Cytokines in Radiobiological Responses: A Review

Abstract

Cytokines function in many roles that are highly relevant to radiation research. This review focuses on how cytokines are structurally organized, how they are induced by radiation, and how they orchestrate mesenchymal, epithelial and immune cell interactions in irradiated tissues. Pro-inflammatory cytokines are the major components of immediate early gene programs and as such can be rapidly activated after tissue irradiation. They converge with the effects of ionizing radiation in that both generate free radicals including reactive oxygen and nitrogen species (ROS/RNS). "Self" molecules secreted or released from cells after irradiation feed the same paradigm by signaling for ROS and cytokine production. As a result, multilayered feedback control circuits can be generated that perpetuate the radiation tissue damage response. The pro-inflammatory phase persists until such times as perceived challenges to host integrity are eliminated. Antioxidant, anti-inflammatory cytokines then act to restore homeostasis. The balance between pro-inflammatory and anti-inflammatory forces may shift to and fro for a long time after radiation exposure, creating waves as the host tries to deal with persisting pathogenesis. Individual cytokines function within socially interconnected groups to direct these integrated cellular responses. They hunt in packs and form complex cytokine networks that are nested within each other so as to form mutually reinforcing or antagonistic forces. This yin-yang balance appears to have redox as a fulcrum. Because of their social organization, cytokines appear to have a considerable degree of redundancy and it follows that an elevated level of a specific cytokine in a disease situation or after irradiation does not necessarily implicate it causally in pathogenesis. In spite of this, "driver" cytokines are emerging in pathogenic situations that can clearly be targeted for therapeutic benefit, including in radiation settings. Cytokines can greatly affect intrinsic cellular radiosensitivity, the incidence and type of radiation tissue complications, bystander effects, genomic instability and cancer. Minor and not so minor, polymorphisms in cytokine genes give considerable diversity within populations and are relevant to causation of disease. Therapeutic intervention is made difficult by such complexity; but the potential prize is great.

Full text

RADIATION RESEARCH 178, 505–523 (2012)
0033-7587/12 $15.00
!2012 by Radiation Research Society.
All rights of reproduction in any form reserved.
DOI: 10.1667/RR3031.1
REVIEW
Cytokines in Radiobiological Responses: A Review
Do¨rthe Schaue,
1
Evelyn L. Kachikwu and William H. McBride
David Geffen School Medicine, University of California at Los Angeles, Los Angeles, California
Schaue, D., Kachikwu, E. L. and McBride, W. H. Cytokines
in Radiobiological Responses: A Review. Radiat. Res. 178,
505–523 (2012).
Cytokines function in many roles that are highly relevant
to radiation research. This review focuses on how cytokines
are structurally organized, how they are induced by
radiation, and how they orchestrate mesenchymal, epithelial
and immune cell interactions in irradiated tissues.
Pro-inflammatory cytokines are the major components of
immediate early gene programs and as such can be rapidly
activated after tissue irradiation. They converge with the
effects of ionizing radiation in that both generate free radicals
including reactive oxygen and nitrogen species (ROS/RNS).
‘‘Self’’ molecules secreted or released from cells after
irradiation feed the same paradigm by signaling for ROS
and cytokine production. As a result, multilayered feedback
control circuits can be generated that perpetuate the radiation
tissue damage response. The pro-inflammatory phase persists
until such times as perceived challenges to host integrity are
eliminated. Antioxidant, anti-inflammatory cytokines then act
to restore homeostasis. The balance between pro-inflammatory
and anti-inflammatory forces may shift to and fro for a long
time after radiation exposure, creating waves as the host tries
to deal with persisting pathogenesis.
Individual cytokines function within socially intercon-
nected groups to direct these integrated cellular responses.
They hunt in packs and form complex cytokine networks
that are nested within each other so as to form mutually
reinforcing or antagonistic forces. This yin-yang balance
appears to have redox as a fulcrum. Because of their social
organization, cytokines appear to have a considerable
degree of redundancy and it follows that an elevated level
of a specific cytokine in a disease situation or after
irradiation does not necessarily implicate it causally in
pathogenesis. In spite of this, ‘‘driver’’ cytokines are
emerging in pathogenic situations that can clearly be
targeted for therapeutic benefit, including in radiation
settings. Cytokines can greatly affect intrinsic cellular
radiosensitivity, the incidence and type of radiation tissue
complications, bystander effects, genomic instability and
cancer. Minor and not so minor, polymorphisms in cytokine
genes give considerable diversity within populations and are
relevant to causation of disease. Therapeutic intervention is
made difficult by such complexity; but the potential prize is
great. !2012 by Radiation Research Society
INTRODUCTION
It would be unwise to try to cover the field of cytokines in
one short review. A book would hardly do it justice. Our aim
is simple. It is to present some general aspects of cytokine
biology that we feel are most relevant for radiation
researchers. Even with this limited discussion we can only
sketch outlines and present examples that inevitably prejudice
content by selection. We will focus primarily on the major
rationale for studying cytokines in radiation responses,
namely that they are induced to orchestrate complex
mesenchymal, epithelial, and immune interactions that
influence tissue damage and restore integrity and homeostasis
through promoting angiogenesis and tissue regeneration or
replacement by fibrosis (Fig. 1). Other less well described
constitutive roles in maintaining tissue homeostasis, and in
development, will not be covered here. We ask to be excused
for numerous omissions caused by space limitations.
A BRIEF HISTORY OF CYTOKINES AND THEIR
CHARACTERISTICS
Cytokine biology grew out of studies in the late 1960s
and 1970s that ascribed specific biological properties to
soluble ‘‘factors’’ secreted into culture supernatants after
cell stimulation. Such factors were often named after the cell
type and the activity affected, for example lymphocyte
activating factor, macrophage chemotactic factor and
macrophage aggregation factor. This nomenclature primar-
ily reflected the researcher’s own field of interest, as in
lymphokines (from lymphocytes), interleukins (inter-leuko-
cyte communication), monokines (from mononuclear
phagocytes), colony stimulating factors (CSFs for hemato-
poiesis), interferons (IFNs that interfered with viral
replication), connective tissue ‘‘growth factors’’, and
chemotactic chemokines. The chaos generated by such
1
Address for correspondence: Room CHS B3-109, University of
California at Los Angeles, 10833 LeConte Ave., Los Angeles, CA
90095-1714; e-mail: dschaue@mednet.ucla.edu.
505

parochial and arbitrary categorizations dissembled a little
when Cohen et al. introduced the term ‘‘cytokine’’ in 1974
(1) to counter the notion that only lymphocytes make
‘‘lymphokines’’ (2), but was only finally averted with the
advent of molecular cloning. This showed some cytokines
to be eponymous but, more importantly, that ‘‘cytokines’’
form a meaningful generic group of multiple structurally-
related families with about 200 member proteins of around a
few hundred amino acids that have a common purpose of
regulating crosstalk between cells and tissues. By virtue of
their crucial role in cellular communication, cytokines are
prime targets for therapeutic intervention in many diseases
including infections, chronic inflammatory and autoimmune
conditions, wound healing and neoplasia. In the past, the
complexity of cytokine networks often thwarted such
efforts, but recently key driver cytokines are emerging in
specific disease situations that can be targeted for
therapeutic benefit (3).
Cytokine Characteristics
The broad characteristics of cytokines that led to their
being considered as a generic group are summarized in
Table I. They are highly potent molecules that are generally
transiently expressed in response to stimuli. Responses are
highly coordinated within a network of molecules that have
interactive and overlapping functionality and whose profiles
change with time to meet challenges. Cytokines extend their
influence by choreographing alterations in cell adhesion
molecules, immune recognition, cell death and survival, cell
cycle arrest/proliferation, and metabolism so as to integrate
cell and tissue responses. Our current cytokine networks
have presumably been sculpted by major periodic epidem-
ics. As a result, redundancy, strong genetic influences and
extensive diversity are characteristics. This is in keeping
with a critical role of ensuring the ultimate survival of the
species by promoting flexibility of response between
individuals. There is still much to be learned about how
coordination is achieved, but it is clear that cytokines hunt
in packs with overlapping and sometime antagonistic
functionality to drive responses forward.
The nature of cytokine biology infers that if the level of a
specific cytokine is elevated in a disease situation or
following radiation, it does not necessarily indicate how
networked cytokines are functioning or if that specific
cytokine is causally involved in pathogenesis. Cytokine
FIG. 1. Cytokines drive the formation of inflammatory lesions working together with DAMPS to generate a pro-inflammatory, pro-oxidant
microenvironment. The vasculature becomes leaky, allowing infiltration by neutrophils, and then macrophages and lymphocytes that migrate
along chemokine gradients. Acute phase proteins, including cytokines, are generated along with a fair measure of cell death. In the periphery, cells
may become more resistant to death and infection. Hypoxia may occur and in time the lesion resolves under the influence of anti-inflammatory
cytokines and cells. Macrophages develop an M2 rather than an M1 phenotype. Angiogenesis and/or vasculogenesis assists either tissue
regeneration or replacement with extracellular materials (fibrosis).
506 REVIEW

