Download Cytokines

Cellular Basis of the Immune
Response
Robert Fleischmann, Ph.D.
Professor
Department of Urologic Surgery
Office: G144 Diehl Hall
Telephone Number: (612) 626-5034
E-Mail Address: [email protected]
Objectives
• To understand the major players in adaptive
immunity
• To understand the nature and general functions of
cytokines
• To understand the identity and functions of the
cytokines that are most important for adaptive
immunity
• To understand the differential regulation for induction
of Th1 and Th2 cells
• To understand the general features of
cytokine/cytokine receptor interactions
• To understand the mechanisms by which interferons,
as model cytokines, activate their target cells
• To understand the positives and negatives of the
clinical use of interferons, as model cytokines
Jose Jimenez, age 3 months, is brought
to the clinic with an extensive fluffy white
growth in his mouth. Jose has a history of
viral and bacterial infections that never
seem to quite go away. Indeed, he has
had diarrheal disease for the last two
months that has caused him to lose
weight.
What do you think is happening to Jose?
What tests would you wish to run?
Jose is diagnosed with Candida albicans. He
is evaluated for immunodeficiency disease.
His total WBC count is low normal.
His differential count shows a preponderance
of PMNs with very few lymphocytes.
He has a normal level of IgG and an absence
of IgM and IgA.
Exposure of his lymphocytes to T cell and B
cell mitogens fails to give proliferation.
What would you wish to do next?
Fluorescence cell sorting of his lymphocytes
shows a complete absence of CD3 cells and of
CD16 cells. All of his lymphocytes appear to be
positive for CD20.
What do these results mean?
The fluorescence cell sorting results indicate
that Jose has non-functional B cells but has no
T cells nor NK cells.
What would you do next?
Cells of the Immune System
Overview of Major Players of
Adaptive Immunity
• Dendritic cells, macrophages, and B cells are
professional antigen-presenting cells.
• CD4+ T cells (Th cells) are exposed to
antigen bound to MHC Class II on antigenpresenting cells and are driven to differentiate
into Th1 or Th2 cells.
• Th1 cells drive the differentiation and
proliferation of CD8+ cytotoxic T cells (CTLs).
• Th2 cells drive the differentiation and
proliferation of B cells to become antibodyproducing plasma cells.
Activated Lymphocytes
Differentiate Into Effector Cells
• Note how the lymphocyte enlarges as it differentiates.
• Note that T and B lymphocytes cannot be visually
distinguished from each other until they are fully
differentiated to their respective effector cells.
Lymphocyte Differentiation
and Proliferation Is Driven by
Cytokines
• Cytokines are proteins that help the
cells of the immune system to
communicate with one another.
• Cytokines are usually soluble mediator
molecules, though some may remain
cell-bound.
Cytokines Are Often Named
for the Type of Cell that
Produces Them
• Macrophages: monokines
• Lymphocytes: lymphokines or
interleukins
• Generic: cytokines
Individual Cytokines Activate
Target Cells by Binding to
Their Specific Receptors
• Each cytokine binds to its unique receptor.
• Binding affinities are very strong, with
dissociation constants in the range of 10-10 to
10-12 (Abs are in the range of 10-7 to 10-10).
• The receptor may be composed of multiple
subunits with one specific subunit and one or
more common subunits (also shared as
subunits of receptors for other cytokines).
Cytokine Receptor Expression
May Be Regulated
• Some cytokine receptors are constitutively
present but may be down-regulated by their
specific cytokine ligand or by other cytokines
(example: IFN receptors).
• Some cytokine receptors are induced by
treatment of target cells with other cytokines
(example: IL-1 induces resting T cells that
express the intermediate-affinity IL-2 receptor
[composed of  and  chains] to express the
high-affinity receptor [composed of , , and 
chains]).
Cytokine Cell Targets
• Autocrine action: the target cell of the
cytokine is the same as the producing
cell.
• Paracrine action: the target cell of the
cytokine a nearby cell that is different
from the producing cell.
• Endocrine action: the target cell of the
cytokine is a distant cell that is reached
through the circulation.
Autocrine Action of Cytokines
1. A Th1 cell is stimulated to
produce mRNA for IL-2.
2. The IL-2 mRNA is transported
to the cytoplasm where it is
translated into IL-2 protein.
3. The IL-2 is secreted.
CD4+ Th1 cell
4. The IL-2 binds to a receptor
on the producing Th1 cell,
activating it.
