A visual review….

A visual review….

physiologic responses that require cytokines

• development of cellular and humoral immune responses, • induction of the inflammatory response,• regulation of hematopoiesis, • control of cellular proliferation and differentiation, • the healing of wounds.• act in an antigen-nonspecific manner.

Cytokines:

• Mediators

• “low” molecular weight proteins• Less than 30 kDa

• Can be classified by recurrent architecture

Functional features• Potent

– Some function at 10-15 Molar

• Local– autocrine– paracrine– (sometimes) endocrine

• Highly interactive– pleiotropic– redundant– synergistic / antagonistic

Functional features• Potent

– Some function at 10-15 Molar

Functional features• Local

– autocrine– paracrine– (sometimes) endocrine

Cytokine Actions• Pleiotropy

– Act on more than one cell type (INF /a b)• Redundancy

– More than one cytokine can do the same thing (IFN /a b and IFN)

• Synergy– Two or more cytokines cooperate to produce an effect

that is different or greater than the combined effect of the two cytokines when functioning separately (IL-12 and IL-8)

• Antagonism– Two or more cytokines work against each other (IL-4

and IL-12)

Functional features• Highly interactive

pleiotropic

redundant

synergistic

antagonistic

How can non-specific cytokines act specifically?

• Only cells expressing receptors for specific cytokines can be activated by them

• Many cytokines have very short half-lives– Only cells in close proximity will be activated

• High concentrations of cytokines are needed for activation– Only cells in close proximity will be activated– May require cell-to cell contact

Cytokine families

I. Hematopoietic family

II. Interferon family

III. Tumor necrosis factor family

IV. Chemokine family

I, II, and III elicit physiological responses.

IV serves as a chemoattractant.

Hematopoietic familyDefined by architecture…

Interferon family….http://www.hon.ch/Library/Theme/Allergy/Glossary/ifn.html

http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?chr=hum_chr.inf&query=Interferon&qchr=&strain=&neighb=off&advsrch=off

http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=147570

Tumor necrosis factor…

• Time to be critical…• Question: What does the text say?• Answer: Not a lot.

– More specifically, there is no text.– There is an entry in Table 12.1 (which

stretches over four pages!) What does it say?

– “Has toxic effects. Induces cytokine secretion…”

So, something’s amiss…

• Try another approach.• Cytokines are soluble immunomodulators.• That means that they are ligands.• Question: Can they be characterized in

terms of their receptors?• Answer: Yes.

Cytokine Receptors

• Expression of cytokine receptors controls the ability of a cytokine to act on a cell.

• Cell activation increases cytokine receptor expression.

Cytokine ReceptorFamilies

5 different families of receptors based on common structural motifs. --> see book for more details

IL-2 Receptor Subfamily Shared common g subunit

Only IL-2 and IL-15 have unique alpha subunit

GM-CSF Receptor Subfamily Hematopoietin Receptors

IL-3, IL-5GM-CSF activate commonb subunit.

Cytokine Receptor Signal through JAKs and STATs

The receptor families:

Of receptors and signal transduction…• Receptors for cytokines have surface

component, single transmembrane component, and cytoplasmic tails.

• Commonly multimeric. The protomers can associate in different groupings; the various associations have different affinities for cytokines; hence, different intensities of signaling.

• The different protomers also have different signaling functions, e. g. phosphorylation sites.

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Signal Transductions often involves phosphorylation:

• Kinase- enzyme that places phosphate groups on proteins.– Tyrosine kinase-*– Serine-threonine kinase-– Histidine kinase

• Either receptor is a kinase OR associates with one.• Phosphatase- enzyme that removes phosphates

– Regulatory- OFF

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Tyrosine kinase associated receptors

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Cytokine signal transduction depends upon a cytoplasmic protein tyrosine kinase:

• Janus Kinase Family (JAKs):– “2 heads”- 2 symmetrical kinase domains.

– Cytoplasmic tyrosine kinases

• Associate with cytoplasmic tails of the receptors.

• Function-

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Who are the activated Transcription Factors?

• STATs- Signal Transducer & Activator of Txn:– Contain SH2 domains:

• Binds phosphorylated tyr.• Permits dimer formation.• Activated dimers function as txn activators (DNA

binding domain).

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SH2 domain

• SH2 domains creates a hole so that the tyr-P from other proteins can plug in!

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• Fig 6-25, p. 165, Parham.

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Model applies to other cytokine receptors: (IL2)

• http://binfo.ym.edu.tw/mb/images/stat_dimer.gif

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How do cytokines mediate specific results in different cell types?

• 1. Use of individual receptors or different subfamily of receptors.

• 2. There are different STAT proteins & different JAKs.

– Recognize different genes!

• 3. Same STATs may recognize different genes in different cell types.

What are the principal signaling systems?

JAK’s

&

STAT’s

OK, let’s look at a complex image

And, an important example…

And, an update…

VI. CD4+ T helper SubsetsTh1/Th2 Cytokine Bias

• CD4+ Thelper cells can be divided into subsets based on their cytokine production.

