar receptors for their activation however members within each family do not all exhibit functional similarity. Cytokine families possess important regulatory cell membrane receptor-ligand pairs, which aid in the upregulation and downregulation of pro- and anti-inflammatory cytokines.

Type-1 cytokines are cytokines produced by Th1 CD4+ T cells and include IL-2, IFN-gamma, IL-12 and TNF-beta. Type-2 cytokines are those produced by Th2 CD4+ T cells and include IL-4, IL-5, IL-6, IL-10 and IL-13.

Cytokines can also be split into family groups depending on their secondary and tertiary structures. For example, the cytokines IL-6, IL-11, CNTF, LIF, OSM (Oncostatin-M), Epo (Erythropoietin), G-CSF, Growth Hormone (GH), Prolactin (PRL), IL-10, IFN-alpha and IFN-beta all form long chain 4 helix bundles.

On the other hand, the cytokines, IL-2, IL-3, IL-4, IL-5, IL-7, IL-9, IL-13, GM-CSF, M-CSF, SCF, IFN-gamma all form short chain 4 helix bundles giving a different structure to the cytokines mentioned above.  

Cytokine Receptors

Cytokines are required to bind to their receptor in order to become activated. Cytokine receptors are categorised into structurally related families and have high-affinity molecular signalling complexes that enables cytokines to execute their functions.

Figure 2: Diagram showing Type I/II cytokine receptor, TNFR (Tumour necrosis factor receptor) and TGF-beta receptor (transforming growth factor beta receptor). (Source)

Type I Cytokine Receptors

Type I cytokine receptors have conserved motifs in their extracellular n-terminal domain however they do not possess intrinsic protein tyrosine kinase activity. This family includes receptors for IL-2 (beta subunit,) IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-12, GM-CSF, G-CSF, Epo, LIF and CNTF. Type I cytokine receptor family is subdivided into three groups based on the ability of the cytokine receptor family members to form complexes with one of three different types of receptor signalling components. These include gp130, common beta, and common gamma which is also known as the gamma chain of the IL-2 receptor.

Type II cytokine receptors

Type II cytokine receptors are multimeric receptors and are made up of heterologous subunits. The type II cytokine receptors are mainly used by the interferons. This family includes receptors for the cytokines, IFN-alpha, IFN-beta, IFN-gamma, IL-10 and IL-22. The extracellular domains of type II cytokine receptors are similar in structure to their ligand-binding domain. Within the various receptor subgroups, there are several conserved intracellular sequence motifs which are proposed to function function as binding sites for the intracellular proteins JAK and STAT proteins in the signal transduction cascade.

Tumour Necrosis Factor Receptor (TNFR) Family

Tumour necrosis factor receptors (TNFRs) are a superfamily of structurally related membrane proteins that enable the activation of cell death pathways or the induction of expression of genes that play a role in cellular differentiation and survival. This occurs when cytokines such as tumour necrosis factor and those related to it bind to the receptor via an extracellular cysteine rich domain. These receptors are present in many organ systems and play a prominent role in the immune system. In order to trigger cell death pathways, the TNFRs when activated stimulate the activation of adaptor proteins such as FADD and TRADD which in turn cleave other inactive procaspases and trigger a process known as the caspase cascade which triggers apoptosis, a form of programmed cell-death.

Transforming Growth Factor beta (TGFβ) Receptor Family

The TGFβ receptors are a superfamily of serine or threonine kinase receptors that are involved in involved in the TGFβ signalling pathway. These receptors can bind the TGF-beta superfamily including TGFβ1, TGFβ2 and TGFβ3. They can also bind growth factor and cytokine signalling proteins such as bone morphogenetic proteins (BMPs), activin and inhibin, myostatin, growth differentiation factors (GDFs), anti-Müllerian hormone (AMH), and NODAL. There are three types of TGF-beta receptors and these include TGFBR1, TGFBR2 and TGFBR3. Each of the receptors are differentiated by their structural and functional properties.

Cytokine Function

Cytokines are essential controllers of cells and they control cell growth, migration, development and differentiation. Their effects may be suppressive or enhancing based on the family which they are divided in to, and their biological structure and function.

Additionally, cytokines can exert cytotoxic effects on infectious agents such as tumour cells, either directly or by activating cells with cytotoxic potential. Any given cytokine may have many different biologic effects, however, two different cytokines from opposing families may have similar or identical activities. Cytokines play crucial roles in tissue damage repair, in tumour cell development and progression, in the control of cell replication and apoptosis (programmed cell-death), and in the regulation of a variety of immune responses.

Some examples of cytokines involved in cellular and systemic activities include those that are involved in the promotion of pro-inflammatory responses which include, IL-1ß, IL-18, IL-33 and IL-17. Those that are involved in the mediation of innate immunity include Type I IFNs, TNF-α and IL-17. Cytokines involved in the regulation of lymphocyte growth activation and differentiation include IL-2, IL-4, IL-5, IL-12 and IL-5. Those involved in the activation of inflammatory cells include Type II IFNs and IFN-γ. Cytokines involved in the stimulation of haematopoiesis, which is the production of the cellular components of blood and blood plasma include IL-3, IL-5, IL-7 and GM-CSF.

