Institute for Radioprotection and Nuclear Safety, Human Health Protection and Dosimetry Division, Independent Section of Radiobiology Applied to Medecine, F-92262 Fontenay-aux-Roses Cedex, France
Submitted 24 February 2003 ; accepted in final form 28 May 2003
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ABSTRACT |
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intestine; inflammation; p65 and p50; c-jun and c-fos
Cytokines are glycoproteins produced by a wide variety of cells. They are
functionally grouped into proinflammatory cytokines (mainly IL-1, IL-6,
IL-8, and TNF-
) and anti-inflammatory cytokines [mainly IL-4, IL-10,
IL-1 receptor antagonist (IL-1ra), and TGF-
]. Investigation of the
balance between pro- and anti-inflammatory events in the gut may provide
important insights into the pathogenic mechanisms of radiation. An imbalance
between IL-1
and IL-1ra is reported to be an important factor in the
pathogenesis of inflammatory bowel disease (IBD) and may explain why the acute
inflammatory response develops into chronic persistent inflammation in some
patients (9).
Cytokines probably play a role in initiating and perpetuating these uncontrolled disease processes. There is, however, a remarkable paucity of information on cellular interactions in complex gut inflammatory diseases such as Crohn's disease and ulcerative colitis, and animal models of IBD have not provided substantial additional data. Some reports suggest that the intestinal muscle layer, including mesenchymal tissue, fibroblasts, myofibroblasts, and muscle cells, may be the source of the inflammatory mediators that account for acute inflammation-induced changes in motor function and later for intestinal fibrosis (24, 40). Intestinal motility dysfunctions with modifications of transit and contractility have been reported after irradiation (16, 46).
Many inflammatory responses, particularly in the gut, are mediated by the
activation of transcription factors such as NF-B and activator
protein-1 (AP-1) (40,
43). NF-
B is an
inducible transcriptional factor defined as a heterodimeric complex of two
subunits, p65 (Rel A) and p50/p105
(2,
3). In unstimulated cells,
NF-
B is sequestered in the cytoplasm as an inactive complex bound to
inhibitor
B (I
B), an inhibitory protein. Activation of
NF-
B by inflammatory cytokines, such as IL-1
and TNF-
,
induces a cascade of reactions leading to I
B phosphorylation and
degradation by proteasomes (2).
Activated NF-
B then translocates into the nucleus, thereby activating
the transcription of a variety of genes
(35). Besides modulating genes
that directly influence cell proliferation and death, NF-
B regulates
the expression of several cytokines, including IL-1
and TNF-
(3,
45). It establishes a positive
feedback loop that amplifies the inflammatory response and increases chronic
inflammation (43). The number
of NF-
B positive cells has been correlated to the degree of
inflammation in human IBD
(39). Until now, ionizing
radiation has been shown to activate NF-
B only in vitro in fibroblasts
(6), endothelial cells
(17), HeLa cells
(28), and astrocytes
(37), and in vivo in
peripheral lymphoid tissues
(52).
Another nuclear target for cytokines is transcription factor AP-1, a homo-
or heterodimeric transcription factor composed of members of the Jun and Fos
families of DNA-binding proteins
(7,
21). AP-1 binding sites are
activated during inflammation in various types of cells and tissues
(21). Radiation-induced
activation of NF-B has recently been related to AP-1 activation.
Cooperative interaction between these factors seems necessary to obtain
inducible expression of proinflammatory cytokines during the prodromal stage
of radiation (4). The in vivo
activation of irradiation-induced transcriptional factors has not yet been
characterized in the intestines.
In the present study, we investigated in vivo the acute effect of ionizing
radiation on the balance between some proinflammatory [IL-1,
TNF-
, IL-6, and IL-8, a cytokine-induced neutrophil chemoattractant
(CINC)] and anti-inflammatory (IL-1ra, TGF-
, and IL-10) cytokines by
quantifying the changes in mRNA levels in the muscularis layer of rat ileum
and monitoring the time course of the activation of NF-
B and AP-1
transcriptional factors.
