Ch 4 Kuby 7e (chapter 12 in 6e)

Receptors and Signaling: Cytokines and Chemokines

Cytokines: a group of low MW proteins/peptides secreted by

leukocytes and other cells in response to stimuli. The principal
producers are dendritic cells, T helper cells and macrophage.

These magical molecules are important because the

cytokine profile determines disease outcome (survival)
and cytokine therapy is increasingly used to save lives.

Human cytokines produced by recombinant DNA

technology are the ultimate natural products- much
more natural than those obtained from plants in rain
forests or organisms that inhabit the sea.

Cytokines are extraordinarily powerful, affecting gene

activation in target cells at picomolar concentrations (10-12
M)!!!! Beyond nanograms (a billionth of a gram) by 1/1000.
Very high dissociation constants= very high affinity.

Interleukins: a group of cytokines secreted by leukocytes that

primarily affect the growth and differentiation of various
hematopoietic and immune system cells.

Chemokines: cytokines that primarily mediate chemotaxis for

different leukocytes and regulate the expression and /or
adhesiveness of leukocyte integrins.

Note interleukin 8, a cytokine that is both a chemokine and

obviously an interleukin.

Chemokine receptor CCR5 that is mutated via deletion to

CCR5 delta 32 ( in homozygous recessive) whites- makes them
resistant to infection by HIV. Why? CCR5 is the co-receptor
for HIV to enter cells.

Normal ligands for CCR5 are, RANTES, MIP-1, and MIP-1,

and they are able to suppress HIV-1 infection in vitro. In
individuals infected with HIV, CCR5-using viruses are the
predominant species isolated during the early stages of viral
infection

News Flash from Immunoleaks!

New HIV drugs, called CCR5 receptor antagonists, have been

designed to interfere with the interaction between CCR5 and
HIV. Examples: Vicriviroc (Schering Plough), Aplaviroc (GW-
873140) (GlaxoSmithKline) and Maraviroc (UK-427857)
(Pfizer). Note: suffix viroc

How did these drug companies develop these antagonists?

Mother Nature taught them- page 114 7e depicts natural

cytokine inhibitors of IL-1 (a proinflammatory cytokine
induces type I anti-viral interferons). Two types:
1. Receptors that do not have signaling domains
2. Soluble forms of IL-1 or other cytokine binding proteins
that the drug companies can mimic.

Why did she develop these? To soak up excess cytokine

and thus fine tune an immune response. IL-1 is highly
proinflammatory (the fever cytokine) and unchecked can
be fatal.
 Classification of cytokines:
A. Autocrine: binds to same cell that secreted it. IL-2
autocrine for CD4+.

B. Paracrine: affects a nearby cell. IL-1 from mac affects

CD4+

C. Endocrine: affects a distant cell. Mac IL-1 affects cells in

hypothalamus of the brain.

III. General Properties/Actions of Cytokines: Figs 4.1, 4.2 4.3

7e (Fig. 12-2 6e)

A. Pleiotropy: many different biological actions, depending on

the target cell
 B. Redundancy: when two or more cytokines have the same
effect.

C. Synergism: combined effects of cytokines is greater than

added effects

D. Antagonism: effects of one cytokine inhibits the action of

another.

E. Cascade effect: one cytokine causes a group of cells to

release other cytokines which can cause other cells to release
other cytokines etc.

Cytokines activate specific T helper cells

Cell Activation via cytokines alters expression of both

Receptors and Adhesion Molecules on a variety of immune
cells

The Immunological Synapse- directs the release of cytokines

from APC to T or Th to B or Tc.

Youtube video of synapse at:

https://youtu.be/gKFXTJ9MjFg
 Methods Used to map the Secretome- the proteins that are
secreted from activated cells.
see advances in pages 111 to 112.

The Cytokine assay technology in Fig 1 on page 112 7e is

commonly called the Cytometric Bead Array or Multiplex
assay. What is the principle of the bead assay? Antibodies
immobilized on beads that fluoresce at different wavelengths.