Although there are is a multitude of cytokines, there are certain groups which carry out the majority of physiological actions in the host. Some examples of these are interleukins, interferons, tumour necrosis factor and chemokines.

Interleukins

Interleukins are glycoproteins that play a role in the activation and differentiation of immune cells. They are produced by lymphocytes, monocytes and various other cells in the body. They are involved in the proliferation, maturation and migration of cells as well as pro- and anti-inflammatory activities. Interleukins stimulate various up-regulatory and down-regulatory mechanisms which impacts the activation and repression of genes. Additionally, interleukins can also influence the synthesis and functioning of other interleukins within the family.

Interleukins belong to a superfamily that is made up of proteins with varying sizes. Currently over 43 members of this superfamily have been identified and these range from IL-1 to IL-43. Within the superfamily of interleukins, the functions of the cytokines differ depending on their structure, biological activity and receptor that they bind to.

Interferons (IFNs)

The interferon family consists of various cytokines that are widely expressed throughout the body. Like other cytokines, interferons are also released by cells of the host's immune system in response to invading organisms such as bacteria and viruses. Interferons are expressed by a variety of cells including viral infected cells (Type I IFNs) and T cells, natural killer cells (NK cells) and macrophages. Interferons aid in the recruitment of effector molecules that protect the cells against infection. For example, as interferons stimulate the activation and production of NK cells and macrophages, they contribute to the destruction of both the viruses and infected cells. Three types of interferons have been identified thus far and these include Type I interferons, Type II interferons, and Type III interferons.

Type I Interferons

Type I interferons are divided into two major groups, IFN-α and IFN-β. There are additional members of the type I IFN family that include IFN-κ, IFN-ω, and IFN-δ, however, these members are less characterised. Only one type of IFN-β exists whereas IFN-α is subdivided into different subtypes including IFN-α1, IFN-α2, IFN-α3, IFN-α4, IFN-α5 and IFN-α6.

The major functions of Type I IFNs (IFN-α and IFN-β) are to protect against viral infection which is achieved through the destruction of viral mRNA in virally infected cells which is required for viral replication. Type I IFNs also inhibit the translation of viral proteins thereby preventing the production of a viral progeny. Additionally, Type I IFNs promote an increase in ligands to the receptors of NK cells which thereby stimulates the cells to attack and lyse infected cells. Type I IFNs also stimulate the activation of pathways that leads to the destruction of infected cells by NK cells and macrophages.

Type II Interferon

Type II interferon is made up of a single cytokine known as IFN-gamma (IFN-γ). This cytokine is mainly produced by Th1 T cells, Natural Killer cells, CD8+ T cells (effector T cells) and in some cases antigen-presenting cells such as dendritic cells and macrophages and B cells.

Type II interferon is different from the other interferons due to the fact that it does not possess a potent antiviral effect, however, IFN-γ can contribute to the long-term control of virally infected cells by activating MHC complexes. Its main function is to activate effector cells giving the cytokine a predominantly pro-inflammatory effect. In innate immunity, IFN-γ is produced mainly by NK cells, whereas in adaptive immunity, it is produced largely by T cells and increased production of IFN-γ is stimulated by the cytokines, IL-12 and IL-18. The cytokines IL-4 and IL-10 are involved in the negative regulation of IFN-γ production. Some other functions of IFN-γ include macrophage activation which promotes the phagocytic and pinocytic activities of these cells. This therefore contributes to pathogen destruction. Additionally, IFN-γ inhibits cell growth and promotes apoptosis of non-self cells or infected cells.

Type III interferons

Type III interferons are divided into three different cytokines that include IFN-λ1 (IL-28a) IFN-λ2 (IL28b), and IFN-λ3 (IL-29). Type III interferons share structural homology to the signalling proteins in the IL-10 family. However, the signalling pathways of Type III interferons is similar to that of Type I interferons due to the fact that activation of Type III interferons are dependent on the actions of interferon regulatory factors (IRFs) and the transcription factor NF-kB. Whilst Type III interferons share a number of similar functions with Type I interferons, they primarily function in mucosal epithelial cells and liver cells where they serve to protect host cells from viral infections.

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Tumour Necrosis Factor

Tumour Necrosis Factor (TNF) consists of a group of proteins involved in a variety of physiological and pathological processes. There are currently 40 members within the TNF superfamily, however TNF-α and TNF-β are the most notable and well characterised.

TNF-α

TNF-α is a multifunctional cytokine that plays a role in immunity and programmed cell death. Its major role in immunity is to attract certain immune cells to the site of infection site by stimulating the expression and production of adhesion molecules by vascular endothelial cells. This allows immune cells to adhere to blood vessel walls which promotes their migration to the infected site and thereby allows for the destruction of invading pathogens. Additionally, TNF-α also stimulates the production of chemokines that are involved in inflammatory responses. This promotes the migration of immune cells to the site of infection.