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MATERIALS AND METHODS |
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RNA extraction and RT-PCR. The mRNA levels of the cytokines,
NF-kB, and AP-1 subunits and of the housekeeping gene hypoxanthine-guanine
phosphoribosyltransferase (HPRT) were measured by real-time PCR. Total RNA was
prepared with the RNeasy total RNA isolation kit (Qiagen, France) according to
the manufacturer's instructions. The cDNA was produced from 1 µg of total
RNA by reverse transcription with 200 U of Superscript reverse transcriptase
(GIBCO) in a 20-µl reaction containing 1 x Superscript buffer
(GIBCO), 1 mM 2-deoxynucleotide 5'-triphosphate, 20 ng random hexamer,
10 mM DTT, and 20 U RNase inhibitor. After incubation for 50 min at 42°C,
the reaction was terminated by a denaturing enzyme for 10 min at 70°C. RNA
integrity was confirmed by denaturing agarose gel electrophoresis and ethidium
bromide staining. For IL-1, TNF-
, and IL-6, we used primers from
the manufacturer to amplify first-strand cDNA through 36 PCR cycles with
TaqMan (Applied Biosystems). PCR amplification of the other cytokines and
NF-kB and AP-1 subunits used Syber PCR master mix; the primer sequences, which
are listed in Table 1, were
designed with Primer Express software (Applied Biosystems). Optimized PCR used
the Abi Prism 7700 Sequence detection system (Qiagen). PCR fluorescent signals
were normalized to the fluorescent signal obtained from the housekeeping gene
HPRT for each sample.
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Preparation of protein extracts. Cytoplasmic and nuclear protein extracts were prepared according to methods described previously (51). Briefly, small aliquots (<0.1 g) were immersed in 1 ml ice-cold lysis buffer (in mM): 10 HEPES, pH 7.9, 10 KCl, 1.5 MgCl2, 1 DTT, 0.5 PMSF, and 5 µl/ml protease inhibitor cocktail (Sigma). They were homogenized on ice with a Dounce homogenizer, kept on ice for 15 min, and then 1% IGEPAL was added to the homogenate. After a brief vortexing, they were incubated on ice for 20 min and then centrifuged at 4°C (12,500 rpm) for 30 s. The supernatant corresponding to this cytoplasmic extract was collected into a new tube. The pelleted nuclei were resuspended into 50-200 µl of extraction buffer (20 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 5% glycerol, 1 mM DTT, 0.5 mM PMSF, and 5 µl/ml protease inhibitor cocktail) and kept on ice for 30 min. The nuclear suspension was centrifuged at 12,500 rpm for 15 min at 4°C to collect the supernatants containing nuclear protein extracts. Protein concentrations of cytoplasmic and nuclear extracts were measured with a modified Bradford method from Bio-Rad (Hercules, CA). The samples were then stored at -80°C.
Western blot analysis. We separated 20 µg of proteins from each
of the samples described above on 12% SDS-polyacrylamide gel and transferred
them onto nitrocellulose membranes. Membranes were blocked for 1 h with 5%
nonfat dry milk. Enhanced chemiluminescence was used to detect specific
proteins with the appropriate antibodies: anti-p65 (F-6, dilution of 1:500;
Santa Cruz), anti-p50 (E-10, dilution of 1:500; Santa Cruz) and
anti-IB
(C-21, dilution of 1:200; Santa Cruz). Densitometric
analyses used the Biocom analyzer (Les Ulis, France).
NF-B transcription factor activation. The DNA
binding activity of NF-
B in the nuclear extract was determined by EMSA.
An aliquot of 2.5 µg nuclear proteins was incubated with a reaction buffer
[in mM: 25 Tris (pH 7.5), 50 KCl, 6.25 MgCl2, 0.5 EDTA, and 0.5
dithiothreitol; and 10% glycerol and 1 µg poly(dI-dC)]. Then 105
counts/min of [32P]-end-labeled double-stranded DNA nucleotides
containing the consensus
B motif
5'-AGTGAGGGGACTTTCCCAGGC-3' and
5'-GCCTGGGAAAGTCCCCTCACT-3' were added to the reaction and
incubated 30 min at room temperature. Specificity of the DNA/protein binding
was determined by competition reactions by using a 10-fold molar excess of
unlabeled NF-
B oligonucleotides. For supershift analysis, nuclear
extracts were incubated with 2 µg of the polyclonal antibodies against the
NF-
B subunit of p65 and p50 (Santa Cruz Biotechnology, Santa Cruz, CA).
Two microliters of 0.1% bromophenol blue dye were then added to each sample.
After electrophoresis (an aliquot of 20 µl through a 6% nondenaturing
polyacrylamide gel for 2 h at 150 volts), gel was dried and the protein-DNA
complexes were visualized by a PhosphoImager.
