Cytokines and Their Functions

Lecture Outline

What are cytokines and what do they do?
How do cytokines exert their effects?
Key cytokines that mediate immunity and hematopoiesis.
Function of cytokine TNF.
Chemokines and chemokine receptors.

Cells of the Immune System

Immune system divided into:
- Innate Immunity (rapid response):
  - Dendritic cell
  - Mast cell
  - Macrophage
  - Natural killer cell
  - Complement protein
  - Basophil
  - Eosinophil
  - Neutrophil
- Adaptive Immunity (slow response):
  - B cell
  - γδ T cell
  - T cell
  - Antibodies
  - Natural killer T cell
  - CD4+ T cell
  - CD8+ T cell
  - Granulocytes

Cell Communication

Cell-Contact Dependent vs. Independent: How do immune cells communicate among themselves and with non-immune cells?
Lymphoid vs. Non-Lymphoid Tissues: Lymph node, Lung alveoli

Cytokines as Regulators of Immune Response

A low-molecular weight (MW) protein produced by one cell that acts on itself or another cell by binding to cell-surface receptors.
Can deliver a positive or negative signal for immune activation.
A messenger that regulates immune responses.

Cytokine Production

Cytokines are not usually stored as preformed molecules.
Cytokines are synthesized and released rapidly after cellular stimulation (i.e., cytokines are usually produced by activated cells).
Cytokine gene transcriptional activation is transient, and most cytokine mRNA is unstable (to ensure cytokine synthesis is brief and self-limiting).
One cell can simultaneously produce multiple cytokines.

How Cytokines Exert Their Effects: Cytokine Receptors

Cytokines act on their target cells by binding specific membrane receptors.
All receptors consist of one or more transmembrane proteins (receptor sub-unit or chain):
- Extracellular domains for cytokine binding.
- Cytoplasmic tails for initiating signaling pathway.

Types of Cytokines

Include Interleukins (IL, from IL-1 to IL-37), Interferons (IFN), Tumor Necrosis Factors (TNF), and growth factors (GM-CSF).
Biological activity:
- Interferons (type I, II, and III).
- Pro-inflammatory cytokines (IL-1, IL-6, TNF…).
- Anti-inflammatory cytokines (IL-10, TGF-β…).
- T helper cytokines (IFN-γ, IL-4, IL-21…).
Grouped based on:
- Receptor structure: Type I vs. type II cytokines.
Cytokines can be defined by cellular source:
- Lymphokines (produced by lymphocytes, IL-2, IFN-γ…).
- Monokines (monocytes/macrophages, IL-1, IL-12…).
- Innate vs. adaptive cytokines.

Cytokine Receptor Families

Type I cytokine (hemopoietin) receptors
- Ligands: IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-12, IL-13, IL-15, GM-CSF, G-CSF
- Features: Conserved cysteines, WSXWS motif, Jak-STAT signaling pathway
Type II cytokine receptors
- Ligands: IFN-α/β, IFN-γ, IFN-I, IL-10, IL-20, IL-24, IL-26
- Features: Jak-STAT signaling pathway
TNF receptor family
- Ligands: TNF-α, TNF-β, LT, CD40, FasL, BAFF, April, Ox40, GITR, NGF
- Features: TRAF signaling pathway
IL-1 receptor family
- Ligands: IL-1, IL-18
- Features: IRAK signaling pathway
G-protein-coupled receptors
- Ligands: Chemokines
- Features: G proteins signaling pathway

Regulation of Cytokine Activity

Three mechanisms that prevent cytokines from activating cells in a non-specific fashion during the immune response:

Regulated cytokine receptor expression
Short half-lives of most cytokines in body fluids
Cytokines and target cells need to be in relatively close proximity to allow effective concentration gradients to be established

Common Properties of Cytokines: Pleiotropism, Redundancy, Synergy, and Antagonism

Pleiotropic: Act on different target cells.
Antagonistic: Inhibition of one cytokine's effects by another.
Synergistic: Cooperative effect of multiple cytokines.
Redundant: Multiple cytokines have the same effect.
IFN-γ dependent host control of intracellular pathogen infection

What do Cytokines Do?

Can classify cytokines into 3 major functional categories:

Mediators and regulators of innate immunity
Mediators and regulators of adaptive immunity
Stimulators of hematopoiesis

Key Cytokines That Mediate Immune Responses

Important component of the early innate immune response to danger, viruses, and bacteria.
Major pro-inflammatory cytokines of innate immunity include:
- IL-12, TNF, IL-1, IL-6, and type I interferons (IFNs).
Major effector cytokines of adaptive immunity include:
- IFN-γ (type II IFN) and IL-4.

Interferons (IFNs)

Interferons were discovered based on their ability to interfere with viral infection.
Now recognized as a family of pleiotropic cytokines that play an essential role in controlling viral, intracellular bacterial, and parasitic pathogens.
There are three major IFN families:
- Type I (α, β, ε, κ, and ω)
- Type II (γ)
- Type III (λ)
Type I and III IFNs: produced by tissue cells and innate immune cells. Essential for anti-viral immunity.
Type II IFN (IFN-γ): produced predominantly by activated lymphocytes. Important for control of intracellular bacterial and parasitic infection.

Biologic Actions of Type I Interferons (IFN)

A large family of structurally related cytokines (single IFN-b and 13 IFN-a) that mediate the early innate immune response to viral infections.
The most potent stimuli for type I IFNs synthesis are viral nucleic acids (RE. innate lecture on RIG-I, TLR etc).
The antiviral action of type I IFN is primarily a paracrine action in that a virally infected cell secretes IFNs to act on and protect neighboring cells that are not yet infected.