TNF-α also promotes the programmed cell death of tumour cells via apoptosis or necrosis. This is carried out by promoting the recruitment of proteins involved in cell death signalling. When produced in large amounts, TNF-α has the ability to reduce blood pressure or shock during events as severe infections. However, when this cytokine is produced in large concentrations, it can result in low blood sugar concentration and intravascular thrombosis (condition caused by the development of small blood clots throughout the bloodstream which can block small blood vessels causing excessive bleeding.)

TNF-β

TNF-β, also known as Lymphotoxin-alpha is a transmembrane protein. It is produced by activated lymphocytes and is involved in the regulation of cell survival, proliferation, differentiation and apoptosis. TNF-β plays a major role in innate immune regulation and its production has been conveyed to prevent tumour growth and destroy cancerous cell lines. However, uncontrolled expression of TNF-β can result in uncontrolled cellular growth and the production of tumours. TNF-β has similar functions to TNF-α in that it is a potent regulator of various immune and inflammatory responses. Additionally, it also plays a role in the coagulation of blood.

Chemokines

Chemokines are a type of cytokine that functions by attracting cells to a site of infection. They are very small in size ranging between 8 and 10 kDa. Their name signifies their function, chemo relates to the fact they use chemotaxis, or movement in response to a chemical stimulus to stimulate the migration of cells from one area to another. Whereas -kines relates to the fact that they are a type of cytokine.

Chemokines play major roles in immunological reactions, in which they are the secondary pro-inflammatory signalling proteins that are produced as a result of the production of primary cytokines. They also function to maintain homeostasis, in which they regulate cell movement in order to ensure that the immune system functions at its optimal state. Chemokines must bind to their receptor in order to become activated. Chemokine receptors are mainly present on the surface of white blood cells (lymphocytes).

Chemokines differ in their structure however they all share similar structural homology. Most chemokines share the presence of four cysteine amino acids, two of which are used to classify all chemokines into four types. Specifically, the two amino acids close to the N-terminal region of the chemokine are used for the classification into CC, CXC, C, and CX3C types. The X symbolizes an amino acid and differs depending on the type of chemokine.CC chemokines have two cysteines that are next to each other, for example, CCL2 contains two cysteines next to each other and two leucine’s at the carboxyl terminus. CXC chemokines have one amino acid between the two cysteine residues, for example CXCL1. C chemokines have two cysteines instead of four, one of which is at the N-terminus and CX3C chemokines have three amino acids between the two cysteines.

Figure 3: Schematic conveying chemokine structure and classification. (Source)

Cytokine Signalling

Cytokine signalling is an essential component of the regulation of the body. In order for the immune system to function effectively, cytokine signalling is essential. For example, macrophages and dendritic cells phagocytose foreign particles and send a cytokine signal to inactivated white blood cells nearby. The cytokines released bind to receptors on the lymphocytes (white blood cells) which in turn allow the lymphocytes to become activated and carry out their designated functions. The combination of the macrophages or dendritic cells releasing cytokines and the activation of lymphocytes through cytokine signalling aid in the regulation of homeostasis in the body.

Cytokine receptors contain one to three chains. The ligand-binding subunit of a receptor is called the alpha chain. Other signal transducing subunits are named beta chains or gamma chains. Cytokine receptors are all associated with one or more members of JAKs, which stimulate tyrosine phosphorylation after the ligand (cytokine) binds to its receptor. This in turn recruits various signalling proteins (STATs) to the receptor complex initiating a response depending on the cytokine.

Figure 4: Schematic showing Jak-Stat signalling pathway stimulated by cytokine receptors. Cytokines bind to cell surface receptors which activate receptor complex formation and the activation of one or more associated Jaks. These phosphorylate the tyrosines that are within the cell, which allows Stats to become activated. The complex moves to the nucleus to upregulate or downregulate gene expression, thus inducing a response. (Source)

There are two main signal transduction pathways that are involved in cytokine activation. Both lead to the activation of protein kinases that stimulate phosphorylation which subsequently stimulate secondary signal transduction and amplification of the signal.

The first pathway, used by mitogenic cytokines such as EGF which induces cell growth and proliferation, contains tyrosine kinases as the main signal transducers which can sometimes be intrinsically built into the receptors for the designated cytokine.

The second pathway involves activation of phospholipases, which stimulate the production small proteins that activate serine-threonine kinases and raise calcium levels inside the cell. This pathway is used by TGF-beta.

Both of the major pathways also involve effector molecules downstream of the signalling pathways such as GDP-binding proteins, calcium-binding proteins, phosphatases and the products of proto-oncogenes (genes that promote tumour production). Although receptors may differ depending on the cytokine family, the respective ligand or cytokine can share similar signal transduction pathways despite the fact that receptor families may be different.

Author: Sarah Donovan MSc

Date Published: 23rd June, 2020