Function of cytokines Table 4-1 in Kuby 7e (12-1 in 6e):

above is table 4-1 in 7th edition
Page 119 Discovery : Interferons (type I antiviral was first
cytokine discovered, purified and produced by
Genentech/Roche.
Discovered by two scientist who published in French, not
recognized at time.
Issacs and Lindenman 1950s chick chorioallantoic membrane
(CAM) for growth of influenza discovered that heat inactivated
influenza induced an interfering molecule when CAM
challenged with live flu virus.
1978- Roche scientists (Dr. Weissman) and others purified IFN
and cloned the gene. Genetech put the human gene into E. coli.
Roche then bought Genetech for 48 billion dollars.

Type II Interferon gamma or immune interferon: page 120

From: activated T and NK cells, activates macrophages and
dendritic cells and elicits a Th1 or cytotoxic T cells response.
NK cells are initially one of largest sources and they in turn
activate macs/dends to produce a lot of it.

IFN gamma induces intracellular killing of microbes in

macs/dendritic and licenses Dendritic cells for cross
presentation of extracellular obtained antigens in MHCII=
licensing of dendritic cells.

All interferons induce high levels of MHC molecules!

Cytokine Receptors: Five Families of receptor proteins-

we will look at only two.

Class I = immune and hematopoietic receptors for majority of

cytokine binding receptors such as most interleukins, GM-CSF,
G-CSF etc.

On their extracellular domain they have:

a. conserved cysteine residues- CCCC

b. Conserved sequence of Trp-Ser-X-Trp-Ser called

(WSXWS)
 They normally have two peptide chains:

a. Cytokine specific subunit that binds the cytokine

b. Signal transducing subunit- present on cytokine

receptors (recall none on B and T cell receptors)

Note: A remarkable variety of receptors is achieved by

different combinations of common signal transducers with other
receptor peptides. Is the immunological synapse thus more
sophisticated and intricate than the neuronal synapse? I
certainly think so, you will too when you study this material.
Which synapse is more critical to your survival?
 They share common signal-transducing subunits, thus
explaining some of the redundancy and antagonism exhibited
by some cytokines
Check fig. 4-9 (12-7a in 6e) and note the shared colors
(subunits) of the signal transducing unit among the family
 Note common signal-transducing subunit in beta blue,
but unique cytokine binding subunit for different cytokines.
Accounts for redundancy (same effects) among different
cytokines.

IL-2 receptor- as key example of a class I receptor:

Importance: IL-2 is key cytokine for proliferation of T

cells
a. It exists as a trimer (alpha, beta and gamma chains)
following binding of TCR to antigen. Trimer form binds
IL-2 with high affinity and T cells are greatly and more
selectively activated. Other cells like NK express
intermediate affinity receptor.
Check out fig. 12-9
 IL-2R alpha (just the alpha chain as receptor) is first seen
in early T cell development/low level activation.
IL-2R alpha is also known as CD25,
High levels on the surface of T cells (bright fluorescence
in flow cytometer) indicates the presence of highly activated T
cells with IL-2R in trimer form ( An antibody to identify CD25
is called anti-TAC

From Fig. 4-8 7e

 Class II Cytokine Family of Receptors: interferons

and the Common JAK-STAT Signalling family.

Cytokines activate Class I and class II receptors through the

the JAK-STAT pathway=Janus Kinase= JAK
Signal Transducers and Activators of Transcription= STAT.

Note: Signal transduction mediated by cytokine receptors-

there are inactive kinases called Janus (Roman God two faces)
tyrosine kinases that are associated with the cytoplasmic tails
of the receptors. So a similar story then follows wherein
phosporylation of DNA binding proteins, in this case STAT
(Signal Transducers ) occurs. STAT then binds to specific
genes and they are transcribed.

Above is also Fig 4-11 in 7edition.

 TNF Cytokine Family- tumor necrosis family of cytokines
(pg. 123).
Unusual: Often anchored in membrane of effector cell, but can
be cleaved to become soluble:

TNF alpha: proinflammatory from macrophage in response to

infection= killing cancer cells and chronic inflammation

TNF beta: also known as Lymphotoxin alpha- from activated

lymphocytes, activates other cells to upregulate MHC and
adhesion molecules.

Puzzling Receptor response: TNF-R1 can deliver either

activating or death promoting signals??????? Mechanisms
described in figure 4-14 which you do NOT NEED to Know)
Response: Depends on the overall cytokine polarizing
environment etc.

