Abbreviations: Antibody Ab, Interleukin IL, Natural Killer NK, Tumor Necrosis Factor TNF

FUN2: 11:00 – 12:00Scribe: Brannon Heape

Monday, October 22, 2008Proof: Ryan O’Neill

Dr. BarnumCytokinesPage1 of 5

Abbreviations: Antibody – Ab, Interleukin – IL, Natural Killer – NK, Tumor Necrosis Factor – TNF, Interferon - IFN

Specific Description of the Topic Being Discussed Today: Cytokines

Introduction [S1] and [S2]: Don’t look at the picture on slide 2; just know that cytokines are critical in the function of immune responses.
There are over 100 different cytokines now and more keep being discovered.
Cytokines are important because: [S3]
Master regulators of the immune response: they are responsible for stem cell cycling, development of progenitor cell lines, growth and differentiation of all immune system cells, and activation of effector cells (B, T, and NK cells).
They are also critical for the initiation and resolution (shutting down) of immune responses once you have a fully matured immune system.
Basically cytokines are important to everything.
Therapeutic reagents:
Arthritis patients take an Ab for TNF so that the TNF cytokine cannot carry out its pro-inflammatory effects in areas such as joints.
Multiple Sclerosis patients take IFN-B, a cytokine that helps down-regulate inflammatory responses.
Cancer patients treated with chemo have blood counts that are low. So they are treated with erythropoietin to get there blood cell count back up.
There is growing use of cytokines in the dental and optometry fields so this is something that may be useful for us one day when treating patients.
Cytokine Nomenclature: [S4] some of these are outdated (monokines and lymphokines) but important to know because they are still used by some
Monokines - cytokines produced by monocytes only
Lymphokines – cytokines produced by activated T cells
Interleukins – were originally described as cytokines produced by leukocytes which acted only on leukocytes but this isn’t really true
However, this term “interleukin” has stuck and today we have approximately 30 different types of interleukins.
Interferons – cytokines important in controlling and clearing up viral infections and augmenting immune responses (particularly IFN-γ).
One of first cytokines to be described.
Colony-Stimulating Factors – cytokines important in the maturation of leukocytes.
Examples are IL-3 and IL-7 and other molecules that help drive progenitor cells down different pathways to full maturity.
Chemokines – cytokines important in directed migration of leukocytes during immune responses
Growth Factors – there are lots of cytokines that are involved in growth such as stem cell factors, fibroblast factors, and others that are still called growth factors but are really just a type of cytokine.
Cytokine Functions Are: [S5]
Pleiotropic: means they do a lot of different things. This allows for a hundred different molecules regulating lots of different cell types and lots of different functions of these cells.
Ex: IL-1 is very important in pro-inflammatory responses helping to eliminate pathogens in the innate immune response, but it also modulates T cells and their responses thus also playing a role in the adaptive immune response.
So almost all cytokines are functioning in several ways. As long as the cell type expresses the receptor for a certain cytokine that cytokine has the ability to modulate that cell in an immune response.
Redundant: TNF-α does a lot of the same things as IL-1 and IL-6 (all of which are part of the acute phase response, the pro-inflammatory response of invading pathogens).
This means you can be missing a certain cytokine and be okay against an infection.
Synergistic: cytokines can function together to give a synergistic response (greater response).
Ex: Cells treated with just IL-1 had a certain response and cells treated with IFN-α had a certain level of response, but cells treated with both IL-1 and IFN-α had a much higher level of response.
So this is not an additive response but a synergistic response.
Antagonistic: this is important because several cytokines function to down-regulate the immune response when it needs to be shut-off.
IFN-γ is a pro-inflammatory cytokine and IL-4 is an anti-inflammatory cytokine. So IL-4 can down-regulate the responses mediated by IFN-γ.

