Transcript The high proliferative potential colony assay

Cytokines Online Pathfinder
Encyclopaedia
 http://www.copewithcytokines.de/cope.cgi
 The term cytokine, or Immunocytokines , was used
initially to separate a group of immunomodulatory
proteins, called also Immunotransmitters , from
other Growth factors that modulate the proliferation
and bioactivities of non-immune cells.
 Some cytokines are produced by a rather limited
number of different cell types while others are
produced by almost the entire spectrum of known cell
types.
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Cyto: cell
-kines: kinesis, 可移動, 分裂
 In the more restricted sense cytokines comprise
Interleukins , initially thought to be produced
exclusively by leukocytes, Lymphokines , initially
thought to be produced exclusively by lymphocytes,
Monokines , initially thought to be produced exclusively
by monocytes, interferons, initially thought to be
involved in antiviral responses, colony stimulating
factors, initially thought to support the growth of cells in
semi-solid media, Chemokines , thought to be involved
in Chemotaxis , and a variety of other proteins.
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 Established cell lines that entirely depend
for their survival and proliferation on the
continuous presence of one or more of
growth factors . Factor-dependent cell
lines are capable of continuous growth in
the presence of the growth factors and
cytokines they require
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Hematopoiesis:
Greek haima for blood and poiein, to make
 Hematopoiesis is the dynamic and complex developmental
process of the formation of new blood cells.
 An early intra-embryonic site of hematopoiesis is found in the
Paraaortic splanchnopleura and in a structure termed AGM
( abbr. for aorta, gonads, and mesonephros) ( Nishikawa et al).
mice and birds
 Hematopoiesis taking place prior to the development of the fetal
liver is referred to as primitive hematopoiesis . The fetal liver is
the site of definitive hematopoiesis early during embryonal
development.
 The bone marrow with its intersinusoidal spaces is the site
responsible for the generation of blood cells in the post-natal
phase.
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www.wsu.edu/~ms523/ ms523s198.html
Hematopoesis
 Sites of blood cell
formation
 Embryo: yolk sac
 Fetus: spleen and
liver
 Adult: “bone marrow”
sternum, ribs, pelvis,
cranium, vertebrae,
long bones (tibia and
femur)
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The high proliferative potential
colony assay

Bone marrow
cells 25000 cells
of Balb/c, after
150 mg/kg of 5Fu.
 Culture s are
incubated under
low oxygen
tension for 12
days and scored
for HPP-CFU.
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Moore, 10:2719
The development of neutrophils
from the stem cell stage
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http://www.whfreeman.com/immunology/CH03/hematopoiesis.htm
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http://www.whfreeman.com/immunology/CH03/hematopoiesis.htm
In the presence of infection, cytokines produced by activated
macrophages and T-helper cells induce additional hematopoietic
activity, resulting in rapid expansion of white blood cells that participate
in fighting infection.
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Mosmann TR et al , J Immunol (1986) 136:2348-2357
 This is underscored by the uniform
phenotype of our anti-CRBC T cell lines,
which were all Th1. Only when we examined
T cell clones specific for FGG or alloantigens
did we discover antigen-specific T cells of
the Th2 cells
 Th2 cells produce activities that strongly
enhance IgE/IgG1 production....., whereas
Th1 cells produce interferon-g, which
strongly inhibits the enhancing activities in
Th2 supernatants .....
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Two types of murine helper T cell
clone Mosmann TR et al , J Immunol (1986) 136:2348-2357
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Cytokine secretion phenotypes of mouse T cells
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Immunosuppresive cytokines
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Th1 and Th2 cell generation is
regulated by the equilibrium between
different groups of cytokines, with
dominant effects of IL-12 and IL-4
(with feed back mechanisms)
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The commitment of TH0 cells to
become TH1 or TH2 is influenced
by cytokines secreted by the 2
subtypes themselves and by
macrophages , NK cells and mast
cells.
http://www.brown.edu/Courses/Bio_160/Projects1999/ies/cytok.html
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Cytokine soup during disease
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A Th1 to Th2 switch ia a critical step in the
etiology of HIV infection
Clerici M and Shearer GM, Immunol Today 14:107-111 (1993)
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Becoming a paradigm for disease management
Susceptibility to Leishmania major infection in interleukin-4deficient mice. Noben-Trauth et al 1996, Science 271:987-989
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Induction of Th1 and Th2 responses: a key
role for the natural immune respones?