levels are generally not good ‘‘end points’’ for disease in
themselves and have to be directly related to functional
events, for example by targeted inhibition. A positive result
may show the cytokine to be a major driver of the pack
causing a condition, but a negative or equivocal result may
simply indicate a high level of redundancy. An additional
interpretative complication is that levels of mutually
antagonistic cytokines may be elevated at the same time.
The balance within each cytokine network and within each
individual has to be characterized if mechanisms are to be
fully understood and if therapeutic intervention is to have
any hope of success.
Finally, most of the information on the roles of cytokines
in human health and disease comes from immunoassay of
levels in the circulation. These are influenced by half-lives
and rates of secretion and may not relate directly or causally
to events within tissues. Analyses of cytokine localization in
cells and tissues by mRNA or immunohistochemical
analyses are generally performed in model systems and
can be more informative, although some cytokines are most
effective in performing certain functions when cell-bound
and detailed knowledge of juxtacrine cell–cell interactions
involving cytokines is sparse.
In cancer, cytokine pathways are often dysregulated.
Causative mutations are sometimes found such as in
receptor-tyrosine kinase fusion genes that cause chronic
myeloid malignancies (8). But, more often than not,
cytokine overexpression results from dysregulated down-
stream control elements that enhance tumor growth,
survival, invasion, and escape from immune surveillance.
The simplest analogy for cancer is as a wound that does not
heal (9). By the time tumors become palpable they have
gone through initial recognition and acute inflammatory
stages and may have been immunoedited so as to escape
these control mechanisms. As a result, infiltrates most often
resemble those in healing wounds with a preponderance of
regulatory cytokines and cells as in the second phase in Fig.
1. As in all chronic lesions, there may be attempts to re-
activate pro-inflammatory responses that can occur in waves
over time (10), as is often seen after irradiation (11).
Cytokine Nomenclature
To this day the nomenclature of cytokine families is
archaic and confusing, often bearing little relationship to
either structure or function. For example, transforming
growth factor-alpha (TGF-a) and transforming growth
factor-beta (TGF-b) were first defined by their ability to
promote a reversible ‘‘transformed’’ phenotype in normal
rat fibroblasts in soft agar, but are in fact unrelated peptides
with very different biological properties. In fact, the major
effect of TGF-bis to inhibit proliferation of many cell types
and it is highly immunosuppressive. One might be equally
excused for thinking that members of the interleukin
families are related. In fact, they share remarkably little
homology even within a family. Many are produced by, or
act on, nonimmune as well as immune cells, while exclusion
of some and the inclusion of others, such as the chemokine
IL-8 in the interleukins, seems arbitrary. Logical order has
been brought to certain cytokine groups by consideration of
common structural traits or common receptor utilization, the
best example being the chemokines and chemokine
receptors (Table III). However, even brief examination of
the enormous size of the cytokine system would lead one to
TABLE I
Features of Cytokines
Potent — effective at very low (pM to nM) concentrations.
Transient — mainly produced in response to stimuli. Low
‘‘background’’ levels of some cytokines are present that may be
required for homeostasis in some tissues.
Local action — most cytokines influence only cells in the
immediate vicinity of production. Some critical variables are the
cytokine concentration, formation of cytokine gradients, and
presence or absence of extracellular matrix that may modify its
persistence and activity. Circulatory levels are normally low, the
main exceptions being the hematopoietic factors, e.g., CSF-1
(macrophage colony stimulating factor), EPO (erythropoietin),
SCF (stem cell factor/c-Kit), or acute phase reactants e.g., IL-6 or
TGF-bthat are latent until locally activated. It follows that high
serum levels of certain cytokines may reflect pathological
processes, but the correlation with tissue events may be poor and
they may not be causally involved.
Cell-bound and secreted forms. The potency and functionality of
secreted and cell-bound cytokines may be different and the switch
may be regulatory. Cell-bound cytokines could interact with
adjacent cells through high avidity juxtacrine interactions, while
secreted cytokines may function more through autocrine or
paracrine mechanisms. Reverse signaling through a ligand may
occur, or signaling by soluble complexes through shared receptor
subunits, e.g., IL-6 (see text).
Cascadic — responses are propagated by progressive changes in
expression patterns of cytokines and their receptors over time.
These coordinate to form a cascade that drives responses forward.
For example, when specific T cells recognize antigen presented
by dendritic cells, the latter can produce IL-1 and TNF-athat aid
in formation of the immunological synapse that activates T cells
(4) to acquire IL-2R, and so that IL-2 can drive their
proliferation, differentiation and activation to produce effector
cells that secrete interferon-c, IL-4, or other effector cytokines.
Pleiotropism — different functions being stimulated depending upon
the cell type. For example, TNF-acan be cytotoxic to certain
cancer and normal cell types, but causes fibroblasts to proliferate.
Multiple regulatory yin-yang mechanisms. These include:
Mutually agonistic and antagonistic cytokines or receptors or
decoys, for example pro- and anti-inflammatory molecules or
nonsignaling ligands such as IL-1Ra.
Modulation of function by shedding or internalization of receptors
and ligands that alters ligand-receptor interplay, such as soluble
TNFRs.
Availability of competing intracellular adaptor proteins, second
messengers or other modifiers of signaling. In other words, one
receptor can send what appear to be contradictory signals because
they are interpreted differently downstream.
Positive and negative feedback loops to terminate or enhance
cytokine production. Multiple mechanisms are available. For
example, suppressors of cytokine signaling (SOCS) families
inhibit STAT activation. Their loss can lead to death due to
excessive cytokine production (5). SOCS1 and SOCS3 can
differentially modulate intrinsic cellular radiosensitivity (6, 7).
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the conclude that bringing order to such apparent chaos
would be a Herculean challenge. Even stunning advances in
genomic sequencing and annotation, crystallography and
NMR have not solved the issues of structural relationships.
Inclusion of ‘‘growth factors’’ as cytokines is controver-
sial, although no one would dispute that some, for example
members of the TGF-bfamily, are bona fide cytokines and
that many ‘‘cytokines’’ act primarily as growth factors, e.g.,
the colony stimulating factors (CSFs). Perhaps the most
persuasive argument for calling some growth factors
cytokines is that they interact within the same networks
and use similar signaling pathways as cytokines. In this
review, the main focus will be the classic cytokines.
Structural Aspects of Cytokines
One might expect clues as to how cytokines evolved such
diversity and redundancy to be embedded in their structures.
There is no universal agreement as to their categorization,
but Table II shows some of the proposed family divisions.
This was a complete mystery until, in a seminal study,
Bazan (12) noted that a large group of cytokines share a
common protein fold, the four a-helix bundle. These
cytokines could be subdivided into short- and long-chain
forms based on the length of their core ahelices (Table II).
How this ‘‘cytokine’’ fold is preserved in the midst of the
huge diversity displayed by the primary structures is still a
mystery. Other structural features within the four a-helix
bundle cytokines are sometimes used to provide finer
categorization. The degree of consensus is not overwhelm-
ing, but the IFN and interleukin-10 (IL-10) families stand
out as having similar yet distinct structures.
Outside the four a-helix bundle cytokines, IL-1 and IL-18
superfamily members are distinguished by shared b-trefoil
structures formed by six two-stranded hairpins. The TNF
superfamily shares b-sandwich trimeric structures. The fairly
recently discovered IL-17 superfamily is distinguished by
four highly conserved cysteine residues and the TGF-b
superfamily is distinctive in having 9 cysteine residues, eight
of which create a characteristic cysteine knot structure, while
the ninth is involved in dimerization. The more than 50
chemokines from 4 distinct subfamilies based on the position
of the conserved cysteine residue. They are involved
primarily in the trafficking of different immune cells to sites
in the body, in particular inflammation. The gradients that
form by their localized secretion are important in directing
such traffic. In general, CC chemokines steer polymorpho-
nuclear leukocytes, monocytes, and NK cells while CXC
chemokines focus more on directing B and T cells (13).
Cytokine Receptors and Signaling
Such is the diversity of cytokines that structural relation-
ships are more easily seen in their receptors where essential
signaling domains are highly conserved (Table II). A general
TABLE 2
Cytokines and Cytokine Receptors
Cytokines
Four a-helix bundle cytokine superfamily - short and long chain
families
Short:
IL-2 family: IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-21
IL-3 family: IL-3, IL-5, GM-CSF, M-CSF, SCF
IL-12 family: IL-12, IL-23, IL-27, IL-35
Long:
IL-6 family: IL-6, IL-11, LIF, OSM, CNTF, CT-1, G-CSF, GH,
EPO, TPO, leptin
IFN family: Type I (IFN-a, IFN-b), type II (IFN-c), type III (IL-
28, IL-29)
IL-10 family: IL-10, IL-19, IL-20, IL-22, IL-24, IL-26
Beta-Trefoil: IL-1 family - IL-1a, IL-1b, IL-18, FGF
Beta-Sandwich: TNF superfamily
IL-17 conserved cysteine family
TGF-bsuperfamily
Chemokine superfamily
CC, CXC, XC, and CX
3
C families
Cytokine receptors
Type I
Heterodimeric
Using cc to signal: IL-2R, IL-4R, IL-7R, IL-9R, IL-13R, IL-15R
Using bc to signal: IL-3R, IL5R, GM-CSFR
Using gp130 to signal: IL-6R, IL-11R, IL-27R, IL-31, OSMR,
CNTFR, LIFR
Homodimeric: G-CSFR, leptinR, EPOR, TPOR
Type II
IFN-a,b,cRs, IL-10R, IL-28R, IL-29R
Ig Superfamily: IL-1R, IL-18R, CSF1R, c-Kit
IL-17R family: IL-17RA to E
TNFR:
Containing death domains: TNF-R1, Fas (CD95), TRAIL-R1
(DR4), TRAIL-R2 (DR5), TRAIL-R4 (DcR2) and TRAMP
(DR3)
No death domains: TNFRII, CD27, CD30, CD40, 4-1BB, RANK
Decoy receptors: TRAIL-R3 (DcR1), DcR3
TGF-bR: TGF-bR1, TGF-bR2
Chemokine receptors
CCR, CXCR, XCR and CX
3
CR families
TABLE 3
Examples of DAMPS and Likely Receptors (in brackets)
DAMPs secreted into extracellular spaces:
ATP, AMP, (P2Rs, NALP3), Adenosine (P1Rs - A1, A2A and B,
A3R), advanced glycation end products (RAGE), oxidation
products (TLR4, CD36), high mobility group protein B1 (TLR2/
4/9, CD44, RAGE), S100 (TLR4, RAGE), monosodium urate
(TLR2/4, CD14, NALP3), heat shock proteins (TLR2/4, CD14,
CD40, CD91), calcium-binding proteins, beta amyloid (RAGE,
NALP3), defensins (TLR4, CCR6), lactoferrin (TLR4),
uromodulin (TLR4), surfactant D, ubiquitin (CXCR4).
DAMPS released on cell death:
HMGB1 (TLR2/4/9, CD44, RAGE), dsDNA and chromatin
(TLR9), RNA (TLR3), mitochondrial DNA and matrix proteins.
DAMPS from enzymic action on extracellular matrix:
Hyaluronan (TLR2/4, NLRP3, CD44), collagen peptides
(CXCR2), elastin/laminin peptides (integrins), fibrinogen/
fibronectin (TLR4, integrins), heparan sulfate (TLR4).
508 REVIEW