Cytokine Properties [S6]:
Low molecular weight proteins or glycoproteins (on average have a molecular weight of 10,000).
They can be monomers, dimers, trimers, or any multimeric molecule.
However, when in the multimeric form they cannot function in the monomer form because they must have all of their subunits to become active.
Synthesized in active and inactive (latent) forms
They may sit on the membrane and require cleaving to become active
There are some cytokines that stick to lipoprotein and are involved in trafficking events. They sit and wait for a T or B cell receptor to activate them so they may enter an organ, such as the thymus or spleen.
Secretion is brief and self-limiting (he said this is very important to know)
They are usually secreted in the time frame of minutes to hours.
This allows for a controlled response to a pathogen.
Active at very low concentrations (10^-9 – 10^-12 Molar). It only takes a small amount of cytokine binding to produce a response.
This implies that the affinities for cytokine receptors are quite high.
For a cytokine to have its effect on a cell, the cell must have that specific cytokine receptor present.
You can have all the cytokines you want but if you lack the receptor nothing will happen.
Cytokine Receptors [S7], [S8]
Every cytokine has a receptor – but sometimes they share receptors.
IFN alpha and beta both share the same cytokine receptor.
Receptors are grouped into 5 families
Cytokine receptors can often be multi-chain complexes (multimeric). So you can have single chain, dimers, and trimers as cytokine receptors.
Efficient signaling through cytokine receptors requires multiple events coming together… he called this “multimerization”.
So for the best response you need a lot of cytokine receptors interacting with each other.
Receptor Families: are classified base on structural differences.
Immunoglobulin Superfamily (Ig Domain): these have immunoglobulin domains that are fairly constant throughout the immune response.
These bind IL-1.
Class I Cytokine Receptors: distinguished based on conserved cysteine residues close to the membrane
These bind IL-2, IL-7, IL-9, IL-5 and a whole family.
Chemokine Receptors: have seven-transmebrane receptors that use a G-protein complex inside the cell for signaling.
These bind IL-8.
Class II Cytokine Receptors: bind IFN’s (alpha, beta, and gamma)
TNF Receptors: bind TNF family member molecules that also have conserved cysteine subunits.

**All he said to remember was that there are multiple types of receptors that are classified based on structural features and conserved residues. He said not to memorize all of the above & structures… just know this main point.**

Cytokine Receptor Subfamilies [S9]:
Cognate Receptor Subunit: binds to the cytokine initially.
Signaling Receptor Subunit: this is the part that sends the signal inside the cell (the γ chain of IL-7 is the one he pointed to on the slide but they all have this function).
This is the very important part because without it you cannot respond internally to any signal that binds externally.
Missing IL-2 and IL-7 signaling receptor subunits would affect T cell development and cause immunodeficiency.
Generalized Cytokine Signaling Mechanism [S10]: don’t worry about memorizing a lot of detail here he said; this is just a simplified version of many different ways signaling can occur.
The α-chain Cognate Receptor is here to bind the cytokine and the β-chain signaling receptor to transduce the signal.
The intracellular portion of a cytokine receptor will self-associate with families, here the JAK family of kinases, which phosphorylates the intracellular portion of the cytokine receptor, can self phosphorylate, and phosphorylate STAT molecules.
STAT molecules that become phosphorylated will dimerize and then move into the nucleus and bind to DNA to initiate transcription.
So you get intracellular phosphorylation events upon cytokine binding that ultimately leads to activation or inhibition of transcription within the nucleus.
Cytokine Effects on Target Cells are: [S11]
Autocrine: they work on the cell that produced them.
IL-2 is a good example. It is an important growth factor for T cells. When T cells get tweaked by an antigen it will cause more IL-2 to be made and more IL-2 receptors to be expressed on the cell surface. The IL-2 will then bind back to its own IL-2 receptors.
Paracrine: act on nearby cells.
IFN’s are good examples.
When you get a viral infection, the virally affected host cells will release IFN to the cells immediately adjacent to prevent the virus from spreading to them.
Endocrine: cytokines acting at a distance
The cytokine here almost acts like a hormone. It can enter circulation like a hormone and trigger the brain to make the necessary adjustments to rid the body of the pathogen.