Paracrine Action of Cytokines
CD8+ T cell
1. A Th1 cell is stimulated to
produce mRNA for IFN-.
2. The IFN- mRNA is transported
to the cytoplasm where it is
translated into IFN- protein.
3. The IFN- is secreted.
CD4+ Th1 cell
4. The IFN- binds to a receptor
on the CD8+ T cell,
activating it.
Endocrine Action of Cytokines
1. A macrophage is stimulated to
produce mRNA for GM-CSF.
Stem Cell
2. The GM-CSF mRNA is
transported to the cytoplasm
where it is translated into GMCSF protein.
3. The GM-CSF is secreted.
Macrophage
4. The GM-CSF travels through
the blood to the bone marrow
where it binds to a receptor on
a hematopoietic stem cell,
activating it.
General Properties of
Cytokines
• Cytokines secretion is generally a brief,
self-limiting event.
• Secreted cytokines generally have their
greatest effects in the immediate area
where they are produced.
• Cytokines are active at picomolar
concentrations.
Cytokine Secretion Is a Brief,
Self-Limiting Event
• Induction of cytokine mRNAs is a
transient event.
• In general, cytokine mRNAs are
unstable and have a short half-life.
• After their translation, most cytokines
are rapidly glycosylated and secreted.
Individual Cytokines May Be
Produced by A Single Cell
Type or by Many Cells Types
• Single Cell Type
– IL-2 is produced by Th1 cells.
– IL-4 is produced by Th2 cells.
• Multiple Cell Types
– GM-CSF can be produced by T cells,
macrophages, endothelial cells, and a wide range
of fibroblasts.
– IFN- can be produced by virtually all fibroblasts
and epithelial cells.
Individual Cytokines May Act
on a Single Cell Type or on
Many Cell Types
• Single Cell Type
– IL-10 acts on macrophages to block IL-12,
subsequently blocking Th1 cells.
– IL-12 acts on Th1 cells.
• Multiple Cell Types (Pleiotropism)
– IFNs act on multiple cell types.
– IL-1 acts on T cells, B cells, macrophages,
endothelial cells, fibroblasts, and epithelial cells.
An Individual Cytokine May
Have Multiple Effects on a
Target Cell: Pleiotrophy
• IFN-
– Induces antiviral proteins
– Induces antiproliferative effects
– Induces MHC Class I antigens
– Stimulates NK cell activation
– Stimulates IL-12 production.
Different Cytokines Can Share
Similar Activities
(Redundancy)
• IFN-, IFN-, and IFN- share similar
activities, while retaining some unique
properties.
• IL-1, TNF, and IL-6 can all cause fever.
• IL-2 and IL-15 share similar abilities to
activate T cell proliferation and differentiation,
while retaining some unique properties.
Individual Cytokines May
Influence the Action of Other
Cytokines
• They may have antagonistic effects.
– IL-12 stimulates Th1 cell activation and proliferation,
while IL-10 blocks macrophage production of IL-12
and therefore blocks Th1 cell activation and
proliferation
• They may have additive effects.
– IFN- plus IFN-have additive effects.
• They may have synergistic effects.
– IFN- plus IFN- or IFN- plus IFN- have synergistic
effects.
Cytokine
Attributes
Cytokines Belong to
Five Families
•
•
•
•
•
IL-1 family
Hematopoietin family
Interferon family
Tumor necrosis family
Chemokine family
Most Cellular Responses to Cytokines
Require mRNA and Protein Synthesis
• Cytokine binds to receptor.
• A transmembrane alteration occurs allowing
the intracellular part of the receptor to be
activated.
• Second messenger molecules are activated.
• Second messenger molecules directly or
indirectly activate transcription of specific
genes.
• Translation of the mRNA for the specific genes
results in new cell function.
Many Cytokines Act as
Growth Regulators
• CSFs stimulate bone marrow stem cells
to grow.
• IL-2 and IL-15 stimulate Th1 cells to
grow and IL-2 stimulates Th2 cells to
grow.
• IL-10 blocks Th1 cell growth by blocking
macrophages from making IL-12.
• IFN- blocks Th2 cell growth by blocking
Th2 from making IL-4.
How Do the Actions of
Cytokines Affect Immunity?
Cytokine Actions
• Some cytokines are involved in mediating
and regulating innate immunity (Proinflammatory cytokines).
