Cytokines and Chemokines

Leo Carr

Immunopharmacology

March 1, 2006

Cellular interaction made simple

Cytokine Basics

• Cytokine or immunocytokine is a generic name used to describe a diverse group of soluble proteins and peptides which act as humoral regulators at nano- to- picomolar concentrations

• Cytokines modulate the functional activities of individual cells and tissues both under normal and pathologic conditions

Cytokine Basics

• The term cytokine is used to separate this group of immunomodulatory proteins from other growth factors that modulate non-immune cells

• Unfortunately, Growth factors and cytokines both act as cellular survival factors by preventing apoptosis - this fact adds confusion to the understanding and naming of cytokines

Cytokine Names

• Interleukins - produced exclusively by leukocytes

• Lymphokines - produced by lymphocytes

• Monokines - produced exclusively by monocytes

• Interferons - involved in antiviral responses

• Colony Stimulating Factors - support the growth of cells in semisolid medias

• Chemokines - promote chemotaxis.

Cytokine Basics

• Cytokines behave like classical hormones in that they act at a systemic level, affecting, inflammation, septic shock, acute phase reactions, wound healing, and the neuroimmune network

• Unlike hormones, cytokines are not produced by specialized cells or in specialized glands - they have no single organ source (this helps clear some of the confusion)

Cytokine Activity

• Cytokine actions may be characterized as:

–Autocrine (self modulating)

–Paracrine (modulating cells in the immediate surroundings)

–Juxtacrine (modulating through cell membrane signaling)

–Retrocrine (modulating to stop host defense)

Cytokine Basics

• Defining cytokines based only on their producer cells or target cells is inaccurate

• The historic cytokine concept of "one producer cell -one cytokine -one target cell" has been falsified for almost every cytokine

Cytokine Basics

• Almost all Cytokines are pleiotropic effectors showing multiple biological activities

• Also, multiple cytokines often have overlapping activities

• A single cell frequently interacts with multiple cytokines with seemingly identical responses - cross-talk

Cytokine Assays

• The biological activities of cytokines can be measured by a variety of bioassays which may employ factor-dependent cell lines, or antibodies (ELISA)

• RT-PCR quantitation of cytokines detects the presence of mRNA encoding specific cytokines

Soluble - Vs - Membrane

• Many Membrane-bound cytokine forms have been characterized

• Some cytokines may also be associated with the extracellular matrix

• Switching between soluble and membrane bound forms of cytokines may be an important immunoregulatory event

Cytokine Regulation

• Most Cytokines are not stored inside cells (exceptions are, for example TGF- and PDGF which are stored in platelets)

• The expression of most Cytokines is strictly regulated - factors are usually produced only by activated cells in response to an induction signal

• Expression is normally transient and can be regulated at all levels of gene expression

Cytokine Action/Timing

• A hierarchical order of cytokine actions has been observed with some early cytokines pre-activating cells so that they then can respond to late-acting cytokines

• Many cytokines induce the synthesis of novel gene products once they have bound to their respective receptors

Cytokine Transport

• Cytokine mediators can be transported quickly to remote areas of a multicellular organism

• They can address multiple target cells and can be degraded quickly. Concentration gradients can be used to elicit specific responses

• These capabilities far exceed the interaction provided by cell-to-cell contacts within a multicellular organism

Pre/Postnatal Cytokines

• Cytokines are important mediators involved in embryogenesis and organ development and their activities in these processes may differ from those observed postnatally

• They also play a key role in neuroimmunological, neuroendocrinological, and neuroregulatory processes

Cytokine - Regulation

• Cytokines are important positive or negative regulators of mitosis, differentiation, migration,cell survival, apoptosis, and transformation-oncogene

Virus Effects• Viral infectious agents exploit the cytokine repertoire

to evade immune responses of the host

• Virus-encoded factors appear to affect the activities of cytokines in at least four different ways: –by inhibiting the synthesis and release of cytokines from

infected cells

–by interfering with the interaction between cytokines and their receptors

Virus Effects

• Virus-encoded factors appear to affect the activities of cytokines in at least four different ways: –by inhibiting signal transmission pathways of

cytokines– by synthesizing virus-encoded cytokines that

antagonize the effects of host cytokines mediating antiviral processes (viroceptor and virokines)

Cytokines In Therapy

• The many specific activities of individual cytokines have been the basis for current concepts of therapeutic intervention - particularly, hematopoietic malfunctions and tumor therapy