Immunol Today 13:379-381 (1992)
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Role of IL-12 in the generation of Th1 cells
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Cytokine responsiveness in a human
volunteer injected with LPS. TNF-a rises
almost immediately and peaks at 1.5 hours;
the sharp decline of TNF-a may be due to
modulation by its soluble receptor sTNF-R.
A second wave of cytokines that peaks at 3
hours activates the acute phase response
in the liver and the systemic pituitary
response (both via IL-6) and the activation
and chemotaxis of neutrophils (via IL-6, IL8 and G-CSF). Pituitary-derived
adrenocorticotropic hormone (ACTH) and
migration inhibition factor (MIF) peak at 5
hours and coincide with peak levels of the
regulatory cytokines IL-Ra and IL-10 that
counteract the release or activity of
proinflammatory cytokines.
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As usual, things may not be so simple
IL-4 instructs TH1 responses and resistance to
Leishmania major in susceptible BALB/c mice
Biedermann T et al. 2001, Nature Immunol 2:11
•
When present during the initial activation of dendritic cells (DCs) by
infectious agents, IL-4 instructed DCs to produce IL-12 and promote
TH1 development. This TH1 response established resistance to
Leishmania major in susceptible BALB/c mice. When present later,
during the period of T cell priming, IL-4 induced TH2 differentiation
and progressive leishmaniasis in resistant mice. Because immune
responses developed via the consecutive activation of DCs and then
T cells, the contrasting effects of IL-4 on DC development and T cell
differentiation led to immune responses that had opposing functional
phenotypes.
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IL-4 instructs DCs to produce increased amounts of IL12 and to promote TH1 development in vitro.
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IL-4 instructs resistance to L. major in susceptible BALB/c
mice. Groups of five BALB/c mice or more were inoculated in
one hind footpad with 2×105 stationary phase L. major
promastigotes either of strain LV39, MRHO/Sv/59/P (a) or
strain MHOM/IL/81/FE/BNI (b).
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IL-4 instructs parasite containment in susceptible BALB/c mice.
BALB/c mice were infected with L. major and treated twice with the
indicated doses of IL-4 or NMS during the first 8 h of infection. After 9
weeks, the number of viable parasites in infected tissues was estimated
using a parasite-limiting dilution assay.
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IL-4 treatment during the first 8 h of L. major infection instructs IL-12–
producing DC1s and suppresses IL-4 expression. Control mice (Control
and IL-4) or mice infected with L. major promastigotes (3×106) were
treated either with NMS or twice with 1.0 μg of IL-4 at 0 and 8 h of
infection.
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Induction of TH1 responses with IL-4 depends on IL-12. BALB/c mice were
infected with L. major and treated twice with IL-4 (at 0 and 8 h) and either NMS
or anti–IL-12. After 8 weeks, popliteal lymph node cells were isolated, RNA
extracted and expression of IL-4 and IFN-γ mRNA determined by semiquantitative RT-PCR. (a) The relative changes in IL-4 and IFN-γ mRNA were
normalized to the values in control infected BALB/c mice, arbitrarily fixed as
100. (b) The ratio of IL-4:IFN-γ mRNA for each experimental group was
determined. L. major infection, cytokine treatment and mRNA analysis were
done.
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Treatment with anti–IL-12 abrogates IL-4–induced resistance to L.
major in BALB/c mice. Mice were infected with L. major
promastigotes and treated with either NMS (open circles) or twice
with 1.0 μg of IL-4 (at 0 and 8 h) and either NMS (filled squares) or
the IL-12 mAbs C17.8 and C17.15 (open triangles) 12 and 3 h before
infection. One of two similar experiments is shown. Infection,
cytokine treatment and weekly monitoring of footpad lesions were
done .
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Extension of IL-4 treatment into the period of T cell priming
reverses IL-4–induced resistance to L. major. Three groups of
mice were infected and treated with either 1.0 μg of IL-4 or NMS.
The first L. major–infected group received NMS (open circles), the
second 1.0 μg of IL-4 at 0 and 8 h (filled squares) and the third 1.0
μg of IL-4 at 0, 8, 16 and 24 h (crosses). Infection, cytokine
treatment and weekly monitoring of footpad lesions were done .
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Timing determines whether IL-4
treatment instructs a TH1 phenotype
and resistance or a TH2 phenotype
and susceptibility to L. major in TCR
Vβ4-deficient mice. (a,b) After parasite
inoculation,Vβ4-deficient BALB/c mice
were treated during the first 64 h with
either eight injections of PBS–1% NMS
or eight injections of 0.1 μg of IL-4
(total amount: 0.8 μg). Alternatively,
mice were treated twice during the
first 8 h of infection with 1.0 μg of IL-4
as in Fig. 2. Eight weeks after infection,
cells from draining lymph nodes were
stimulated in vitro with ultravioletirradiated parasites and cytokines
were determined in the supernatants
as described (see Methods). The IL4:IFN-γ ratio was 3.71 in mice that
received IL-4 over 64 h and 0.37 in
mice that received two injections of IL4 at 0 h and 8 h. (c).