Ex: TNF-α or IL-1 (involved in fever responses)

*So depending on the size and nature of the infection, cytokines can be released and function very locally (which is good when the infection is very localized) or act at an infection that is far away. This allows for the proper control of immune cell responses.*

Hematopoietic Cytokines in the Immune System: [S12]

Cytokines are important for the development of all types of immune system cells.
Once you’ve formed the progenitor cells, certain cytokines will allow for the continuation of that cell type.
How many of these cytokines affect the cell type is still not understood, especially with NK cells.

Cytokines are critical for lymphocyte development: [S13]
Cytokines are very important in B and T cell development.
Ex: Without IL-7 or the IL-7 receptor you will not make B cells and will be deficient in that lymphocyte.
Other factors like stem cell factors and certain adhesive factors are needed as well, but you have to have the cytokines to get an adaptive immune response.

T Cell Maturation and Cytokines: [S14]
You have to have IL-2 for activation, growth, and proliferation after Ag binding.
Differentiation to helper T cells subsets (Th1 and Th2) depends on cytokines.
Th1 cells produce cytokines that are critical in cell-mediated immunity (IFN-γ, IL-2, others); where you’re pushing CD8+ cells to become cytotoxic T cells to fight off viruses.
Th2 cells produce cytokines like IL-4, IL-5, and others that aid in humoral immunity (or Ab production).
B Cell Maturation and Cytokines [S15]: are dependent on multiple cytokines for activation, growth, and proliferation after Ag binding.
IL-6 is a major growth factor for B cells and it is also very important in innate immunity.
So IL-6 is part of the acute phase response where inflammation is trying to recruit many cells.
So this again is an example of a pleiotropic cytokine playing an important role in innate and adaptive immunity.
Multiple cytokines are involved in isotype switching; such as TGF-β which pushes cells to make IgA and IL-4 which makes IgE.
Cytokines in Viral Infections [S16]: this is done by interferons.
Viruses infect the host by binding to a receptor, getting inside the cell, and then taking over the host machinery and duplicating itself numerous times. It will then leave the cell or just cause cell lysis.
Viral genetic material is recognized as foreign by TLR’s (TLR3 recognizes double stranded RNA and TLR8 recognizes single stranded RNA).
When TLR’s bind viral PAMP’s it leads to production of “interferons” which are anti-viral cytokines critical to clearing viral infections.
Viral Responsive TLR’s [S17]:
This is just a diagram showing a viral TLR. They are quite homologous structurally outside the cell and have a common signaling domain inside the cell. This is an old mechanism of recognizing invading pathogens.
Interferons [S18]: there are two major types of interferons: types I and II
Type I: produced by all cells (IFN-α/β); there’s between 20-30
These help get rid of viruses.
Type II:these are produced by active T cells and NK cells (IFN-γ)
Functions of these interferons are in both innate immunity in viral clearance and in adaptive immunity in activation and maturation of lymphocytes.
Interferon anti-viral mechanisms [S19]
Interferons also induce the expression of MHC molecules. MHC molecules are critical in antigen presentation.
There are 2 classes of MHC molecules
MHC Class I: important in viral infections and are expressed on all cells within your body. When a virus gets internalized its proteins are chewed up and placed as a peptide on an MHC Class I molecule and this allows the cytotoxic T lymphocyte to recognize the virally infected cell & destroy it.
MHC Class I allows Ag presentation to cytotoxic T cells.
Some viruses have figured out how to turn off MHC molecule expression.
However, loss of MHC expression by a cell allows targeting by NK cells because the NK cell recognizes any cell in our body without an MHC Class I molecule as foreign.
The NK cell will destroy the virally infected cell.
There are two different mechanisms to destroy virally infected cells: with cytotoxic T cells & NK cells.
One requires the MHC Class I molecule and the other doesn’t.
Interferons also induce the production of cells that degrade RNA. This keeps viral RNA from being made but unfortunately also keeps host RNA from being made. It also stops protein synthesis so that viral proteins can’t be made, but again host proteins can’t be made either. Your body makes the trade off of killing one viral cell and host cell in an effort to prevent the virus from spreading to many host cells.
Two main mechanisms that interferon uses to stop viral infections:

1) induce the expression of molecules (MHC class I) that allow the adaptive immune response to recognize the virus

2) shut down the infected host cell so that the virus cannot replicate.