principle is the need for receptor dimerization and aggrega-
tion for signaling, although this is often by itself insufficient
and additional adaptor molecules are needed (14).
Both type I and 2 receptor families bind 4-helix bundle
cytokines. Type I cytokine receptors recognize many
interleukins and CSFs. They bind ligands through a shared
conserved 200 amino acid cytokine homology domain
(CHD) (12) and require a WSXWS motif for activation
(15). Type II cytokine receptors are similar to type I
receptors but lack the WSXWS motif. They bind IFNs, IL-
10 homologs and IL-28-29 family members. Both type I and
2 receptors lack intrinsic tyrosine kinase activity and use
Janus tyrosine kinases (Jak) and STAT proteins as
intracellular signaling mediators.
A remarkable feature of the type I cytokine receptors is the
potential for promiscuity and pathway interconnectivity that
is brought about by sharing of signaling chains ( II). In all,
about 27 cytokines signal through 3 common chains (16). In
addition, IL-12 and IL-23 cytokines have unique IL12Rb2 or
IL-23Rb3 chains, but share an IL12Rb1 signaling chain that
has structural similarity to gp130 (17). There are many
unanswered questions as to why this sharing evolved (18),
but it is an efficient use of common signaling pathways while
retaining ligand specificity. For example, there are more than
twice as many chemokines as receptors; some receptors will
bind more than one chemokine while some chemokines can
bind several receptors. This functional pleiotropy and
redundancy also indicates the degree of control exerted by
spatial and temporal cytokine and receptor gene expression.
In other words, ‘‘specificity’’ resides not solely at the level of
ligand-receptor interaction, but by what cells express what
and where (19). This organization reinforces the notion that
cytokines form very tight, socially interconnected groups
while retaining their own functional identities.
A remarkable example of controlled promiscuity is seen
in IL-6 signaling. IL-6R is expressed in a limited fashion,
primarily by macrophages, neutrophils, hepatocytes and
some T cells. ‘‘Classic’’ signaling requires association of
IL-6R with gp130. However, IL-6Rs can be cleaved to an
alternative soluble form, sIL6R; as can some other
receptors, e.g., sIL-1R, sTNFR1, and sTNFR2. IL-6R is
cleaved by disintegrin and metalloprotease ADAM17,
which is activated by many signals, including IL-1, TNF-
aand apoptosis (20). Interestingly, soluble IL-6/IL-6R
complexes can ‘‘trans-signal’’ through gp130 and since
gp130 dimers are ubiquitously expressed on all cells, the
spectrum of IL-6 targets and the cytokine’s functional
impact is greatly expanded. Gp130 expression can be
further enhanced, e.g., by IL-10, to increase cellular
sensitivity to trans-signaling (21). Importantly, ‘‘classic’’
IL-6 signaling has regenerative and anti-inflammatory
consequences, while trans-signaling is responsible for many
of IL-60s pro-inflammatory effects (20). Given the role of
IL-6 in inflammation and cancer, targeting soluble com-
plexes with soluble gp130 is being developed as a potential
therapy (22) that may be relevant to RT, as sIL-6R has been
shown to act with radiation-induced IL-6 to protect cells
from radiation cytotoxicity (23); although IL-6 may affect
radiation responses differently in different cancers.
IL-1R and IL-18R are prominent members of the
immunoglobulin (Ig) superfamily that includes CSF-1R,
PDGF-Rband stem cell factor receptor (c-Kit). IL-1 and IL-
18 are structurally closely related cytokines whose receptors
of which have signaling Toll/IL-1R (TIR) domains, also
present in toll-like receptors (TLRs), indicating a common
ancestry. Signaling in response to IL-1 requires IL-1RAP in
addition to IL-1R binding, while so-called ‘‘decoy’’
receptors such as IL-1Ra have an inhibitory role.
Other distinct cytokine receptor families are the TNFR
family, the TGF-bR family and the chemokine receptors.
The TNFR superfamily has in excess of 27 members (often
called TNFRSF1-27) that share partial homology in their
extracellular cysteine-rich domains. TNFRs can be subdi-
vided into whether or not they contain so-called cytoplasmic
‘‘death domains’’ (DD). The ten that do can recruit adaptor
proteins, in particular Fas-associated protein with death
domain (FADD) that bridges receptor activation to the
caspase 8 cascade and apoptosis, a process that can be
inhibited by FLICE-inhibitory protein that binds to FADD
and caspase 8. Blocking the apoptotic pathway can result in
programmed necrosis (necroptosis) that is regulated by
activation of receptor-interacting serine/threonine protein
kinase 1 (RIPK1) and RIPK3. Another outcome of TNF-a
binding to TNFR1 is when dynamic endosome-associated
complexes form containing TNFR-associated death domain
(TRADD) and RIPK1 along with numerous other proteins,
whose activities are regulated by ubiquitination, proteolysis
and phosphorylation (24). Under such circumstances,
MAPK and NF-jb pathways are activated and generate
downstream inhibitors of apoptosis (IAPs) such as survivin,
IAP-1, IAP-2 and X-IAP; effector caspases are blocked, cell
survival and inflammatory cytokines are produced. These
signaling outcomes by death receptors vary depending on
the TNFR and cells that are involved. In contrast, the
TNFRs that do not have DDs can bind TNFR-associated
factor (TRAF) interactingmotifs(TIMs)tosignal
MAPKp38, extracellular signal related kinase (ERK) and
phosphoinositide 3-kinase (PI3K) as well as NF-jB and
JNK. They generally function as regulators of the DD
pathways, as do 5 decoy receptors.
Members of the TGF-bsuperfamily of receptors (types 1,
2, 3) have intracellular serine/threonine kinase domains and
can form homo- or heterodimers. TGF-bsignaling is
through activation of SMADS. There are 8 SMADS
belonging to 3 functional classes. Receptor-regulated
SMADS are directly phosphorylated by type I TGF-bR
through the intracellular kinase domain. These bind to a
common mediator co-Smad4 to initiate gene transcription.
Inhibitory iSMADS6 and iSMADS7 compete with SMAD4
to regulate transcription. The chemokine receptors are G-
protein coupled with 7 transmembrane domains and their
nomenclature reflects that of the chemokines (Table II).
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The Early Response to ‘‘Danger’’
Important for our understanding of radiobiological respons-
es is how spatially and temporally integrated cytokine gene
expression patterns unfold with time to direct tissue responses
(Fig. 1). Regulation of gene expression is exerted transcrip-
tionally and post-transcriptionally through a multilayer
composite of genetic elements and processes, including
DNA methylation, chromatin structure and remodeling,
DNA sequence variants, RNA binding proteins, and micro-
RNAs (miRNAs) so that rapid primary relatively promiscuous
responses are mechanistically distinct from later more
restricted responses. Immediate early gene responses are
under the control of promoters that often have CpG islands.
They involve constitutively active chromatin and are
independent of nucleosomal remodeling complexes and
contrast with later gene expression programs that may require
gene demethylation and/or chromatin remodeling, with
control being programmed into DNA structure at an early
developmental stage and during lineage commitment (25).
Many inflammation-related cytokine genes (e.g., TNF-a,
IL-1, IL-6, IL-8, IFNs, G-CSF, VEGF, and EGFR) fall into
this category, being activated within minutes to hours after an
exogenous signal without de novo protein synthesis. Control
is exerted primarily by adenylate\uridylate (AU) elements in
their 30UTR regions (26, 27). Binding at such sites by cell-
type-specific trans-acting binding proteins or microRNAs
(28) causes immediate changes in mRNA abundance by
transcript stabilization, although redox-sensitive proteins (29)
and chromatin structure (30) also regulate expression.
Importantly, polymorphisms and mutations within the 30UTR
have been associated with various diseases including
radiation-induced cancer (31)andmaycontributeto
inflammatory carcinogenesis. Other major groups of genes
with AU rich elements are proto-oncogene transcription
factors (e.g., c-jun, c-fos) or are involved in metabolism (e.g.,
GLUT1) or in redox regulation (iNOS, thioredoxin reductase,
COX-2). Not surprisingly, radiation induces an immediate
early gene response with rapidly increased expression of
some proto-oncogenes and cytokines (32, 33). Since
radiation-induced cytokines reappear much later (34), it is
likely that these later responses are more cell-type restricted
than those for initial ‘‘danger’’ responses. Examples of more
restricted responses might be maturation of antigen present-
ing dendritic cells (DC) to present antigen, induction of
regulatory T cells (iTregs) to terminate responses (35, 36)or
radiation-induced senescence in keratinocytes (37). In the last
example Bmi-1, a polycomb group protein, was shown to
epigenetically silence NOX genes and mitigate radiation-
induced genotoxicity.
Radiation-induced rapid changes in redox-sensitive pro-
teins could be important in many aspects of ‘‘danger’’
signaling, although this aspect of radiobiology is little
studied. However, it is easy to see how, for example,
cysteine oxidation may modulate the action of multiple
redox-sensitive proteins (38) and one could imagine that
there may be distinctive redox requirements for triggering of
such molecules, which would be impacted by the basal
redox status of the cell and the amount and type of ROS/
RNS generated and their intracellular location. Importantly,
the oxidative stress that follows irradiation can also result
from the actions of pro-inflammatory cytokines.
Radiation-inducible redox-sensitive transcription factors
include NF-jb, early growth response factor (Egr1), and
AP-1 (39) that are heavily involved in inflammatory
cytokine production. Other ROS-responsive molecules of
importance in radiobiology would include the protein
mutated in ataxia-telangiectasia (ATM) (40), redox-sensi-
tive phosphatases that may be responsible for rapid
phosphorylation of EGFR and PDGFR (41) after irradiation
and that can lead to further ROS generation (42), and
phosphatase and tensin homolog deleted on chromosome 10
(PTEN) that reacts to oxidative stress to activate the
powerful mediator of cell survival and proliferation, Akt,
and other kinases (43). Downstream of Akt, mTOR along
with AMPK pathways that sense cellular nutrient and
energy levels are also receptive to redox changes, and can
downregulate biosynthetic processes, including proteasome
function (44), often results in further ROS production,
autophagy and eventual cell death. The redox-sensitive
transcription factor HIF-1, which is a major contributor to
angiogenic cytokine production, is also activated by pro-
inflammatory cytokines and is another example of their link
to oxidative stress (45).
Cytokines and Radiation Converge in Free Radicals
Ionizing radiation effects converge with pro-inflammatory
cytokines in that both generate free reactive oxygen and
nitrogen species (ROS and RNS) such as superoxide, nitric
oxide, hydroxyl radicals, peroxynitrite and their derived
products (46). Some cytokines and growth factors, includ-
ing those as diverse as TNF-a(47, 48) and EGFR (49),
generate cellular ROS and actually require ROS for signal
pathway activation. Conversely, anti-inflammatory cyto-
kines, such as TGF-b, IL-10 and IL-4, tend to inhibit ROS/
RNS-mediated effects and display anti-oxidative properties
(50–52), although as always this may vary with the cell type
and circumstances. This yin-yang feature is intrinsic to
cytokine networks and suggests that redox is the fulcrum on
which pro-inflammatory and anti-inflammatory responses
are balanced (Fig. 2). This might explain why free radical
scavengers, such as N-acetyl cysteine (NAC) (53), amifos-
tine (54), or superoxide dismutase mimetics (55), can lower
pro-inflammatory cytokine expression.
Questions arise as to the extent to which radiation
responses and inflammatory cytokines mutually influence
each other through ROS/RNS generation. Given the short
half-life of ROS in cells, where, when, and how much of
ROS is generated will impact their persistence and their
abilities, for example, to deplete free radical scavengers, to
change cellular redox status, to impact transcriptional
510 REVIEW