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How Th1/2 cytokines are regulated?
• Transcriptional regulation of Th1/Th2 polarization, Jyothi
Rengarajan J et al 2000, Immunol Today 21:479
The two polarized T helper (Th) subsets Th1 and
Th2 are identified by their signature cytokines,
interferon-g (IFN-g) and interleukin 4 (IL-4)
respectively. Understanding the transcriptional
regulation of cytokine expression is therefore critical
for elucidating the process of Th cell differentiation.
Ubiquitous and tissue-specific transcription factors,
as well as chromatin remodeling of genomic loci
have been implicated in IL-4 and IFN-g regulation.
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Class I cytokine receptor family
(Hematopoietin recetor)
 this family includes receptors
for growth hormone and
prolactin. There are conserved
amino acid sequence motifs in
the extracellular domain - 4
positionally conserved cysteine
residues (CCCC) and a
conserved sequence of TrpSer-X-Trp-Ser (WSXWS)
where X is a nonconserved
amino acid. The receptors
consist of 2 polypeptide
chains: a cytokine-specific
subunit and a signaltransducing subunit which is
usually not specific for the
cytokine.
http://www.umdnj.edu/pathnweb/genpath/lec_1/Class_I_Cytokin
e_Receptors/class_i_cytokine_receptors.htm
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Class II cytokine receptor family
(Interferon receptor family)
 The ligands for these
receptors are the three
interferons a, ß, and
g. These receptors
possess the conserved
cysteine motifs, but lack
the WSXWS motif
present in class I cytokine
receptors.
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Overview of Th cell differentiation. A naive CD4 T
cell is activated via the TCR when it encounters
antigen presented by an antigen presenting cell.
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Regulation at transcriptional level
 GATA3, a zinc finger protein that was
originally identified as binding the TCRa
gene enhancer via a WGATAR sequence.
 T-bet (T-box expressed in T cells) is a
member of the T-box family of
transcription factors that regulate
several developmental processes. T-bet
expression strongly correlates with IFNg expression.
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 (a) IL-4 regulatory regions: the IL-4 proximal promoter with cis
and trans elements and map of IL-4 locus showing DNAse I HSs.
 (b) IFN-g regulatory region and map of locus with DNAse I HS
sites.
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 Striking a balance:
GATA3 and T-bet –
model for Th1/Th2
polarization. Signals
through the TCR and
cytokine receptors can
lead to the initiation of a
Th1 program (via Stat4
activation) and the
induction of T-bet,
which promotes Th1
lineage commitment.
Signals that favor the
activation of Stat6
induce GATA3 leading
to Th2 differentiation. cMaf is then upregulated
leading to increased IL4 production and Th2
polarization.
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More than immunoregulatroy actions
 Cytokine regulation of neuronal differentiation of hippocampal
progenitor cells (Mehler MF et al. 1993, Nature 362:62-65.)
 The signalling mechanisms governing haematolymphopoiesis and
those regulating neural development may be closely related, as
indicated by similarities of higher-order structure and function of
the cytokines involved, of the regional and temporal regulation of
their transcription and translation, and of their bioactivity.
 The mechanisms regulating lineage commitment and cellular
differentiation in the neural and haematopoietic systems are
similar.
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Electrophysiological analysis of the effects of
growth factor treatment on neuronal maturation
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Morphological and immunocytochemical indices
of progressive neuronal maturation
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Immune System and the Brain
Estrogen, microglia and the IBB The microglial-cytokine--estrogen
mediated network is described as a major player in the brain including
the Immune Brain Barrier. One of the functional areas involved maybe
the role that microglia could play in synaptic plasticity.
http://info.med.yale.edu/obgyn/reproimmuno/projects/immune.html
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Combinatorial signal by inflammatory cytokines and
chemokines mediate leukocyte interactions with
extracellular matrix
Vaday GG et al. 2001, J leukoc Biol 69:885-892
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limitations of cytokines for
therapeutic use
 High local concentrations of cytokines
during immune response cannot be
mimicked clinically
 Short half-life of cytokines
 Pleiotropic effects can cause
unpredictable side-effects
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