Biologic Actions of Type II Interferons (IFN)

IFN-γ is the only member of type II IFN.
Mainly produced by activated lymphocytes.
A major phagocyte-activating cytokine.
Upregulates MHC class I and II molecules.
Induces antimicrobial substances, such as nitric oxide.

Cytokines: Regulators of Immune Response - Mode of Action

Autocrine
Paracrine
Endocrine

Tumor Necrosis Factor (TNF)

Principle mediator of acute inflammation produced in response to bacteria and other infectious agents.
Large amounts of TNF can be produced in response to gram+ and gram- bacteria.
LPS is the most potent stimulus for eliciting TNF production.
TNF is produced by macrophages, DCs, Th1 cells, and other cell types.
Preformed TNF is quickly released from mast cells.
IFN-γ produced by T cells and NK cells augments TNF production by LPS-activated macrophages.

Local and Systemic Actions of Cytokines in Inflammation

Local Inflammation:
- Endothelial cells: Increased adhesion molecule expression and permeability
- Leukocytes: Activation
Systemic Protective Effects:
- Brain: Fever (IL-1, IL-6, TNF)
- Liver: Acute phase protein synthesis (IL-1, IL-6, TNF)
- Bone marrow: Leukocyte production (IL-1, IL-6, TNF, chemokines)
Systemic Pathologic Effects:
- Heart: Low output (TNF, IL-1)
- Endothelial cells/blood vessel: Thrombus formation and increased permeability (TNF, IL-1)
- Multiple tissues: Insulin resistance (TNF, IL-1, IL-6)
- Skeletal muscle: Wasting

Functions of TNF: Local Inflammation

Stimulates the recruitment of neutrophils and monocytes to sites of infection and activates these cells to eradicate microbes.
Induces endothelial cells (ECs) to express adhesion molecules, making them more adhesive for leukocytes.
Stimulates ECs and macrophages to secrete chemokines, enhancing the affinity of leukocyte integrins for their ligands.
Acts on mononuclear phagocytes to stimulate IL-1 production, which functions much like TNF.
Induces DC migration to lymph nodes.

Functions of TNF: Systemic Protective Effects

In severe infections, TNF is produced in large amounts and causes systemic effects.
Large amounts of TNF can enter the bloodstream and act at distant sites as an endocrine hormone.
Can act on the hypothalamus (brain) to induce fever.
Acts on hepatocytes (liver) to increase synthesis of serum proteins (e.g., serum amyloid A & fibrinogen).
Prolonged production of TNF causes wasting of muscle and fat cells (cachexia).

Functions of TNF: Pathogenic Effect (Septic Shock)

High concentrations of TNF cause the pathologic abnormalities of septic shock.
Reduction in myocardial contractility.
Disseminated intravascular thrombosis, leading to organ failure.
Causes severe metabolic disturbances, including a fall in blood glucose (hypoglycemia).

Cytokines Involved in Hematopoiesis

Stem cell factor promotes self-renewal of stem cells.
IL-3, GM-CSF, IL-1, and IL-6 promote the differentiation of myeloid progenitors.
IL-7 is important for the development of lymphoid progenitors in the thymus.
Erythropoietin promotes the development of erythroid CFU.
Thrombopoietin and IL-11 promote the development of megakaryocytes.
IL-5 promotes the development of eosinophil CFU and basophil CFU.
G-CSF promotes the development of granulocytes.
M-CSF promotes the development of monocytes.

Chemokines (chemotactic cytokine)

Divided into 4 families (CC, CXC, C, and CX3C) based on the number and location of two of four conserved cysteine residues.
The chemokines of the CC and CXC subfamilies are produced by leukocytes and by several types of tissue cells, such as endothelial cells and epithelial cells.
Induced by microbial products and cytokines (IL-1, IL-17, and TNF).
Chemokine receptors are expressed on all leukocytes. The receptors exhibit overlapping specificity for chemokines within each family.
The pattern of cellular expression of the receptors determines which cell types respond to which chemokines.

Biological Functions of Chemokines

Interaction of chemokine and chemokine receptor stimulates cytoskeletal changes, leading to increased cell motility.
Critical in the development of lymphoid organs.
Required for the migration of dendritic cells from the sites of infection into draining lymph nodes.
CCR5 and CXCR4 are co-receptors for HIV.
Essential for recruiting circulating leukocytes from blood vessels into extravascular sites, partly through enhancing integrin affinity.

Integrin Activation by Chemokines

Integrins: heterodimeric cell surface protein composed of 2 polypeptides.
Respond to intracellular signals (e.g., chemokines) by rapidly increasing their affinity to their ligands.
Mediate adhesion of cells to other cells or the extracellular matrix.
Essential for the recruitment of leukocytes into the sites of infection or injury.

Leukocyte-Endothelial Interactions and Leukocyte Recruitment into Tissues

Leukocyte recruitment from the blood into tissues requires adhesion of the leukocytes to the endothelial lining of postcapillary venules and then movement through the endothelium and vessel wall into the extravascular tissue.
This is a multistep process in which each step is orchestrated by different types of adhesion molecules and chemokines:
- Selectin-mediated rolling of leukocytes on endothelium.
- Chemokine-mediated increase in affinity of integrins.
- Stable integrin-mediated arrest of leukocytes on endothelium.
- Transmigration of leukocytes through the endothelium.

Positioning of Lymphocyte Populations by Chemokines in Lymphoid Tissues

CCL21 is a ligand of CCR7 expressed on naïve T cells.
B220: a marker for B cells.
CD3: a marker of T cells.

Summary

Main types and functions of cytokines.
Major functional categories based on their principal biological functions:
- Mediators of innate and adaptive immunity.
- Stimulators of hematopoiesis.
Cytokines are soluble “messenger” proteins that play a key role in the host defense against pathogens and provide links between innate and adaptive immune responses.