• Applications involve the support of chemo- and radiotherapy, bone marrow transplantation, and general immunostimulation - adoptive immunotherapy

Cytokine Shape/Type

• Among the many different cytokines, the Type I cytokines share a similar four -helical structure and correspondingly, their receptors also share characteristic features that have led to their description as the cytokine receptor superfamily, or Type I cytokine receptors

Cytokine Shape/Type

• Despite extremely limited amino acid sequence similarities between different Type I cytokines - all Type I cytokines have similar three-dimensional structures

Cytokine Type I Subgroups

• Type I cytokines can be further divided into either short-chain or long-chain

• The short-chain cytokines include: –IL-2, IL-3, IL-4, IL-5, IL-7, IL-9, IL-13, IL-15

–Granulocyte-macrophage colony-stimulating factor (GM-CSF)

–Monocyte-CSF (M-CSF),

–Stem cell factor (SCF),

Cytokine Type I Subgroups

• Long-chain cytokines include:–IL-6, IL-11

–Erythropoietin

–Thrombopoietin

–Leptin

–Leukemia inhibitory factor (LIF)

–Oncostatin M (OSM)

–Ciliary neurotrophic factor (CNTF)

– Cardiotrophin-1 (CT-1)

–Granulocyte colony-stimulating factor (G-CSF)

Cytokine Receptors

Cytokine Receptors

• The receptors for five different immunologically important cytokines, IL-2, IL-4, IL-7, IL-9, and IL-15, share the common cytokine receptor chain, c (CD132) p.515 in Abbas

• These cytokines are all short-chain Type I cytokines

Cytokines Receptors

IL-2

IL-2 Production

• Mature IL-2 is a peptide 133 amino acids long

• Produced mainly by mitogen activated CD4+T-lymphocytes

• Originally called T-cell growth factor (TCGF)• Transformed T-cells and B-cells, leukemia

cells,LAK cells (Lymphokine-activated killer cells) and NK-cells also secrete IL-2

IL-2 Actions/Modulation

• Secondary signals are required for maximal expression of IL-2

• Resting cells do not produce IL-2

• Induces cell cycle progression in resting cells

• Allows clonal expansion of activated T-lymphocytes

• Growth/expansion effect is modulated by hormones such as prolactin

IL-2 Actions/Modulation

• In vitro synthesis of IL-2 is inhibited by dexamethasone or CSA

• Vitamin E can enhance IL-2 production

• With increasing age, the antigen/mitogen-stimulated synthesis of IL-2 (T-cell-mediated immune response) decreases

IL-2 Facts

• IL-2 damages the blood-brain barrier and the integrity of the endothelium of brain vessels

• IL-2 does not have a saturable transport system across the blood-brain barrier

• Electrophysiological alterations may cause neuropsychiatric side effects such as fatigue, disorientation, and depression, frequently observed under IL-2 therapy

IL-2 Summary• IL-2 important actions:

–It can increase immunoglobulin synthesis and J-chain transcription

–Proliferation in B cells (with IL-4)

–potently augment the cytolytic activity of natural killer (NK) cells

– induce the cytolytic activity of lymphokine-activated killer (LAK) cells

–Due to its effects on T-cells and B-cells IL-2 is a central regulator of immune response

–Passes BBB

IL-4

IL-4 Production

• IL-4, like IL-2, is produced principally by activated CD4+ T cells

• It is also produced by natural killer cells, and by mast cells and basophils

IL-4 Actions/Modulation

• IL-4 is the major B-cell growth factor

• Vital for immunoglobulin class switch IgG to IgE and inhibits the synthesis of IgM and other IgG subtypes

• IL-4 induces expression of class II major histocompatibility complex (MHC) molecules on B cells and increases cell surface expression of FceRII (CD23)

IL-4 Actions/Modulation

• IL-4 can inhibit responses of cells to IL-2

• IL-4 can also exert actions on:–T-cells (proliferation)

–Macrophages

–Hematopoietic precursor cells

–Stromal cells

–Fibroblasts

–Myelomas (inhibiting IL-6 - myeloma growth factor)

IL-7

IL-7 Production

• IL-7 is not a lymphokine but instead is produced by stromal cells

• IL-7 is secreted constitutively into bone marrow stroma, and thymic cells

• Murine and human keratinocytes have been shown also to express and secrete IL-7

