Cytokine Receptor Proteins

 Cytokine Receptor Proteins Background


Cytokines are a series of highly localized soluble messenger proteins produced by cells of the immune system, such as macrophages, lymphocytes, neutrophils, basophils, and eosinolphils. The molecular weights of cytokines range from 8 to 80 kDa, and many of them are found to be biologically active in their oligomeric forms. Once the cytokines are produced within the cytoplasm of the cells, they are secreted and present in the tissue extracellular fluid (ECF). Cytokines are a category of signaling proteins, like hormones and neurotransmitters, and function as chemical communicators between cells. They are responsible for regulating a variety of immunological and inflammatory responses during host defense against pathogen.

The signal transduction process is initiated from the binding of cytokines to their specific receptors on the surface of target cells and coupled to subsequent cascades of intracellular signaling via second messengers, often tyrosine kinases, to alter the cell functions. Generally, the binding of one cytokine to its cell-surface receptor can stimulate the release of many other cytokines. Many cytokines can modulate the synthesis or the actions of other cytokines in a complex network of interactions. The released cytokines can have effect via autocrine, if they act on the cell that secretes them; paracrine, if the target cell is in the vicinity of their secretion; or endocrine, if they diffuse to distant regions of the body. What’s more, cytokines are highly active molecules and their concentration in body fluids is usually at low picomolar level. Elevated expression of cytokines indicates activation of cytokine pathways associated with inflammation or disease progression.


Cytokine Receptor

The cytokine receptor family performs a wide variety of essential biological functions including the growth and development of multiple cell types (e.g., blood cells) or regulation of the immune system. Members of this family of receptors commonly feature at least one cytokine receptor homology domain (CHD) composed of the fibronectin derived D1 and D2 domains; the D1 domain has 4 conserved cysteines and the D2 domain has a conserved WSXWS motif. The receptors that have functional intracellular domains (ICD) share homology as well, containing the conserved Janus kinase (Jak) binding sites Box 1 and Box 2.

Each receptor shows high conservation across species. They are subdivided into 5 subgroups based on sequence homology, receptor structure, and type of functional oligomer (homo- or heterooligomer). Group 2 is the largest subgroup and has the most diverse extracellular domain (ECD), with a varying number of fibronectin and immunoglobulin domains in addition to the CHD. This group contains the gp130 common signaling chain receptor used by several of the Group 2 members. Group 3 also has a modified ECD containing an immunoglobulin domain in addition to the CHD; several of its members also use gp130 as a common signaling chain. Groups 4 and 5 are also heterocomplex receptors, but instead of using gp130 as the common signaling chain.

The diverse array of biological processes regulated by the cytokine receptors make them targets for disease-causing mutation or other modulation resulting in pathology. Mutations can cause gain-of-function or loss-of-function, leading to physiological states that may be characterized as being within normal limits or pathologic, depending on severity. Interestingly, each receptor does not seem to have an equal chance of acquiring a pathologic mutation. For instance, the Tpo receptor has several described mutations, but the Epo receptor does not. This may reflect a difference in the structural requirements for signaling, a difference in tolerance of variability in the ultimate physiological response, or differences in importance of the pathway either overall or at different stages of organism development. The most common mutation associated with increased cytokine receptor function occurs in the associated Janus kinase, in EpoR/TpoR, Jak2 (JAKV617F).


Cytokines and inflammation

In response to injury/infection, cytokines are secreted by inflammatory cells over several hours to days, and in some instances even weeks. It is important that investigation of such a complex phenomenon as inflammation includes careful and focused attention on its fundamental mediators. Since inflammation is a well-orchestrated and tightly regulated process, it was important to first evaluate a broad scope of mediators, and then narrow the focus on those which were specifically altered by treatment with anti-inflammatory reagents. A diverse selection of mediators including pro-inflammatory cytokines, anti-inflammatory cytokines, and chemokines was investigated.

Tumor necrosis factor: Tumor necrosis factor (TNF) was originally identified in 1975 by Lloyd Old as a macrophage-derived factor that could "necrotize" tumors in mice. It is a classic pro-inflammatory cytokine secreted by a number of cells including macrophages/monocytes, mast cells, and tumor cells. TNF is induced in response to a variety of stimuli including, but not limited to, bacterial endotoxins, oxygen radicals, and viruses. In response to LPS (lipopolysaccharide) stimulation, PBMCs (peripheral blood mononuclear cells) obtained from whole blood rapidly secrete a significant amount within the first three to four hours. In addition to being produced in response to direct stimulation, TNF production can also induce various other cytokines including IL-1β andIL-8.