• Th1 cells produce IL-2, IFN-g, TNF-b CKs which activate cell mediated immunity

• Th2 cells activate IL-4, IL-6, IL-10

CKs that activate humoral immunity These Th subsets were originally identified using mouse T cell clones.

Mouse Th Subset

Cytokine Th1 Th2

Table 12-4 from Goldsby

Th0 ---> Th1 or Th2Original mouse experiments on Th cells

(Mosmann et al (DNAX) 1986 J Immunol)

Antigen specific T cells placed in culture with antigen and APCs to make T cell lines.

• Spleen cells (Th0) add IL-12 Th1 cells IL-2, IFN-g, TNF-b

• Spleen cells (Th0) add IL-4 Th2 cells IL-4, IL-6,IL-10

• (Th0 --precursor cell that produces IL-2, IL-4, and IFN-g.)

Th1/Th2 Naïve Th0 IL-2, IL-4, IFN-g

IL-4IL-12

EffectorTh1 cellIL-2, IFN-g

EffectorTh2 cellIL-4, IL-6, IL-10

Th1/Th2 AntagonismIL-4 blocks Th1 IFN-g blocks Th2

IL-4IL-12

Th1 cellIL-2, IFN-g

Th2 cellIL-4, IL-6, IL-10

IFN-gIL-4

Th1/Th2 RegulationT-bet is a transcription factor that is required

for Th1 specific genes such as IL-12Rb

IL-12

Th1 cellIL-2, IFN-g

Th2 cellIL-4, IL-6, IL-10

IFN-gEnhances T-betIL-4 blocks T-bet

IL-4 vs IFN-g• T-bet (Th1 associated)

activated by IFN-g and turned off by IL-4.

• Conversely in Th2 transcription factor GATA-3 activated by IL-4 turned off by IFN-g.

Role for Th1 vs Th2 in Immune Response

• Both subsets activated in lymph nodes (LN) immune responses to complex antigens.

• Th1 cells leave LN to find activated endothelium tissue to activate macrophages.

• Th2 cells can stay in LN to activate B cells.

What controls Th1 vs Th2?

1) Amount of antigen. Mouse experiments originally showed high dose for Th1.

2) MHC and TCR affinity. High affinity TCR = Th1.

3) Dendritic cell subsets during activation. APC subsets activate Th1 or Th2 preferentially.

4) Toll-like receptor activation.

Influence of APC Subsets on Th1/ Th2Dendritic cell

• Myeloid-like dendritic cells produce abundant IL-12 and drive Th1.

• Lymphoid-like dendritic cells produce low levels of IL-12 are permissive for Th2.

Toll-like receptors (TLRs) Influence of APCs on Th1/ Th2

• Evidence for TLR activation influencing Dendritic cell maturation. – TLR9 binds bacterial CpG DNA – TLR4 binding to bacterial heat shock proteins

• TLR activation induces APC expression of IL-12, IL-23, IL-27 Th1

TLR vs IL-12 in Th1/ Th2 development

• New evidence suggests that TLR activation influencing Th1 outcome through initiation of TLR adapter molecule MyD88.

• May be more important than IL-12 for Th1.

Th Cytokine Bias in DiseaseExamples

Leishmania in mice (Richard Locksley)• C57Bl.6 mice mice have Th1 immune response and

resolve infection. • BALB/c mice produce Th2 cytokines unable to control

Leishmania lesions.

Leprosy in Humans (Robert Modlin)• Tuberculoid form has Th1 response and limits disease

(healing). • Lepromatous form has Th2 response and uncontrolled

disease (leprosy).

Th Cytokine Bias in Disease:LeprosySkin disease caused by Mycobacterium leprae

Lepromatous: has Th2 response and uncontrolled disease (leprosy).

Tuberculoid: has Th1 response and limits disease (healing).

Cytokine Bias in Leprosy

RNA from skin lesions of patients

X-Linked SCID Common g chain Deficiency

• Mutation in g chain so unable to signal through IL-2, IL-4, IL-7, IL-9, IL-15.

• No T cells abnormal thymus.

• Immunocompromisedsusceptible to infections.

SCID Patient with severe Candida in mouth.

Cytokine Receptor SignalingJAKs and STATs (Model of Signal)

Binding of cytokine ligand brings together receptor subunits

JAKs (Janus Associated Kinases) are tyrosine kinases that phosphorylate tyrosines.

STAT (Signal Transducers andActivators of Transcription ) dimerize for function.

JAKs and STATsUsage by CK Receptors

Overlapping and Unique

JAK1 is commonly used by CKs from completelydifferent CKR families

STAT6 is ONLY usedIL-4.

The Yin and Yang

of Th1 and Th2

Immune Responses

Measuring Cytokines

• Protein amount by ELISA. Good for in vitro experiments.

• Protein amount by bioactivity assay using CK dependent cell lines.

• RNA message by PCR.

Antigen Capture ELISA for IL-2