Quantitation of NF-B activation was assayed with Trans-AM
NF-
B kits (Active Motif; Rixensart, Belgium) that included a 96-well
plate with an immobilized oligonucleotide containing the NF-
B
consensus-binding site (5'-GGGACTTTCC-3'). The active form of
NF-
B contained in nuclear extract specifically binds to this
oligonucleotide. Primary antibodies that detect NF-
B recognize an
epitope on p65 that is accessible only when NF-
B is activated and bound
to its target DNA. A horseradish peroxidase conjugated secondary antibody was
used for the spectrophotometric quantification.
Cytokine immunoassays. For the IL-1, TNF-
, and IL-6
analyses, the tissue samples were weighed and then homogenized in 10 mM PBS
(pH 7.4) supplemented with protease inhibitors: 2 mM PMSF, 10 µg/ml
pepstatin A, 1 µg/ml aprotinin, 10 µg/ml leupeptin, and 0.5 mg/ml EDTA.
Samples were then centrifuged at 10,000 g for 10 min, and the
supernatants were stored at -20°C for later measurement. The IL-1
,
TNF-
, and IL-6 assays used ELISA kits (R&D Systems, Minneapolis,
MN). The rabbit anti-rat IL-1
polyclonal antibody of the IL-1
kit
recognizes both recombinant and natural rat IL-1
. The manufacturer
reports that this antibody does not cross-react significantly with recombinant
(r) human (rHuman) IL-1RI, IL-1RII, or IL-1ra or rRat IL-1
, IL-2, IL-4,
IFN-
, or TNF-
, or rMouse IL-1
or IL-1ra. The rabbit
anti-rat TNF-
polyclonal antibody of the TNF-
kit recognizes
both recombinant and natural rat TNF-
. No significant cross-reactivity
was observed between this antibody and rHuman TNF-
or rRat IL-1
,
IL-2, IL-4, or IFN-
. The anti-rat IL-6 antibody recognizes both
recombinant and natural rat IL-6. No significant cross-reactivity of this
antibody was observed with rat IL-1
, IL-2, IL-4, IFN-
, or
TNF-
.
Result expression and statistical analysis. We used the
comparative CT-method
(8) for the relative mRNA
quantitation. The relative quantitation of target, normalized to an endogenous
reference (HPRT) and a relevant unirradiated control, is given as relative
quantitation = 2-
CT, where
CT is defined as the difference between the mean
CT (irradiated sample) and the mean
CT
(unirradiated sample), and
CT is the difference between the
mean CT (threshold cycle; cytokines, NF-
B subunit, or AP-1)
and the CT (HPRT) is the endogenous control.
All data are expressed as means ± SE for five animals. Comparisons among groups used one-way ANOVA, the Bonferroni's t-test (applied to test the rationale gene expression), and Student's t-test for nonpaired data.
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RESULTS |
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Effects of irradiation on mRNA levels of proinflammatory
cytokines. mRNA levels of the proinflammatory cytokines IL-1,
TNF-
, IL-6, and IL-8 (CINC) were quantified and expressed as a ratio to
a reference gene, HPRT, in the ileal muscularis layer
(Fig. 2). The HPRT mRNA level
in the ileal tissue was unchanged after irradiation (data not shown).
Abdominal irradiation (10-Gy) induced a significant increase of IL-1
(2.5-fold, P < 0.05), TNF-
(4.1-fold, P <
0.005), and IL-6 (2.9-fold, P < 0.05) mRNA levels at 6 h. Levels
of IL-1
and IL-6 mRNA remained significantly higher (3.5- and 2.9-fold,
respectively) in the irradiated than in the control tissue at 3 days after
irradiation. IL-8 (CINC) mRNA appeared only at 3 days at a level 10.8 times
greater than in the control tissue (P < 0.001).
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Effects of irradiation on mRNA levels of anti-inflammatory
cytokines. Analysis of the temporal patterns of the IL-1ra mRNA levels
showed increases by factors of 3.7 (P < 0.05) and 4.4 (P
< 0.01) at 6 h and 3 days postirradiation, respectively
(Fig. 3A). At day
1, on the other hand, the IL-1ra mRNA level did not differ from that in
the control. The IL-1ra/IL-1 ratio, which is an indicator of the
inflammatory balance, was significantly lower than the control at 1 day after
exposure (-41%; P < 0.05), although there was no significant
change at either 6 h or 3 days (Fig.