Interleukin 1β: Interleukin 1β is a member of the IL-1 family of cytokines which consists of IL1a, IL-1β, and IL-1 receptor antagonist (IL-1ra), all of which are encoded by separate genes. The IL-1 genes are induced by a wide variety of stimuli including LPS, viruses, and TNF. Like IL-6 and TNF, IL-1 is most prominently produced in monocytes/macrophages. Upon endotoxin stimulation, for example, IL-1β is rapidly induced in whole blood. Although IL-1a and IL-1β are both induced upon stimulation of the inflammatory response, IL-1a remains in the cytosol, whereas IL-1β is processed and cleaved into its active form by IL-1β converting enzyme (ICE). Additionally, direct stimulation with IL-1β can activate the transcription of specific inflammatory genes such as TNF and IL-8. Finally, once bound to its receptor, IL-1 transmits a downstream signaling cascade similar to that observed with TLR binding.

Interleukin 6: Although widely studied throughout the 1980s, IL-6 was not identified as such until long after having first been cloned in 1980 by Weissenbach et al. While nearly all nucleated cells have been reported to express IL-6, it is most often produced by monocytes/macrophages in response to endotoxin stimulation. During the acute phase response, IL-6 can induce B cell proliferation as well as regulate hepatic production of acute phase proteins.

Chemokines: Leukocyte migration dictates the initiation, maintenance, and repair process of inflammation. These processes employ positive/start signals as well as negative/stop signals. Chemokines are start signals which are secreted by resident and circulating immune cells during injury/infection. In response to these signals, adhesion molecules are upregulated and leukocytes are recruited from the circulation. The subsequent sequence of events includes the rolling of leukocytes along the endothelium and culminates with transendothelial cell migration into the injured/infected tissue. Chemokines are specialized chemotactic cytokines which, upon release into peripheral blood circulation, recruit leukocytes to the sight of injury. Chemokines secreted by PMNs and Mo at the sight of injury form a concentration gradient towards which additional immune cells migrate. They are characterized by four conserved cysteine residues near their amino termini which form disulfide bonds. Specifically, CC chemokines have two cysteine residues and induce migration of Mo while CXC chemokines have two cysteine residues separated by one amino acid and induce migration of PMNs. CXC chemokines can be further classified as Glutamic acid Leucine-Arginine (ELR)-positive or negative. ELR-positive CXC chemokines contain a Glutamic acid-Leucine-Arginine amino acid sequence immediately before the first cysteine residue in the CXC motif which is critical for activity.


Cytokines and viral infections

Antiviral defense mechanisms are numerous and range from relatively primitive, constitutively expressed, non-specific defenses to sophisticated mechanisms that are specifically Induced in response to viral antigens. The large number of such mechanisms, with distinct yet overlapping roles, attests to their importance in host survival in the face of virus infection. Cytokines and chemokines, produced largely by macrophages and T lymphocytes, play a pivotal role in antiviral immune responses. An important aspect of cytokine/chemokine activity is the induction and orchestration of the antiviral response. A wide range of mechanisms are involved, including alteration of the expression of MHC molecules, adhesion molecules and co-stimulatory molecules, and direct activation or deactivation of immune cells. These changes may lead to the activation of cellular antiviral responses involving NK cells and CTL, and antibody-mediated virus clearance.

In addition to regulating antiviral cellular and humoral immune responses, cytokines and chemokines may have more direct antiviral effects. Thus, the addition of cytokines, such as TNF-a or IFN-g, to virus-infected cells directly suppresses virus replication.

Methods for studying the roles of cytokines and chemokines in virus infection are numerous and two broad approaches are described. Firstly, we constructed recombinant viruses which encode factors of interest. During replication in vivo, these viruses produce the encoded factor which is then secreted from infected cells. The location and extent of virus replication, therefore, determine the levels and sites of cytokine or chemokine production. This approach has enabled us to investigate some of the antiviral and immune-regulating functions of many of these molecules. The second approach involves the study of viral pathogenesis in cytokine-deficient mice produced by targeted gene disruption (gene knockout mice), often in combination with our recombinant viral constructs.