IL-7 Actions/Modulation

• Its major role is to enhance thymocyte growth, survival, and differentiation

• It also has some activity for the growth of mature T- cells

• Although important for growth of murine pre-B cells, it is now clear that human B cells can develop in the absence of IL-7

IL-7 Actions/Modulation

• IL-7 stimulates the proliferation of pre-B and pro-B-cells without affecting their differentiation

• IL-7 can replace murine bone marrow stromal cells in supporting the extended growth of both pre-B-cells and pro-B-cells

• It does not act on mature B-cells

IL-9

IL-9 Production

• IL-9 was originally described as a murine T-cell growth factor. Human and murine IL-9 are 126 amino acids long

• IL-9 is produced by activated T-cells and supports the growth of T-helper (Th) clones but not CD8+ cytolytic clones

• In contrast to IL-2, its production is much more delayed, suggesting late/secondary signaling

IL-9 Production

• IL-9 can be isolated from culture of mitogen- or antigen-stimulated T helper cells

• In primary lymphocyte cultures it is produced predominantly by cells expressing CD4

• The synthesis of IL-9 can be induced by calcium ionophores (Ionomycin)

IL-9 Actions/Modulation

• IL-9 stimulates the proliferation of a number of T helper cell clones in the absence of antigens or antigen-presenting cells

• It does not promote the proliferation of freshly isolated T-cells or cytolytic T-cells

• The activity as a growth factor seems restricted to a distinct subpopulation of cells or to a particular late activation state

IL-15

IL-15 Production

• IL-15 is the most recently identified Type I cytokine

• Although IL-15 messenger RNA (mRNA) is produced by a range of non-lymphocytic cell types, it is difficult to detect IL-15 protein production

IL-15 Actions/Modulation

• IL-15 receptors are widely expressed, and it is becoming more clear that IL-15 plays a major role related to NK cell development and cytolytic activity

Type I Cytokines

• IL-2, IL-4, IL-7, IL-9, and IL-15 collectively exhibit overlapping roles related to T cells, NK cells, B cells, and mast cells, and together would be expected to play vital roles in normal development and function of these cells

Break

Chemokines

• Chemoattractant Cytokines

• Small (8-10kd) secreted heparin-binding

• Promote recruitment and activation of leukocytes

• Can be divided into subclasses by virtue of structural properties

Chemokine Subclasses

• Most chemokines have 4 cysteine residues which form disulphide bonds

• CC class – The first two cysteines are adjacent (example: MCP-1, RANTES)

• CXC class- The first two cysteines are not adjacent (example: IL-8)

• C class – Only has 2 cysteines not 4 (example: Lymphotactin)

• CX3C class – Has 3 amino acids between the first two cysteines and a different N-terminal

Chemokine Subclasses

Chemokine Function

Chemokine Function

• Recruitment of inflammatory cells to infection

• Provide immune homeostasis

• Important in host defense against bacteria, parasitic and viral infection

• Role in wound healing

• Role in hematopoesis and angiogenesis under study

Chemokine Function

• Chemokines promote chemotaxis in the direction of highest concentration

Chemokine Effects

• Chemokines promote shape change in cells

• Polymerization and breakdown of actin

• Breakdown of lamellipodia (cell anchoring arms/legs)

• Upregulate integrins causing cell adhesion in vascular endothelium

• Promote cell diapedesis

Chemokine Effects

Chemokine Signaling

7 transmembrane g-coupled signaling on 3rd loop

Chemokine Receptors

• Specific receptors bind specific chemokine–CXCR1 binds IL-8

• There are also shared receptors–CCR1 binds MIP-1a,RANTES, MCP-2 and MCP3

• Promiscuous receptors bind any class

• Viral encoded receptors–Encoded by cytomegalovirus- CMV U28 binds CC

chemokines

Chemokine Receptors

Chemokines in Disease

• MIP-1, and RANTES were discovered to be HIV suppressor factors

• CCR2 Ko mice and MCP-1 Ko show reduced atherosclerotic lesions

Chemokines in Transplantation

• Many chemokines are expressed in transplantation (RANTES, MIP-1, IP-10)

• CXCR3 Ko mice show reduced graft rejection

• Antibodies to CXCR3 ligands imiprove graft survival

Chemokines in Transplantation

• Chemokine receptors are differentially expressed on effector T-cells

Chemokine/Cytokine Interaction

• Allergic reaction

Chemokine/Cytokine Interaction• Dermatitis

Therapeutic Trials

Questions?