signaling and to cause cell death. At clinical doses,
radiation-induced ionizing events occur more or less
randomly, unlike most oxidative stresses that act primarily
at membranes. Classical radiobiology indicates that ROS
generated per Gy through radiolysis of water will be short-
lived and low in number in comparison with most oxidative
stresses, at least for equivalent cell kill. The high cytotoxic
efficacy of ionizing radiation is attributed largely to ROS
generated within 2 nm of DNA and the formation of
complex DNA DSBs, whereas the main sources of cellular
ROS generated in response to oxidative stresses are
generally mitochondria, membrane-bound nicotinamide
adenine dinucleotide phosphate oxidases (NOX), or other
oxidases. These may be secondarily linked to DNA damage
response pathways (56–58). However, there is growing
evidence that radiation also can generate ROS from these
sources without nuclear intervention by damaging mito-
chondria, activating NOX or other oxidases (57, 58), or
causing ATP release, ion channel activation (59) and
purinergic signaling (60) (Fig. 3).
The consequences of secondary ROS production from
pro-inflammatory cytokines after radiation exposure can be
profound. High levels are likely to cause cell death and
further perpetuate DNA damage, while lower levels may
activate redox-sensitive signaling pathways such as those
directed by nuclear factor-jb- (NF-jB) and mitogen-
activated protein (MAP) kinase (61, 62). These pathways
lead to the additional production of pro-inflammatory
chemokines, such as IL-8 (CXCL8) and MIP-2 (CXCL2),
and cytokines such as TNF-aand IL-1 both in vitro and in
vivo (63–66). Interestingly, EGFR signaling can both induce
inflammation and DNA damage through the generation of
pro-inflammatory cytokines (67) and can also enhance
DNA repair (68). Ultimately, cytokines may alter intrinsic
radiosensitivity (69) by linking damage with cell fate
decisions, including DNA repair, genomic instability, cell
proliferation, differentiation, and death.
The radiation dose required to activate transcriptional
responses by NF-jB and pro-inflammatory pathways is of
interest. Even low doses of radiation can be effective, for
example in causing NF-jB-mediated immune cell differen-
tiation, although the optimal activation dose tends to be in
the region of 7–10 Gy (70), doses that are surprisingly also
optimal for pro-inflammatory cytokine responses, at least in
some studies (32). Radiation-induced bystander effects may
be ascribed, in part, to these mechanisms (71).
DAMPS and their Receptors in Cytokine and ROS
Production
The most obvious, though not the primordial, role of
cytokines is to orchestrate mesenchymal, epithelial and
immune cellular communications so as to restore homeo-
FIG. 2. The yin-yang of cytokines. The balance between pro-inflammatory cytokines and anti-inflammatory cytokines is critical in determining
outcome. Chemokines have preferred partners that link cell trafficking to function, as indicated. Angiogenesis, tissue replacement (fibrosis) and
regeneration predominantly fall within the influence of the more anti-inflammatory axis.
REVIEW 511