Cytokines and diabetes

Cytokines are believed to play a role in the loss of β-cell function and viability. Mandrup-Poulsen demonstrated that treatment of rat islets with IL-1 results in the inhibition of glucose-stimulated insulin secretion. The inhibitory effects of IL-1 on β-cell function are both time- and concentration-dependent and require mRNA transcription and new protein synthesis. Nitric oxide has been identified as the effector that mediates the deleterious effects of cytokines on β-cell function and viability. Inhibitors of nitric oxide synthase (NOS), such as aminoguanidine and NG-monomethyl-L-arginine (NMMA) prevent the inhibitory effects of IL-1 on glucosestimulated insulin secretion by rat islets, and the effect of IL-1 and IFN-g on mouse and human islets. Furthermore, cytokines fail to inhibit glucose-stimulated insulin secretion in iNOS-deficient mice, and mice expressing iNOS under control of the insulin promoter develop diabetes that can be attenuated by daily administration of aminoguanidine.

IL-1 in rat islets and in combination with IFN-g in mouse and human islets induces iNOS expression and the subsequent production of nitric oxide. Insulin containing β-cells are the primary islet cellular source of nitric oxide production following cytokine treatment. The mechanisms by which nitric oxide impairs β-cell function is by the targeted disruption of iron-sulfur center containing enzymes such as the Krebs cycle enzyme aconitase and electron transport chain complexes I and II This results in decreased mitochondrial function, decreased glucose oxidation to CO2, and reduced cellular ATP content. Importantly, glucose-stimulated insulin secretion is dependent on β-cell depolarization and Ca2+ entry due to the closing of ATP-sensitive K+ channels. This targeted disruption of mitochondrial function is one mechanism by which nitric oxide inhibits insulin secretion.


Cytokines and tumor

Chemokines consist of a superfamily of 50 human ligands and 20 receptors that play an important role in regulation of cell migration. The function of chemokines and their receptors in regards to cancer can be divided into three main categories. The first category is providing directional cues for migration/metastasis, secondly shaping the tumor microenvironment and lastly survival and/or growth signals. The tumor microenvironment contains a large number of cells from the innate and adaptive immune system which become activated by a profile of chemokines, cytokines, growth factors and proteases. Chemokines and their receptors play an important role in modulating angiogenesis, cell recruitment, tumor survival and proliferation resulting in the progression of cancer.

On the other hand, tumor cells can express functional chemokines receptors to induce proliferation, survival, angiogenesis and organ specific metastasis. A recent study has shown a critical role of various cytokines, such as IL-6, IL-8, CCL2, and TGF-β, in the tumor microenvironment and plays an important role in regulating cancer stem cell (CSC). The majority of cytokines are inflammatory chemokines that are inducible and control cell recruitment to sites of inflammation. Cytokines can signal to the microenvironment in an autocrine, paracrine or endocrine fashion to regulate tumor growth, migration/invasion and metastasis.

An inflammation response has a significant impact on the different stages of tumor development such as initiation, promotion, malignant conversion, invasion and metastasis. There is clear evidence showing that inflammation plays a key role in tumorigenesis. It has been clearly evident that the inflammatory microenvironment is necessary for all tumor survival, proliferation and homeostasis. Most cancers are correlated to somatic mutations and/or environmental factors such as chronic inflammation. Roughly, 20% of cancers are linked to chronic infection. Due to the different types of inflammation the tumor microenvironment contains a vast number of different cell types including the innate (neutrophils, macrophages, mast cells, dendritic cells, myeloid derived suppressor cells and natural killer cells) and adaptive immune cells (B and T lymphocytes) along with the surrounding stroma (fibroblasts, endothelial cells, pericytes and mesenchymal cells) and cancer cells.

The different cytokines and chemokines expressed in the tumor microenvironment can significantly affect the tumor’s ability to develop and progress. The cytokines and chemokines can either promote or inhibit tumor growth regardless of the source that is releasing them. The release of anti-tumor cytokines, including IL-12 and IFN-g, can activate downstream effectors such as AP-1, STAT, SMAD and NF-KB in addition to causing the release of IL-6, IL-7, IL-23. Meanwhile TNFα and TNFβ can enhance tumor progression and survival. Many cytokines have been previously shown to activate transcription factors that are crucial for inflammation and tumor growth. Cytokines such as IL-1 and IL-6 can activate downstream transcription factors such as Zeb1 and STAT3 respectively that result in increased tumorigenesis in colorectal cancers. Pharmacological intervention can alter cytokine signaling and decrease tumorigenesis in addition to cancer growth and development. These pharmacological interventions can work to achieve preventative and therapeutic approaches to decrease tumorigenesis and promote cancer survival rates.