• Some cytokines are involved in mediating
and regulating specific immunity.
• Some cytokines are involved in mediating
and regulating both innate immunity and
specific immunity.
Most Important Cytokines
Principle Cytokines in
Acquired Immunity
M
IL-1
IL-12
IL-1
CD4 Th1
CD4 Th2
IL-2
IL-3
GM-CSF
IFN-
IL-4
IL-5
IL-6
IL-9
IL10
IL-13
CD8 T cell
Cytolysis:
FAS/FAS ligand
TNF
Granzyme
Perforin
B cell
Antibody
Triggers for Th1 Cell
Predomination
•
Naïve helper T cells are strongly stimulated
to become Th1 cells and cell-mediated
immunity predominates:
– When IL12 production by macrophages is high.
•
IL-23 and IL-27, relatives of IL-12 that share some
similarities to IL-12, also play a role to activate Th1
cells.
– When IFN- levels are high.
•
IL-18, a relative of IL-1 that shares some
similarities to IL-1, also activates Th1 cells.
Triggers for Th2 Cell
Predomination
• Naïve helper T cells are strongly
stimulated to become Th2 cells and
humoral immunity predominates:
– When IL-4 levels are high.
– When IL-10 levels are high.
Who Holds the High Trumps?
• If IL-4 levels are low and IL-12 and IFN levels are high, Th1 cells develop.
• If IL-4 levels are high, Th2 cells
develop.
• Thus, IL-4 holds the high trumps.
Is There Cross-Regulation?
• If naïve T cells are triggered to become Th1
cells, they produce IFN- which shuts down
Th2 proliferation.
• If naïve T cells are triggered to become Th2
cells, they produce IL-10 which shuts down
macrophage production of IL-12, removing
the stimulus for naïve helper T cells to
become Th1 cells.
• Thus, each local environment establishes its
own balance of Th1 and Th2 cells.
Principle Cytokines in
Acquired Immunity
M
IL-1
IL-12
IL-1
CD4 Th1
CD4 Th2
IL-2
IL-3
GM-CSF
IFN-
IL-4
IL-5
IL-6
IL-9
IL10
IL-13
CD8 T cell
IFN-
Cytolysis:
FAS/FAS ligand
TNF
Granzyme
Perforin
B cell
Antibody
Treg Cells
• Another subset of CD4+ T cells has
been identified: Treg cells.
– Treg cells produce IL-4, IL-10, and TGF-
– Treg cells suppresses T helper cell activity
through contact with the T helper cell.
– Limits autoimmune T cell activity.
Why Is T helper Cell
Regulation Important?
• Ex., Mycobacterium leprae infection
– If Th1 cells predominate, tuberuloid leprosy
develops.
• Most of the Mycobacteria are walled off in granulomas,
with slow tissue destruction and nerve damage caused
by a few “free” Mycobacteria.
• The patient survives.
– If Th2 cells predominate, lepromatous leprosy
develops.
• The Mycobacteria become widely disseminated in
macrophages and the patient has infection of the bone
and cartilage, with extensive tissue and nerve damage.
• The patient dies.
Cytokines Activate Their Target Cells
by Binding to Specific Receptors
• Cytokines bind to specific receptors on the
cell surface.
• Cytokines have their signal passed through
the cell membrane by changes in receptor
configuration.
• Cytokines have their signal passed through
the cytoplasm by the generation of “second”
messengers.
• Cytokine activated transcription factors upregulate and down-regulate transcription.
Cytokine Families
and the Receptors
To Which They Bind
• IL-1 family
– Ig Domain Receptor Superfamily
• Hematopoietin family
– Type I Cytokine Receptor
Superfamily
• Interferon family
– Type II Cytokine Receptor Family
• TNF family
– TNF Receptor Family
• Chemokine family
– Seven Transmembrane Helix
Family
Role of the
Different Receptor Subunits
•
Specific subunit ( subunit,
low affinity subunit)
– Binds specifically to a
particular cytokine
– Does not transduce a signal
through the membrane
•
Common subunit ( subunit,
 subunit, gp130)
– Binds to two or more
cytokines that are members
of the same group, when
they are bound to the
specific subunit
– Transduces a signal through
the membrane
•Note that the loss of the common subunit eliminates the activity of all cytokines of the family.
–Example, loss of common  chain causes loss of IL-2, IL-15, IL-7, IL-9, and IL-4 activities.