Fas Receptor FasL interaction (page 125) member of TNF

family-more on this later: Think death, death, death of cancer
cells BUT ALSO activated T cells through activation of the
Caspase cascade= apoptosis.

Fas L induces formation of : the death-Inducing Signaling

Complex or DISC) which consists of FAS+FADD+Procaspase 8
(fig. 4-13)

Programmed cell death For shutting down the immune

response! Sounds counterproductive?
Activation induced cell death: Activated lymphocytes up-
regulate Fas receptor containing death domains-
See fig. 4-13. Why? One of many mechanisms to shut down an
immune response.
Note: Cancer cells express PDL-1 or programmed death
ligand. Who do you suppose is their target?

Th17- Recently discovered Cytokine:

Important in inflammation, especially asthma colitis, Crohns

Disease and other immune diseases. Also a family A-F. see table
4-5 but do not memorize.

TGF- + IL-6 is a crucial cytokine for Th17 cell

development and for T 17 cells.
This same cytokine TGF beta released from T reg cells
shuts down an immune response when IL-10 is present!
Concept: polarizing mixtures of cytokines have different
effects depending on the microenvironment and cells
present.

Cytokine Related Diseases- page 134.

a. Bacterial Septic Shock: infection with gram negative
bacteria possess LPS which causes macrophages to
release IL-1 and TNF alpha as an overproduction. =
disseminated intravascular coagulation (DIC) or lots of
blood clots in the vascular system, A drop in blood
pressure, inability to each hospital food and then
the patient dies (over 70,000 patients/year die )-$5
billion market for biotech firms making monoclonal
antibodies to TNF or finding other ways to block
runaway inflammation.
b. IL-1Ra or receptor antagonist has been developed
(page 133)
It looks like the receptor and lowers mortality from
45% to 16%!
c. Inflammatory cytokine levels may be increased in
disease states

a. Bacterial toxic shock also induced by

superantigens
b. Potential involvement in rheumatoid arthritis and
Type 2 diabetes
c. Implication in lymphoid and myeloid cancers
d. 1918 H1N1 influenza pandemic and cytokine
storms
e. Evidence of cytokine involvement in Severe acute
respiratory syndrome (SARS)

Viruses Exploit Cytokine Activity- virokines and viral

encoded cytokine receptors!!

Most of these receptor homologous proteins are either

secreted glycoproteins or are located at the infected cell
surface. It is believed that the function of these viral
receptor mimics is to intercept the activity of the cognate
cytokine, thus effectively short-circuiting host immune
responses to the viral infection.
Cytokine Therapy: Clinical Focus Therapy with Interferons on
page 322 6th edition, page 120 and 121 in 7e. Read the Clinical
Focus especially use of :
Interferon alpha in Chronic myelogenous leukemia,Kaposis

Interferon beta or Tysabri in MS therapy.

If cytokine levels are related to disease, therapies to

reduce cytokine levels can treat disease
Rheumatoid arthritis
Soluble TNF- receptor (Enbrel)
Monoclonal antibodies against TNF-
(Remicade and Humira)

Prevention of transplantation rejection- Thanks to a
student in the class, I looked this up:
Monoclonal antibodies against the chain of
IL-2R (Simulect and Zenapax)
Alton had asked why we cant use antibody
against IL-2 to block T cell? We block the
receptor, not the cytokine.
Note: we do NOT block the gamma chain
common to many cytokine receptors. Clever.

Note: Interferons alpha and beta cause depression. They

stimulate an enzyme on macs/dends called IDO which
converts/depletes tryptophan. If low tryptophan then no
serotonin.

Conversion of tryptophan to Kyurenine is toxic to T cells and

thus could benefit Tc mediated diseases like multiple sclerosis.
Cytokines, Obesity, Adult Diabetes : Clinical Focus page 136

1. Adipocytes produce TNF alpha constituitively

2. TNFalpha levels drop with weight loss
3. TNF signaling in adipocytes inhibits insulin receptors in
fat/liver cells so no glucose is taken up= sweet urine
4. Interleukin 6 inhibits insulin response in hepatocytes
where glucose is stored and stimulates Th17 to secrete IL-
17.
5. Interleukin 17 induces monocytes to produce more IL-6
Maintenance of an inflammatory state as fat cells lyse and
release inflammatory lipids and other DAMPS