stasis, as after radiation exposure. To fulfill these roles,
cytokines network with endogenous and exogenous ‘‘dan-
ger’’ signals released from damaged tissues, as after
irradiation. Tissue damage causes secretion or release of
molecules that express damage-associated molecular pat-
terns (DAMPS) into extracellular spaces that signal through
conserved receptors that recognize broad features of
molecules (Table III and Fig. 3). In this respect, DAMPS
are similar to pathogen-associated molecular patterns
(PAMPS) release during infection. Some of these DAMPS,
like ATP, are secreted rapidly after irradiation and mediate
cellular responses through activation of purinergic receptors
that activate calcium channels (Fig. 3) (60). Others are
secreted later or come from dead cells or the action of
enzymes on the extracellular matrix. Mitochondrial peptides
and DNA can act as DAMPS and if the damage is sufficient
can cause systemic inflammatory response syndrome. The
type of DAMP released with time may be important in
defining the response that is made. Because the intracellular
environment is generally a reducing one, molecules outside
cells may be subjected to conformational change, making
oxidation-specific moieties a particularly interesting source
of DAMPS in response to radiation (72).
DAMPs and PAMPS signal through pattern recognition
receptors (PRRs) (73). PRRs include the transmembrane
TLRs and C-type lectin family receptors (Table III),
endosomomal TLRs (TLR3, TLR7, TLR9), cytosolic
retinoic acid-inducible gene-I-like helicases (RIGs) and
receptors with nucleotide-binding domain (NOD) and
leucine-rich repeats (NLR). Selectivity is broad but
meaningful. For example, CpG-rich DNA from bacteria
and viruses activate TLR9 in an endosomal compartment to
generate type I IFN and other cytokines with antiviral
properties. The link between receptors and cytokines is
provided by adaptor molecules, such as myeloid differen-
tiation primary response protein 88 (MyD88) and TIR
domain-containing adaptor-inducing interferon-b(TRIF).
These act through NF-jB, MAP kinase, IRF and other
down-stream signaling pathways so as to induce TNF-a, IL-
1, IFNa/b, IL-10, and other cytokines (74). DAMPS
therefore can initiate self-propelled cytokine cascades that
primarily initially cause inflammatory tissue damage (11).
PRRs at epithelial surfaces are equally important as those in
immune cells in combating or facilitating entry of organisms
into the body, including bacterial translocation from the gut
after irradiation (75). In a radiation-damaged gut, various
types of DAMPS may work in cohort with PAMPS to
generate inflammatory infiltrates and activate innate im-
mune defenses.
FIG. 3. ROS can be generated from many sources following irradiation. Released nucleotides including ATP can activate P2X purinergic
receptors to open the cation pore and trigger calcium-dependent intracellular processes. This is required for activation of NADPH oxidases that
can also be activated by TLR signaling to generate superoxide. Radiation damage to mitochondria is another potential source of ROS. Further
DAMP and pro-inflammatory cytokines signaling, the DNA damage response through Bax, and the formation of inflammasomes can all
perpetuate ROS generation by forming positive feedback circuits. Adenosine can be generated from nucleotides by ectonucleotidases such as
CD39 to signal through the adenosine receptors (AR) to negatively regulate inflammation, as does the production of anti-inflammatory cytokines.
512 REVIEW

An example of a DAMP released after irradiation is the
high-mobility group box 1 (HMGB1) protein, a chromatin-
binding nuclear protein that signals through TLR4 to
generate further ROS production (76) (Fig. 3). Through
DAMPS, ROS and cytokine production multiple layers of
self-amplifying feedback control circuits are created that
prolong responses for long after the initial radiation-induced
ionization events are completed (57). The consequences
include vascular damage, interstitial fluid accumulation,
inflammatory cell infiltration and creation of a lesion with a
pro-oxidant microenvironment that is hostile to pathogens
and cells alike and with a spatially and temporally expanded
‘‘danger’’ zone (77, 78) (Fig. 1). One consequence of this
‘‘danger’’ microenvironment is maturation of dendritic cells
(DCs) that acquire the ability to present antigen so that
adaptive immunity can develop. Other consequences may
include certain radiation-induced ‘‘bystander’’ effects.
Effects are likely to change spatially. At a distance from
the lesion, increased cell proliferation and survival may be
promoted (79), which may be a mechanism to limit the
spread of infection. One would also expect cytokine-
mediated changes in cellular radioresistance (69).
Remarkably, some of PRRs form higher order oligomeric
structures in the cytoplasm of some cells (74). In most
cases, how and how often this process occurs is not well
known. What is known is that NLR family members can
assemble in response to appropriate ‘‘danger’’ signals to
form inflammasomes (80). These can also be activated by
microbial and host DNA independent of TLRs (81), where
DNA binding proteins seem to play a major role. This
mechanism may underlie systemic lupus disease.
The formation of the inflammasome autocatalytically
activates caspase I (ICE) to cleave IL-1band IL-18, which
are ‘‘leaderless’’ pro-cytokines unable to exit the cell
through normal secretory paths. The activation of IL-1bin
this way may lead to cell death by ‘‘pyroptosis’’, which has
characteristics of both necrosis and apoptosis. Pro-inflam-
matory NF-jB driven synthesis of high levels of IL-1 in
concert with an inflammasome secretory mechanism must
send a strong ‘‘danger’’ signal to the body and could
mediate many auto-inflammatory disease states (82).
Recombinant IL-1R antagonist (Anakinra) or soluble IL-
1R (Rilonacept) have been used to identify patients with
such diseases and to distinguish them from those that
respond better to anti-TNF therapies, such as infliximab
(anti-TNF) or etanercept (TNFR2-IgG1 fusion product)
(83). The role of inflammasomes in radiation-induced
responses has yet to be defined but irradiated tissues often
show a very strong IL-1bsignal (84, 85) and this form of
catalytic processing may be particularly important in the
irradiated gut (86).
The involvement and impact of any cytokine will vary
with the cell type/tissue and with time, but it is easy to see
how ROS, pro-inflammatory cytokines, and DAMPS can
mutually reinforce their relationship with time. Not
surprisingly, after irradiation cells and tissues can express
pro-inflammatory cytokines within minutes and re-expres-
sion can follow in waves for long thereafter (34). Survivors
of Hiroshima (87) and Chernobyl (88) continue to have
dysregulated cytokine expression. It also follows that many
early and late manifestations of radiation damage can be
cytokine-mediated. For example, early radiation-induced
microvascular destruction after irradiation can be abrogated
by an anti-TNF antibody (89), in keeping with the natural
role of TNF-ain vascular effects that are manifest clinically
in skin as erythema. TNF-acan also contribute to radiation-
induced DNA damage, including c-H2AX-staining double-
strand breaks that may occur late after exposure and that
often are associated with genomic instability (67). Protec-
tion against late radiation-induced demyelination in the
brain is conferred by TNFR2 (90), suggesting a dichotomy
between TNFR1 and TNFR2 pathways in mediating cell
survival after irradiation. TNF-aand IL-1 therefore often
appear as ‘‘driver’’ cytokines in inflammation.
Breaking the Free Radical-Cytokine Circuit
The fate of ROS in a cell may be short-lived but their
effects are far-reaching and complex by virtue of their link
to cytokines, cell signaling, and other interactive pathways.
The production and activities of ROS need to be controlled,
and this is generally achieved with high constitutive levels
of free radical scavengers and antioxidants such as
glutathione. Antioxidants are also generated in response to
ROS that is induced by pro-inflammatory cytokines,
radiation, and other oxidative stresses.
At the first level of protection, manganese superoxide
dismutase (MnSOD/SOD2) can be generated in the
mitochondrial matrix to control superoxide production in
the site. Hydrogen peroxide results can require further
degradation, for example, by catalase. Radiation can induce
MnSOD expression through NF-jB dependent pathways, as
can cytokines and microbial products, most notably IFN-c
and LPS and enhanced expression of SOD2 protects cells
and tissues against radiation damage (91). NF-jB therefore
serves as a transcription factor for both pro- and antioxidant
programs (92). Inducible nitric oxide synthase (iNOS) that
generates nitric oxide (NO) can be produced by similar
pathways after high-dose irradiation, or after low doses by a
paracrine cytokine-dependent mechanism (93). NO can
negate ROS, forming RNS with nitrosation and nitration. In
rare cases, NO and RNS are able to promote apoptosis
through inhibiting NF-jB(94) but more often activate anti-
apoptotic pathways (95). At the tissue level NO can directly
effect blood flow and metabolism (96).
Although antioxidant enzymes involved in glutathione
(GSH) synthesis can be generated rapidly through NF-jB
(97),this is generally part of a second level pathway that is
controlled through another redox-sensitive transcription
factor: NF-E-2-related factor-2 (Nrf2) (98). Nrf2 is normally
bound in the cytoplasm by its redox-sensitive inhibitor
Keap1. In response to oxidative stress, Nrf2 is released and
REVIEW 513