3B).
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Other cytokines with anti-inflammatory effects include IL-10 and
TGF-. Analysis of the time course of IL-10 expression in the irradiated
tissue showed that its mRNA levels were dramatically lower than in the
control: 87% lower (P < 0.05) at day 1 and 93% lower
(P < 0.01) at day 3 postirradiation
(Fig. 4A). In
contrast, the TGF-
expression was weak but higher than in the control at
1 day (1.5-fold) and significantly higher at 3 days (1.8-fold, P <
0.005) (Fig. 4B).
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Influence of irradiation on levels of NF-B
subunits. The predominant form of NF-
B is a dimer of p50 and p65
subunit proteins that binds to I
B inhibitory proteins in cytoplasm.
Cytokines stimulate the release of I
B and the consequent translocation
of NF-
B to the nucleus
(39,
43). Nuclear levels of
NF-
B p65 and p50 proteins were evaluated by Western blot at 6 h, 24 h,
and 3 days after radiation exposure (Fig.
5). Analysis of protein levels showed that irradiation induced
NF-
B translocation. The nuclear p65 protein concentration was five
times greater than in the control at 6 h and 24 h postirradiation (P
< 0.005) (Fig. 5A).
Irradiation also induced p50 translocation, and the p50 protein level in the
nucleus was greater than in the control tissue by a factor of 1.8 at 6 h
(P < 0.05), of 1.3 at 24 h (P < 0.01), and of 1.4 at 3
days after irradiation (P < 0.005)
(Fig. 5B). In
parallel, analysis of cytoplamic protein levels showed that irradiation
induced a fourfold decrease of p65 level at 6 h (P < 0.005) and a
2.5-fold increase 3 days after irradiation (P < 0.01)
(Fig. 5C). No
modification was observed on cytoplasmic p50 level induced by irradiation
(Fig. 5D). After the
NF-
B p65 and p50 proteins translocate into the nucleus, I
B is
ubiquitinated and rapidly degraded by proteasomes; the cytoplasmic I
B
content is thus substantially modified
(2). New I
B
synthesis is NF-
B dependent
(47). I
B
is the
only inhibitor protein regulated by NF-
B, and its level increased
progressively after the first day, reaching five times the control level
(P < 0.001) at 3 days after irradiation
(Fig. 5E).
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Effect of irradiation on NF-B activation.
NF-
B dimers, after translocation into the nucleus, activate appropriate
target genes. Involvement of p65 and p50 subunits in NF-
B activation
was determined by electrophoretic mobility shift assays of nuclear extracts.
Activation of NF-
B peaked at 6 h and declined at day 1 and
day 3 (Fig.
6A). The composition of the NF-
B complex was
determined with p65 and p50 antibodies. The anti-p65 produced a supershift
band, indicating that the activated complex contained predominately the p65
subunit. The p65 DNA-binding activity was confirmed by the Trans-AM
NF-
B analysis (Fig.
6B) showing a 3.5-fold increase of activity (P
< 0.01, n = 5) 6 h after irradiation. This activity disappeared in
the presence of an excess amount of soluble oligonucleotide containing a
wild-type NF-
B consensus-binding site (data not shown).
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Effect of irradiation on gene expression of NF-B and
AP-1 complex components. The relative p105 mRNA level was half that of
the control at 6 h after irradiation (P < 0.001). A small but
significant decrease in p65 mRNA expression was observed at 1 day (P
< 0.05) and 3 days (P < 0.01) after irradiation
(Fig. 7A).
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AP-1 is a homo- or heterodimeric transcription factor composed of members of the Jun and Fos families of DNA-binding proteins (7, 21). The level of c-fos mRNA in the ileal muscularis layer was double that for control tissue (P < 0.005) at 3 days after irradiation, the only time point with a significant difference (Fig. 8A). The c-jun mRNA level of the tissue from irradiated and control rats did not differ (Fig. 8B).
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DISCUSSION |
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Although early cytokine response after radiation exposure has been reported
previously in alveolar macrophages
(34) and in organs such as the
lungs, spleen, and brain (11,
20,
37,
52), this study is the first
to demonstrate in vivo that abdominal irradiation induces alterations in the
expression of genes involved in acute intestinal inflammatory response and
thereby modifies the balance between pro- and anti-inflammatory cytokines.