Cytokines in Host Defense against Bacterial Infection [S20]
Bacteria infect by a number of routes- ingested, inhaled, or through cuts.
The bacteria takes over a niche in your body and will replicate rapidly.
Bacterial PAMPs (such as LPS and flagellin) are recognized as foreign by toll-like receptors (TLR’s)
Bacterial PAMPs binding to TLR’s leads to production of pro-inflammatory cytokines and the acute phase response, a cumbersome process that is critical in clearing the bacterial infection.

Bacterial PAMP Responsive TLRs [S21] : just look at slide, he didn’t say much about it.
Acute Phase Response [S22]: when you get a good bacterial infection your body starts the acute phase response which is a well orchestrated sequence of events to mobilize a metabolic response of the organism to:
Eliminating invading pathogens
Preventing on-going tissue damage
Activate repair processes of damage tissue
Because like he has already said, if you get a heavy bacterial infection your innate immune response will not discriminate your tissue from the pathogen and will cause damage to your own tissue.
The acute phase response is mediated by “proinflammatory” cytokines including TNF-α, IL-1, IL-6, IL-8, and IFN-γ. These are all critical to the response.

Tumor Necrosis Factor-α (TNF-α), IL-6, and IL-1 [S23]: all 3 of these work together
Production of these is induced by LPS and other PAMPS and they are made by macrophages, fibroblasts, and other cells.
Activates myeloid cells, epithelium, and endothelium
Induces production of multiple cytokines
Initiates acute phase response (fever)
Induces adhesion molecule expression
These are however toxic at high levels. They can cause septic shock which will kill you.

Acute Phase Proteins [S24]: causes an increase in lots of molecules like:
Host Defense Proteins:
C reactive protein (CRP), complement, and fibrinogen
Proteinase Inhibitors
C1 inhibitor which limits the complement system damage to host tissue
Alpha1-proteinase inhibitors will block enzymes that damage the pathogen and the host
Anti-oxidants: will tie-up or block the damaging oxidizing molecules produced by the acute phase response
Haptoglobin
Ceruloplasmin
Chemokines [S25]:
There are close to 60 different chemokines that are grouped into multiple families.
Families are classified based on different conserved cysteine residues.
Just know that there are multiple families and how they are classified.
Involved in multiple immune functions including inflammation, cell recruitment, lymphocyte trafficking, lymphoid organ development and wound healing.
As you can see chemokines are very important in several aspects of the immune system response.
Expressed in primary and secondary lymphoid organs
Clinical Applications of Cytokines [S26]: cytokines are becoming much more applicable in therapeutic settings.
Some of your patients will be receiving cytokine therapy for non-dental/optometry conditions and we must know this and realize it when we decide how to treat them.
Cytokine-specific therapies are in use for some dental/optometry conditions.
Cytokine Therapy [S27]:
Interferon-α therapy for chronic myeloid leukemia – puts disease into remission
Soluble TNF-α receptor (Infliximab) is used in therapy for rheumatoid arthritis
It stops TNF from binding to its receptor.
However, the downside of this is that some patients using this as treatment develop multiple sclerosis.
Interferon-β therapy for multiple sclerosis is effective in about 30% of patients.
Procrit (erythropoietin) is being used to boost RBC levels in patients undergoing chemotherapy.
Cytokines in the Eye
Anti-TNF-αAb in refractory posterior uveitis restore activity
Nerve growth factor is used on corneal ulcers
IFNα works with Beckett’s Disease
Cytokine Therapy for the Oral Cavity
Thalidomide (anti-TNF-alpha) therapy for orofaical granulomatosis – clinical resolution
Used to be a drug given to pregnant mother’s in the 50’s but caused birth defects.
Soluble TNF-alpha receptor to fight root resorption.
IFN-α therapy combined with surgery clinically resolves aggressive oral giant cell tumors