binds the antioxidant response element (ARE) in the
nucleus to transcribe numerous Phase II detoxification
enzymes and antioxidant proteins (99). Under at least some
conditions, radiation-induced Nrf2 activation does not kick
in until several days after exposure, perhaps in response to
late ROS generation and the depletion of antioxidant
reservoirs (100). Nrf2 activation generally plays an
important protective role in limiting the upregulation of
NF-jB activity and pro-inflammatory cytokine production
and its depletion causes autoimmunity (102) and sensitivity
to radiation (100).
There are many other examples of the importance of cross
talk between redox-sensitive proteins and cytokine net-
works. For example, the antioxidant thioredoxin (Trx) in its
reduced form binds and inhibits the MAP kinase kinase
apoptosis signal-regulating kinase (ASK1). Oxidation of
Trx-ASK1 by oxidants such as ROS drives the release and
activation of ASK1 (103), leading to sustained activation of
JNK, P38 and apoptosis (62). Alternatively, induction of
Trx by cytokines such as IFN-c, or oxidative or radiation
stresses, blocks ASK1 activation and protects cells against
apoptosis (104). Trx-ASK1 is therefore a molecular switch
that converts a redox signal into kinase activation. This
ASK1 pathway is required for pro-inflammatory cytokine
production through TLR4 and p38 signaling pathways
(105). Interestingly, the TNF-related adaptors TRAF2 and
TRAF6 also form part of the ASK1 signalosome, linking
the TNFR superfamily and TLR/IL-1R family to this ROS-
responsive pathway (106).
Cytokines Hunt in Packs
The essential purpose of ‘‘danger’’ signaling is to alert the
body so as to cause inflammatory host cell infiltration into
the site. This varies in composition and function with time
in a programmed manner. Initially, primarily neutrophils
form a pathophysiological lesion to remove debris and
pathogens if present. A balanced measure of cellular self-
sacrifice by stressed local tissue cells and by the
inflammatory cells [the ‘‘grateful dead’’ (73)] contributes
and lymphocytes and macrophages follow. With time, cell
proliferation and resistance to invasion and death in
nonimmune ‘‘bystander’’ cells is enhanced, progenitor/stem
cells are recruited from local stem cells by epithelial-
mesenchymal cell transitions and from the bone marrow,
and angiogenesis and vasculogenesis are stimulated. The
transition from a pro-inflammatory, pro-oxidant environ-
ment to one that is more anti-inflammatory and antioxidant
is critical for the tissue recovery processes. Later infiltrates
of regulatory cells complete the regenerative process, or
encourage replacement of damaged areas with fibrotic
extracellular materials (Fig. 1). The ability of a tissue to
recapitulate its original structure, which is present during
prenatal life, is lost in adults, with some exceptions, and
fibrosis and scarring driven by TGF-bis a common
outcome (107). While fibrotic responses may have the
advantage of immediacy in maintaining tissue integrity, it
comes at a cost of long-term loss of function and may
inhibit the regenerative process.
Vascular damage after irradiation is a potentially
important aspect of normal tissue and tumor responses that
is compounded by a failure of angiogenesis, as can be
demonstrated by the ‘‘tumor bed effect’’, where tumor
growth in an irradiated site is slower than normal (108). In
fact, hypoxia may be a general switch within any
inflammatory site to drive factors like hypoxia inducing
factor 1 (HIF-1) to reprogram a pro-oxidant, pro-inflamma-
tory microenvironment to one supporting angiogenesis and
wound healing through HIF-dependent cytokines, such as
VEGF (Fig. 1). This has implications for irradiated tumors,
where alternatively activated macrophages with an M2
phenotype accumulate under the influence of radiation-
induced CSF-1 and stromal derived factor 1 (SDF-1) in
areas of hypoxia that are generated by loss of microvascu-
lature (109). Such macrophages produce large amounts of
TGF-band VEGF and can enhance tumor growth and
wound healing. Hypoxia caused by irradiation of normal
tissues may elicit similar consequences. The concept is that
M1 pro-inflammatory macrophages ‘‘switch’’ into, or are
replaced by, M2 macrophages with a change in the cytokine
profiles (Figs. 1 and 2). The lack of angiogenesis and
reliance on vasculogenesis in irradiated sites could lead to a
vicious cycle of chronic activation of macrophages,
fibroblasts and worsening hypoxia (110), more tissue
damage and fibrosis; a nonhealing wound.
Amovingpictureemergesofdiversecelltypes
interacting with a common purpose and with a high level
of control being exerted over their functions and their
existence. Control is in large part the purview of mutually
antagonistic, cytokine-driven processes with pro-inflamma-
tory, pro-oxidant pathways being opposed by, and giving
way in time to, anti-inflammatory, antioxidant forces (Figs.
1 and 2). Loss of control has serious consequences, often
ending in debilitating disease and even death. It is easy to
see how snapshots, which do not take account of temporal
aspects of responses, often paint cytokines as two-edged
swords with roles in both pathogenesis of and recovery from
disease.
Orchestration of these responses by cytokines requires
considerable functional integration to drive them forward.
To achieve such integration, cytokines are elaborated as
functionally interactive cohorts that change in composition
with time. These cohorts can be grouped in a very general
way as: pro-inflammatory such as TNF-a, IL-1aand b, IL-
17; angiogenic/vascular VEGF, TNF-aand FGF; anti-
inflammatory IL-4, IL-10 and TGF-b; pro-fibrotic IL-6 and
TGF-b; immune IL-2, IL-4 and IL7, and hematopoietic
CSF1, GM-CSF, IL-3, EPO (Fig. 2). In fact, the cohorts
should be viewed as interlocking, cross-talking networks
that coordinate with other molecular and cellular systems to
orchestrate tissue responses through changing redox,
514 REVIEW

extracellular matrix, cell adhesion, cell cycle proliferation
and cell migration to focus on the challenge at hand.
While cells of the immune system elaborate high levels of
cytokines to effect host defense and maintain tissue
integrity, this is not anarchy. Resident mesenchymal and
epithelial cells are in the frontline of defense and they
instruct immune cells how to behave in a site, in part
through shared ligands and receptors and juxtacrine/
paracrine interactions (75). Many, perhaps all, cell types
share PRR recognition systems for DAMPS and PAMPS,
though with differential expression.
Balancing Opposing Forces to Maintain Homeostasis
Moving beyond the acute phases of inflammation to later
more directed responses, the most illuminating and dramatic
example of coordinated expression and action of cytokines
and division of labor comes from the discovery of distinct
patterns of cytokines being produced by different antigen-
specific helper/regulatory T cell subsets (Th/Tregs) (111).
CD4
þ
Th cells recognize antigenic peptides 15–24 amino
acids in length in association with MHC class II molecules
on DCs through their T cell receptor-CD3
þ
complexes. T
cells must also receive a second verification signal through
CD28 or a similar co-accessory molecule, or they will
become anergic; a mechanism for maintaining peripheral
tolerance to ‘‘self’’. DCs gain such molecules and other
properties required for efficient antigen presentation by
maturing in a ‘‘dangerous’’ microenvironment; a process
that is switched off during healing. The potency of co-
accessory stimulation was dramatically seen when volun-
teers were given an agonistic antibody to CD28
(TGN1412). They developed a cytokine storm and severe
multi-organ damage (112).
Based on the signals received, CD4
þ
naı¨ve cells (Th
0
) can
differentiate along one of at least 4 pathways to form Th1,
Th2, Th17 or iTregs, each with distinct cytokine profiles
(Fig. 4). Antigen-specific responses in this way translate
into broader effector mechanisms through cytokine secre-
tion, affecting bystander immune cells and nonimmune cells
that have the appropriate receptor profiles. For example, the
M1/M2 profiles can be directed by the Th cell cytokines
secreted and can feed back to either stimulate or inhibit
lymphocyte responses. Th1 cells respond primarily to IL-12
to produce IFN-c, GM-CSF and TNF-a, and cooperate with
CD8
þ
T cells and M1 macrophages to make cell-mediated
responses that focus on elimination of intracellular viruses,
bacteria and tumors, and that may also play a role in organ-
specific autoimmune damage. Th2 cells, in contrast, are
stimulated primarily by IL-4 to produce IL-4, IL-5, IL-6, IL-
13 and IL-25. They assist B cells in the generation of
antibodies that form allergic responses and are critical for
expelling extracellular parasites and worms. Th17 cells
differentiate in response to IL-6 or IL-22 to produce IL17,
IL-21, IL-22, IL-23, and GM-CSF. Th17 cells have been
implicated in the pathogenesis of many chronic inflamma-
tory and autoimmune diseases (113) and they appear to be
in a dynamic equilibrium with Tregs, as IL-6 can drive
naive Tregs to become Th17 cells (114), a process that may
be under HIF-1 control (115).
Tregs (116) are the other side of the immunological coin
from Th cells. They are activated by antigen to maintain
peripheral immunological tolerance and exert homeostatic
control over inflammation. The presence of T cells that
could suppress antigen-specific inflammatory T cell activity
was recognized in 1971 by Gershon and Kondo, who called
the phenomenon ‘‘infectious immunological tolerance’’
(117). The field fell into disrepute for many years, but re-
emerged with the discovery of two subsets of natural and
induced Tregs with mainly nonoverlapping antigenic
repertoires that focus on controlling immune responses to
‘‘self’’ and on dampening inflammation. iTregs are induced
by TGF-band IL-2. They are distinct from the majority of
Tregs that are naturally occurring thymus-derived nTregs.
Although the respective roles of these subsets have yet to be
fully elucidated, iTregs may be more important than nTregs
in controlling inflammation at mucosal surfaces, while
nTregs are more involved with tolerance to ‘‘self’’ (118).
Tregs display specificity through their T cell receptors but
secrete anti-inflammatory and immunosuppressive effector
cytokines, such as IL-10 and TGF-b, and collaborate with
M2 macrophages to diametrically oppose Th1 and M1
cellular responses (Fig. 2). Another arrow in their quiver is
their ability to convert pro-inflammatory extracellular ATP
FIG. 4. Antigen-specific Th cells differentiate under the influence
of cytokines into subsets with distinct cytokine profiles and functions.
Two classes of Tregs (iTregs and nTregs) produce immunosuppressive
effector cytokines that work by juxtacrine and paracrine action. nTregs
from the thymus can be influenced by IL-6 and TGF-bto develop into
auto-inflammatory Th17 cells, while blocking iTreg development.
Other Treg subsets have been described, but are less well established.
REVIEW 515