Increase in IL-1, TNF-
, and IL-6 expression occurred early at 6 h
after irradiation and the high levels of IL-1
and IL-6 mRNA persisted
for 3 days. Increased cytokine expression and protein production were observed
simultaneously.
A cascade of inflammatory events ensued, with the proinflammatory cytokines
(IL-1, TNF-, and IL-6) increasing the ability of endothelial cells,
macrophages, smooth muscle cells, and fibroblasts to secrete IL-8
(15). The accumulation of
neutrophils at the inflammatory site is known to be caused mainly by the
chemotactic cytokine IL-8
(36). Recently, by using
immunohistological analysis, we reported
(31) a marked neutrophil
infiltration characterized by an increase of myeloperoxidase-positive cells
and myeloperoxidase activity in the ileum 3 days after 10-Gy abdominal
irradiation. The IL-8 (CINC) was highly expressed 3 days after irradiation
(Fig. 2). Although the exact
role of IL-8 in the inflammatory pathogenesis is not totally clear
(25), its level (mRNA and
protein) has been positively correlated with the intestinal inflammation
score.
The intestinal immune response is carefully regulated in normal tissue so
that an inflammatory event is quickly and appropriately counterbalanced by
local anti-inflammatory mechanisms. An imbalance between pro- and
anti-inflammatory cytokines leads to intense inflammation and tissue
destruction. The biological effects of IL-1 are regulated by naturally
produced inhibitors, including IL-1ra
(9). In our study, the
IL-1ra/IL-1
ratios calculated at 6 h and 3 days after irradiation were
nearly identical to those for the nonirradiated rats, suggesting that at these
times, ileum produces appropriate amounts of IL-1ra to counterbalance the
IL-1
excess. However, 24 h after irradiation, the IL-1ra/IL-1
ratio was half that of the control. In human IBD, both tissue IL-1ra and
IL-1
levels increase with inflammation, but the IL-1ra/IL-1
ratio
decreases (9). The deficit of
endogenous IL-1ra production is an important factor in IBD pathogenesis and
may explain why the acute inflammatory response in some individuals develops
into chronic persistent inflammation rather than resolving. Our results
suggest that the imbalance observed here is not specific to IBD but extends to
irradiation-induced inflammatory effects.
TGF-1 is a cytokine with pleiotropic properties and local
proinflammatory effects. Among other things, it stimulates chemotaxis of
granulocytes and macrophages as well as the release of proinflammatory
cytokines (IL-1, TNF-
, IL-6). Turner et al.
(49) show that it induces IL-6
as well as IL-1ra production. However, analysis of the temporal pattern of
cytokine expression in our study shows that IL-6 and IL-1ra expression begins
some hours before TGF-
1 expression. In our study, TGF-
1 expression
increased on and after day 1 postirradiation. TGF-
1 is a
critical profibrogenic factor that induces the synthesis and deposit of
collagen and other matrix components. It appears to play a particularly
prominent role in the chronic phase of injury and is consistently
overexpressed in areas of the intestinal wall that have histopathological
lesions. Our data corroborate the increase in TGF-
1 protein observed by
immunohistochemistry in the small intestines of the rat 1 day after
fractionated exposure to X-radiation
(38).
IL-10 is a potent anti-inflammatory cytokine that downregulates the
synthesis of TNF- and IL-1
and upregulates IL-1ra synthesis
(10). It probably counteracts
the production of proinflammatory cytokines. In contrast to the substantial
expression and concentration of IL-10 in IBD patients during the acute phase
(42), IL-10 mRNA levels fell
drastically 1 day after irradiation. This response seems to be specific for
radiation exposure. This IL-10 gene repression is important, because the
cytokine has a direct anti-proliferative effect through its modulation of
T-cell functions and antifibrotic properties. Increased TGF-
expression
associated with decreased IL-10 levels characterizes a fibrotic state. The
importance of these anti-inflammatory cytokines on the pathophysiology of
acute radiation-induced inflammatory processes is underlined by findings that
IL-10 gene knockout mice develop gastrointestinal inflammation
(27) and that exogenous IL-1ra
improves colitis in animal models
(13).