–Loss of common  chain causes severe combined immunodeficiency disorder (bubble baby)
Low Affinity Versus High Affinity Receptors
Consequences of Signaling
Through a Common Subunit
Explains redundancy:
(1) IL-3, GM-CSF & IL-5 induce
bone marrow proliferation
(2) all three induce
eosinophil proliferation
and basophil degranulation
Note, however, that not all
cells express  subunits
for the three different
cytokines, so there can
still be some specificity.
Consequences of Signaling
Through a Common Subunit
Because of a limited
number of  subunits,
IL-3 and GM-CSF
exhibit antagonism.
This is particularly
apparent if IL-3 is
added before GM-CSF.
How Does a Cytokine Bind
to Its Receptor?
• Example: The IL-2 receptor is
composed of three subunits.
  chain: binds IL-2
  chain: binds IL-2
  chain: signal transducing chain (common
 chain for IL-2, IL-4, IL-7, IL-15, IL-21)
Affinities of IL-2 Receptor Multimers
The strength of
cytokine binding
increases with the
addition of the
common subunits.
Note: (1) IL-2 binds to a pocket formed by the  and  subunits
(2) the  subunit binds to the  subunit but not to the  subunit
Signal Transduction Across
the Cell Membrane
• Example: IFN signal transduction.
The IFN-/ Receptor
•JAK kinases (Jak1, Tyk2) bind
to intracellular domain of
receptor.
•JAK kinase phosphorylates
tyrosines on receptor and
itself (autophosphorylation).
•Activated JAK kinase
phosphorylates tyrosines on
STATs (signal
transducer/activator of
transcription).
•Activated STATs become
active transcription factors.
Differences in the Receptors for Different IFNs
Vilcek, J. Nat Immunol. 2003 Jan;4(1):8-9
Permission for use: Nature Immunology
Janus kinases
are used by a
number of class I
and class II
cytokine receptors.
Jose Jimenez
Jose is tested for the presence of the
common  chain of IL-2, IL-4, IL-7, IL-15, and
IL-21. He is also tested for the presence of
JAK1 and JAK3.
He is found to have JAK1 and JAK3.
However, he does not express the common 
chain.
Why would a lack of common  chain lead to his
condition?
Jose Jimenez
IL-7 is needed for maturation of B cells in the
bone marrow. The common  chain is the
signal transducing part of the IL-7 receptor.
Thus, only non-functional immature B cells are
present in the blood.
The common  chain is needed for early
steps in the differentiation of the precursor to T
cells and NK cells. This may relate to a need
for IL-7 for differentiation of NK and T cells.
However, it may also reflect a lack of function of
the IL-2 receptor.
Clinical Focus:
Therapy with Interferons
Rationale for Interferon
Therapy
• Antiviral agent
– Block replication of viruses in viral
diseases
• Antiproliferative agent
– Block replication of cancer cells
• Immunomodulatory agent
– Enhance macrophage activity
– Enhance NK cell activity
– Enhance cytotoxic T cell activity
Types of Interferons
• Type I interferons
– IFN-
– IFN-
– Also, IFN-, IFN-, IFN-, IFN-, IFN-,
IFN-
• Type II interferon
– IFN-
Diseases Successfully
Treated with IFN-
• Hepatitis
– Hepatitis B
– Hepatitis C
• White blood cell cancers
– Hairy cell leukemia (now purine nucleoside
analogs are better)
– Chronic myelogenous leukemia
• Solid tumors
– Melanoma
– Kaposi’s sarcoma
– Renal cancer
Diseases Successfully
Treated with IFN-
• Multiple sclerosis
– IFN- therapy
reduces the
number of
exacerbations
(attacks) and the
size of the lesions
in multiple
sclerosis patients.
Diseases Successfully
Treated with IFN-
• Chronic granulomatous disease
– Characteristics of chronic granulomatous
disease
• Impaired ability of phagocytes to kill ingested
microbes.
• Suffer recurrent infections
– IFN- treatment
• Increases ability of phagocytes to produce
reactive oxygen and nitrogen molecules
• Decreases number and severity of infections
Side Effects of IFN Therapy
• Bone marrow suppression
–
–
–
–
•
•
•
•
Leukopenia
Neutropenia
Anemia
Thrombocytopenia
Hepatic disorders
Renal disorders
Hypertriglyceridemia
Psychiatric considerations
– Impaired concentration and decreased alertness
– Deficits in verbal memory
– Depression