‘‘danger’’ signals into immunosuppressive adenosine
through induced expression of cell surface ectonucleoti-
dases (Fig. 3), a process that is enhanced by radiation
therapy (119), and in which HIF-1 and hypoxia might play a
role (120). This is in keeping with the thesis that an
antioxidant/adenosinergic microenvironment is generated
that is tissue-protective which is the antithesis of pro-
oxidant acute inflammation, and controls and neutralizes
inflammation to promote healing. Recently, RT has been
shown to increase Treg representation in mice and humans,
perhaps to control radiation-induced inflammation (35,
121–125).
This dramatic T cell polarization leads to an important
interpretation of disease progression that is based on the
cross talk between Th subsets and their cytokines that form
balanced opposing forces. The cytokine-driven switch from
a pro- to antioxidant environment suggests that the fulcrum
of this balance is redox (Fig. 2). In T cells, this polarization
is orchestrated by the prevailing cellular microenvironment
through a network of transcription factors: T-bet for Th1,
GATA-3 for Th2, RORgammat for Th17 cells, and Foxp3
for Tregs (126). Thus, loss of the forkhead transcription
factor Foxp3 results in Treg deficiency and multi-organ
fulminating autoimmunity in humans and mice (127), while
the IL-10 knockout mouse is an excellent model for chronic
inflammatory disease (128).
The concept that distinct functional T cell subsets exist as
balanced forces to maintain homeostasis within and outside
the immune system has established validity. However, its
extension to CD8
þ
T cells and ‘‘classically’’ activated M1
and ‘‘alternatively’’ activated M2 macrophages, with the
former being pro-inflammatory and anti-microbial and the
latter anti-inflammatory, wound healing and pro-angiogen-
esis (129) is less firmly established. DC1/DC2 subsets have
also been proposed that selectively stimulate different Th
subsets (130). Although they express distinct phenotypes
and cytokine profiles, there is some controversy as to how
‘‘fixed’’ these subsets are and the degree to which they can
be reprogramed. They may be more ‘‘plastic’’ than Th
subsets that seem set in their lineages. Alternatively, even
some T cell subsets show some evidence of plasticity as
nTregs, but not iTregs, can be converted into Th17 cells by
IL-6 with a distinct change in function (Fig. 4) (114).
In spite of these caveats, the concept of functional
polarization of many cell types, whether transient or
permanent and the cytokines they produce is critical for
understanding many biological processes including the
switches that drive progressive wound healing and the
factors that establish the tumor microenvironment, with and
without therapy. At any given time, what appear to be
mutually antagonistic forces may be observed simulta-
neously. This is to be expected from a system that relies on
the balance between opposing forces, expressed spatially
and temporally, to maintain and restore control. As the
battle ebbs and flows, one or the other aspect of events will
be displayed, as is seen late after radiation exposure or in
any other chronic condition. The tissue damage response to
radiation will depend upon the same forces and redox
responses.
‘‘Radiation-Induced’’ Cytokine Gene Expression In Vitro
and In Vivo
As has already been mentioned, exposure of cells and
tissues to ionizing radiation in vitro and in vivo induces
expression of many cytokines and growth factors. A few
examples shown in Fig. 2 are: TNF-a, IL-1aa, IL-1b,(84,
131–133), type I IFN (134), GM-CSF (135, 136), IL-4, IL-5
(137, 138), IL-6 (136, 139), IL-10 (137), IL-12 and IL-18
(140), VEGF and bFGF (141), and TGF-b(142). Many
appear as immediate early genes and legitimately qualify as
‘‘radiation-inducible’’. However, many confounders can
alter the cytokine profiles produced after radiation exposure.
Other factors will influence the late transcriptional,
developmental and lineage-specific hierarchies that respond
to tissue damage. The genetic make-up of the host, the
influence of microbial products, tumors and other ‘‘extra-
neous’’ stimuli are possible influences. This begs the
question as to what is meant by ‘‘radiation-induced’’.
Several general points are worthy of consideration.
Obviously, radiation dose is important. One does not
expect to see a straight linear dose-dependency for cytokine
production. As mentioned previously, NF-jB and pro-
inflammatory responses generally require moderate doses of
around 7–10 Gy to be optimal (11), but low-dose effects
have also been observed (143). The strength of the signal
and its persistence (e.g., dose fractionation/low-dose rate)
would be expected to strongly influence the outcome.
Indeed, one of the rationales for developing the standard 2
Gy fractionation protocol may have been to minimize the
levels of pro-inflammatory cytokine expressed over a short
time period. Unfortunately, highly detailed analyses over
wide dose ranges and times in multiple systems are difficult
to perform and complete dose-response datasets are lacking.
It is important to realize that cytokine expression profiles
change if cells are cultured in vitro, and are different from
what is observed in vivo (144). Serum factors and even
adherence to plastic can activate cytokine expression by
macrophages (145). Furthermore, after whole-body irradi-
ation the cytokines observed might be in response to
microbes that have translocated across the gut or invaded
the host due to radiation-induced immune suppression
rather than being genuinely ‘‘radiation-induced’’. Microbial
PAMPS, such as LPS, are far stronger pro-inflammatory
stimuli than are radiation-induced DAMPS.
In vivo, the pathogenesis of radiation damage has a clear
genetic element. The genetic bias in cytokine profiles
demonstrated by different mouse strains in models of
parasitic and autoimmune disease is well known. As a
result, BALB/c and DBA/2 mice are often designated as
Th2-oriented strains, and C57Bl/6 and C3H are Th1 strains.
There is no reason to believe that this does not influence the
516 REVIEW