NF-B and AP-1 activity play critical roles in the activation of
several cytokines (IL-1, TNF-
, IL-6, IL-8, and IL-10). In particular,
we have reported that in vivo the treatment by the caffeic acid phenethyl
ester, an inhibitor of the NF-
B-DNA binding properties
(33), inhibited totally the
increase of the IL-6 and its specific receptor expressions induced by
-irradiation, demonstrating the implication of NF-
B
(30). In the present study,
NF-
B activation induced by irradiation appeared to be fully
supershifted by the p65 antibody, suggesting that the NF-
B complex
implicated in the irradiation effect concerned more particularly the p65
subunit. However, Zhou et al.
(52) used
p50-/- mice to demonstrate the involvement of
the p50 subunit in the irradiation-induced expression of cytokines in vivo.
There was a minimal NF-
B activation in these knockout mice and low mRNA
levels for IL-1
, TNF-
, and IL-6 after irradiation (8.5-Gy), but
NF-
B activation in intestine was not detected. In the intestinal
ischemia model, however, Yeh et al.
(50) showed that the
activation of p50/p50 homodimers is unique to the intestine, whereas p50/p65
dimers are activated in other tissues. The time course of NF-
B nuclear
translocation and activation after irradiation (Figs.
5 and
6) is in agreement with
previous observations of cells in which p65 binding activity was maximal 2 to
3 h after irradiation and decreased after 5 h
(28). Western blot studies
(Fig. 5) showed that
cytoplasmic p65 levels decreased at 6 h, whereas nuclear levels increased.
However, although p65 nuclear levels return to control values at 1 and 3 days
after irradiation, a marked increase in cytoplasmic p65 levels was observed,
but it did not correlate with p65 mRNA levels
(Fig. 7). In fact, p65 and p105
(p50 precursor) expression decreased slightly 1 and 3 days after irradiation.
Taken together, our results showed that 1) despite the low
NF-
B subunit expression, there are enough subunit proteins already
present in the cytoplasm to produce the early irradiation response as
suggested earlier (28); and
2) the expression of the NF-
B subunits increased later after
irradiation, but their activation is limited by the I
B system.
Interestingly, Beg et al. (5)
reported that NF-
B is constitutively inhibited by I
B
,
activated by I
B
degradation, and then inhibited once again by
the resynthesis of I
B
. NF-
B rapidly induces
I
B
synthesis in an effective negative feedback loop that
controls the inflammatory process. Indeed, we observed an increase of
I
B
levels in the cytoplasm 24 h after irradiation, suggesting
that NF-
B activation induces the synthesis of its inhibitor
I
B
to regulate cellular activation, providing a negative
feedback loop that regulates the inflammatory process induced by irradiation.
The precise mechanism of NF-
B activation by
-radiation remains
unknown. The release of reactive oxygen species (ROS) that characterizes the
response to ionizing radiation occurs very early, and in vitro studies
(29) showed that ROS could
activate NF-
B in some cell types. However, direct involvement of ROS in
radio-induced NF-
B activation has been challenged
(28).
AP-1 transcription factors consist of the Jun and Fos protein families.
Studies of several tissues show that c-jun and c-fos can be
induced by ionizing radiation
(19). In this study, only the
level of c-fos mRNA was elevated on day 3 postirradiation,
whereas no effect on c-jun was observed from 6 h through 3 days.
However, the time course of expression may differ according to the tissue. For
example, expressions of c-fos and c-jun increase in vivo in
skin 2 h after an 8-Gy -irradiation, with a maximal effect at 6 h
(32), but do not increase in
the gut (1). Sherman et al.
(44) showed that the increase
in c-fos expression peaked at 3 h and was associated with
downregulation of c-fos RNA levels 24 h after irradiation.
Irradiation induced a very early (15 min after) expression of c-fos
in the brain, whereas the c-jun level was not changed up to 24 h
(19). Accordingly, in our
study, the absence of increased expression between 6 h and 3 days may be
explained by a repression of expression. Indeed, expression of c-fos
does not appear to parallel that of c-jun
(19).
The molecular cascades initiated by ionizing irradiation are complex and involve more molecules than those studied here. In particular, cytokine function is mediated through cytokine receptors that can be also secreted in a soluble form and that contribute to limiting cytokine action. The effect of radiation on these pathways must also be studied, because they are potential targets for therapeutic action. The existence of chronic cytokine-driven cascades raises the question of what perpetuates the response. The critical question that needs to be answered now is whether the acute responses we observed are responsible for acute and late intestinal damage, and if so, how efficient would the modification of proinflammatory cytokine expression be on the development of late complications after radiotherapy?
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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