response to radiation. This may be why Th2-type cytokine
mRNAs, such as IL-4, IL-5 and IL-10, in addition to pro-
inflammatory cytokines, were found to be increased after 5
Gy irradiation of Balb/c splenocytes (137). This simple Th1/
2 designation is insufficient to describe all responses. For
example, C3H mice develop potentially lethal pneumonitis
following thoracic radiation, while C57Bl/6 mice resist this
outcome and instead develop fibrosis (146). Both are
‘‘Th1’’ strains but distinct gene loci are involved (147).
Both strains develop inflammatory infiltrates but in C3H
mice pulmonary Mac1
þ
macrophages increase dramatically
as does pro-inflammatory cytokine production just before
death (Fig. 5). C57Bl/6 mice control this macrophage-
related cytokine response only to later develop IL-6/TGF-b
associated fibrosis.
These ‘‘waves’’ of responses are seen in several different
tissues and strains following radiation exposure (34). It
appears most likely that the tendency to develop radiation-
induced pneumonitis or lung fibrosis is determined by
nonimmune host genetics, not by the genetics of the
immune cells. The crossbred C57Bl/6 3C3H strain
develops fibrosis, not pneumonitis, even if they have
undergone a bone marrow transplant to give them a C3H
immunohematopoietic system (McBride, unpublished data).
A parallel for the interaction between immune and
nonimmune cell types can be found in the way that tumors
dictate the nature of their host cell infiltrates and modify
systemic immunity through the release of cytokines and
other modulators (148). Given these responses, it is not
unreasonable to consider radiation-induced late effects as
forms of chronic inflammatory responses that fluctuate in
severity over time, much the same as in rheumatoid arthritis.
Immune cells, including lymphocytes, are an integral
feature of many radiation late effects. Their role remains
rather a mystery but we know that thymectomy reduces
radiation-induced pneumonitis and fibrosis in mice, sug-
gesting that there is an autoimmune T cell component and
that immune homeostasis is dysregulated (149).
Finally, it should also be remembered that all mouse
strains have genetic features that might affect the cytokines
they produce. C57Bl/6 have defects in phospholipase A II
that is responsible for arachidonic acid release leading to
production of eicosanoids (150). C57Bl/6 and DBA/2 have
mutations in the P2X7R that governs responses to
FIG. 5. Within the first month, the cytokine response of C57Bl/6 mice that develop fibrosis in response to 20 Gy local thoracic irradiation is not
markedly different from that of C3H/HeN mice that develop pneumonitis, as assessed by an RNase protection assay of whole lung. However,
macrophage (Mac1þve) infiltration increases with time in irradiated C3H/HeN lungs, followed by large increases in pro-inflammatory cytokines
that leads to their death by pneumonitis. C57Bl/6 mice control this pro-inflammatory response, but later develop high levels of IL-6 and TGF-b
that lead to lung fibrosis.
REVIEW 517

extracellular ATP (151). C3H/HeJ mice have a natural
mutation in TLR4 that limits TNF-aproduction and LPS
fails to protect them against radiation hematopoietic failure
(152). Humans will express similar diversity.
Cytokine-Driven Responses in Irradiated Tissues
Radiation damage to many tissues ultimately culminates
in fibrosis. Macrophages and other cells that accumulate in
the damaged tissue that may previously have been pro-
inflammatory, switch to elaborating pro-fibrogenic cyto-
kines like PDGF and TGF-b. TGF-b, which initially
dampens macrophage and lymphocyte activation, begins
to drives senescence of progenitor fibroblasts to fibrocytes,
with consequent collagen synthesis and deposition (153–
155). Thus, radiation-induced fibrotic remodeling of tissues
represents a multi-cellular process, with initiation and
sustenance of the fibrotic cascade by many different cell
types.
In the central nervous system, cytokines and growth
factors such as IL-1, FGF, PDGF, ciliary neurotrophic
factor (CNF), NGF, and TGF-bappear to have a major role
in regulating normal development and homeostasis (156). In
addition, pro-inflammatory cytokines, especially IL-1 and
TNF-a, have been implicated in the pathogenesis of CNS
injury in multiple studies, including after irradiation (157,
158). In the brain, these pro-inflammatory cytokines also act
as neuromodulators of sleep, neuroendocrine secretion and
other functions (159).
The early effects of brain irradiation are due to damage to
the cerebral microvasculature, leading to increased vascular
permeability and loss of integrity of the blood-brain barrier
(160). Edema and immune cell infiltration often lead to
clinically significant nausea, vomiting and headaches,
occurring in the acute (the first 24 h) and sub-acute (weeks
to months) stages. Pro-inflammatory cytokines play a
central role in all of these effects (84, 131). For example,
micro-vascular changes occur in both irradiated and non-
irradiated hemispheres as early as 3 days after irradiation
that were abrogated when the mice were treated with anti-
TNF-amonoclonal antibody prior to irradiation (89).
Late effects in the brain occur from six months to several
years after radiation treatment. The resultant damage to the
white matter can be very severe and interfere with the
patient’s quality of life. Chiang et al. showed increased
TNF-aand IL-1 expression 2 weeks, 2–3 months and 5–6
months after mouse brain irradiation. This correlated with
subacute and late loss of oligodendrocytes and demyelin-
ation (161). Loss of neural precursor cells residing in the
subventricular zone and hippocampal dentate gyrus also
occurs and may be implicated in somnolence and the
inability to learn new tasks (131). Pro-inflammatory
cytokines also drive the gliotic response to radiation (162–
164). Loss of TNFR2 exacerbates radiation-induced brain
demyelination (131), indicating the dual nature of the roles
of TNF-ain the brain depending on receptor expression.
Kim et al. also reported elevated levels of TNF-aweeks and
months after unilateral mouse brain irradiation and TGF-b
production (165).
In the lung, type II pneumocytes have been considered the
traditional targets of irradiation. Electron microscopy
reveals large-scale ultrastructural changes in the endoplas-
mic reticulum, mitochondria and plasma membranes of
these cells, as well as in endothelium and type I
pneumocytes. This leads to inflammation, desquamation
of epithelial cells from the alveolar surfaces, edema,
exudation into the alveolar spaces, thickening of the
alveolar septa and alteration of the capillaries. An initial
decrease in cell numbers is followed by an influx of
neutrophils and lymphocytes into the alveoli (166, 167),
although cells obtained by brochoalveolar lavage (BAL) do
not have the same cytokine profile as do interstitial cells and
are relatively inert (168). The final outcome is often
genetically determined (Fig. 5), as noted above, with
pneumonitis being associated with high levels of pro-
inflammatory cytokines and fibrosis with IL-6 and TGF-b
production (169). It has been suggested that in rats there is a
switch to CD4 Th2 phenotype cells with TGF-b, IL-4, IL-
10, and PDGF production that leads to activation of
fibroblasts and increased collagen production, although this
may be strain-specific (170). Antibodies to ICAM-1 and
TGF-b, or transfer of soluble TGF-btype II receptor can
block radiation pneumonitis (171–173). However, clinically
this approach has yet to be shown effective.
In the rat intestine, within hours after irradiation, the ileal
muscularis layer expresses high levels of IL-1b, TNF-aand
IL-6 (174). The epithelium may regenerate normally but
ulceration leads to accumulation of TGF-band membrane
thickening (175). Intestinal mesenchymal cells, mainly
smooth muscle and subepithelial myofibroblast cells, are
released from quiescence to begin the wound healing
process and chronic fibrosis results with TGF-bdriving the
process. Radiation enteritis can be extensive, resulting in
dysfunction, dysmotility, fibrotic structures with obstruc-
tion, fistula formation or bleeding, up to 8–12 months after
irradiation. Injection of lipopolysaccharide, with induction
of IL-1 in mice, prior to abdominal irradiation greatly
increased peritoneal adhesions 2–4 months afterward
suggesting a role for inflammation in this process (176).
In skin, a transient erythema is seen soon after irradiation.
IL-1, IL-6, and TNF-aproduced by activated dermal
macrophages and Langerhans cells mediate proliferation
and activation of keratinocytes, with early desquamation
and late hyperkeratosis, and proliferation of fibroblasts, and
the resultant fibrosis (177). Again, TGF-bstimulates
fibroblast production of collagen in dermal layers (178,
179). Fetal wounds heal with minimal scarring: there is no
acute response, and dermal fibroblasts are rare. TGF-b
levels are low in fetal wounds as compared to adults, and
injecting TGF-binto fetal wounds causes scarring, while
injection of neutralizing antibodies to TGF-b-healed adult
wounds causes minimal scarring (180). The suggestion is
518 REVIEW

that this cytokine may initially be immunoprotective,
dampening down the inflammatory response of macrophag-
es and lymphocytes to radiation damage, but it negatively
modulates regeneration by stimulating proliferation and
activation of fibroblast collagen synthesis and deposition
later on.
CONCLUSIONS
In spite of their complexity, links between cytokine and
cytokine receptor structures and function are obvious with
multiple family members overlapping to create diverse,
socially interconnected networks that impact multiple
aspects of radiobiology. While it is not easy to causally
link increased cytokine levels to pathogenesis, the use of
specific inhibitors has shown that in certain disease
situations ‘‘driver’’ cytokines are critically important
players and form useful targets for intervention. As a result,
the number of clinically useful cytokine inhibitors has
grown in recent years. Since the evidence that cytokines are
intimately involved in radiation responses at all levels is
irrefutable, manipulation of cytokine pathways is likely to
be important in future radiation research and therapy.
ACKNOWLEDGMENTS
The authors would like to thank Natalia Mackenzie for proofreading the
manuscript and, for funding support, the NIH 2U19 AI67769 (WMcB) and
DOD W81XWH-10-10424 (DS).
Received: April 30, 2012; accepted: July 16, 2012; published online:
October 29, 2012
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