Patent Abstract:
a method for the prevention of and therapy for radiation pneumonitis , dermatitis , soft tissue fibrosis and central nervous system toxicity in patients undergoing therapeutic radiation . in addition , the invention provides for pre - treatment of those responding to nuclear bio terrorism or other nuclear or radiological accidents . thus , with the present invention , subjects may be treated in order to prevent toxicity from nuclear bio terrorism or other nuclear or radiological accidents . more particularly , we have discovered a method for prophylactically treating radiation toxicity in normal tissue of subject comprising administering an anti - radiation toxicity effective amount of a cytokine blocking agent through the subject . more specifically , we have discovered a method for prophylactically treating radiation pneumonitis , dermatitis , soft tissue fibrosis or central nervous system toxicity in a subject comprising administering an anti - radiation pneumonitis , dermatitis , soft tissue fibrosis or central nervous system toxicity effective amount of a cytokine blocking agent to the subject .

Detailed Description:
materials and methods : prospective blood sampling , scoring of respiratory symptoms , and chest imaging were conducted for patients receiving thoracic radiation for malignancy . serial plasma specimens were analyzed for circulating cytokine changes before , during radiation , and up to 12 weeks post - radiation . radiation pneumonitis was diagnosed using nci common toxicity criteria . cytokine analysis was assayed for interleukin a ( il - 1α ), interleukin 6 ( il - 6 ), monocyte chemotactic protein 1 ( mcp - 1 ), e - selectin , l - selectin , transforming growth factor β1 ( tgf - β1 ), and basic fibroblast growth factor ( bfgf ) using enzyme linked immmunosorbant assay ( elisa ). patients who were to receive thoracic radiation for malignancy were eligible . blood , thoracic imaging , and respiratory symptom scoring were collected prospectively . twenty - four patients had follow - up longer than 12 months and their characteristics are shown in table 1 . history and physical examinations emphasizing the respiratory symptoms were performed periodically . clinical evaluation for pulmonary symptoms was evaluated and graded using the lent / soma scoring system for the lung . this system includes the rtog treatment side effect scoring of subjective clinical symptoms , and an objective assessment of serial chest x - rays and ct scan changes . pneumonitis grading was also defined according to nci common toxicity criteria . plasma samples were collected before therapy and weekly , during therapy . specimens were collected in sodium heparin as well as edta up to 12 weeks post - therapy . platelet - free plasma was produced by centrifugation at 1200 rpm at 0 ° c . for 10 minutes . the plasma was stored in aliquots at − 20 ° c . heparinized plasma was used for the analysis of most cytokines and edta plasma was used for the analysis of bfgf . cytokines were analyzed using enzyme - linked immunosorbent assay ( elisa ). the methodology of elisa analysis was according to manufacturers &# 39 ; instructions as previously described . twenty - four patients had clinical follow - up longer than 12 months after radiation . thirteen developed symptomatic pneumonitis ( nci grade 2 ). the peak incidence of symptoms was between 6 - and 13 weeks post radiotherapy . six patients had only radiographic infiltrates . ( nci grade 1 ). five patients did not have clinical or radiographic pneumonitis . both il - 1α and il - 6 levels were significantly higher before , during , and after radiation for those who developed pneumonitis . the pattern of changes of mcp - 1 , e - selectin , l - selectin , tgf - β1 , and bfgf varied but none of these cytokines correlated with radiation pneumonitis . analysis of a panel of circulatory cytokines with different putative function in radiation pulmonary injury showed that pre - treatment iλ - 1α and il - 6 , as well as mid and post - treatment levels were significantly higher for patients who subsequently developed radiation pneumonitis . symptomatic radiation pneumonitis is characterized by an annoying cough that is either non - productive or with clear sputum . this period is generally accompanied by markedly worsening dyspnea in an otherwise healthy appearing individual . generally there are also radiographic infiltrates on chest x - ray and ct scan that usually conforms to radiation ports . the individual in general is afebrile or has a low - grade temperature , and is without an increase of blood neutrophil counts . clinical symptoms are rapidly relieved with low dose steroid treatment . of the 24 patients with follow - up longer than 12 months , 13 developed clinical symptoms consistent with radiation pneumonitis ( nci grade 2 pneumonitis ). six had radiographic infiltrates only , without clinical symptoms ( nci grade 1 ). five did not have either radiographic infiltrates or clinical symptoms . the timescale of occurrence of pneumonitis is shown in fig1 . as demonstrated in fig1 , asymptomatic infiltrates occurred at random time points after radiation , while symptomatic pneumonitis occurred most commonly between 6 weeks and 13 weeks after completion of radiation . for all symptomatic pneumonitis , the first episodes all occurred within 6 months post - radiotherapy . the outliers beyond 6 months in fig1 were those with recurrence of symptomatic pneumonitis . in all these cases , however , the first symptoms had occurred within 6 months after therapy . we analyzed pro - inflammatory cytokine il - 1α , and il - 6 levels before radiation treatment , weekly during treatment , and up to 12 weeks following radiation . fig2 shows the absolute cytokine level ( in pg / ml ) ( a1 for il - 1α , and a2 for il - 6 ) and the relative cytokine changes normalized to individual pre - treatment value ( b1 for il - 1α , and b2 for il - 6 ), as well as the comparison of absolute values between the groups with and without radiation pneumonitis ( c1 for il - 1α , and c2 for il - 6 ). the data showed a very wide range of individual circulatory il - 1α levels ( a1 ), but a relative lack of changes with radiation treatment ( b1 ). in contrast to il - 1α , il - 6 levels were not as variable among individuals ( a2 ), but they fluctuated somewhat with radiation . of note , after completion of radiation treatments , there is a trend toward an increase of il - 6 in both absolute levels and relative changes ( a2 and b2 , p = 0 . 065 ). both il - 1α and il - 6 absolute levels were significantly higher before radiation , at multiple time points during radiation , and after radiation ( c1 , and c2 , p & lt ; 0 . 05 ) in patients who subsequently developed radiation pneumonitis . fig3 demonstrates results of circulatory cytokine changes of fibrotic cytokines bfgf and tgf - β1 . basic fgf levels fluctuated during treatments and showed no correlation with pneumonitis ( a1 , b1 , and c1 ). in contrast , there were many individual variations of circulatory tgf - β1 ( a1 ), but there was much lesser degree of relative changes during radiation and after radiation up to 12 weeks post - therapy . similar to bfgf , circulating tgf - β1 did not show an appreciable difference between the group with and the group without pneumonitis ( c2 ). plasma levels of mcp - 1 ( monocyte chemotactic protein 1 ), l - selectin , and s - selectin ( fig4 ) were also measured . fig4 a demonstrates the absolute levels of mcp - 1 ( a1 ), relative changes of mcp - 1 ( b1 ), and the comparison of the groups with and without pneumonitis ( c1 ). our data showed a decline of mcp - 1 levels during the last week of radiation and up to 8 weeks after radiation ( p & lt ; 0 . 04 ). data on l - selectin demonstrated a marked and significant decline of the circulatory levels of l - selectin ( a2 , p & lt ; 0 . 01 ) and the relative changes ( b2 , p & lt ; 0 . 01 ), and a lack of difference between the pneumonitis group and the non - pneumonitis group . there was some decline of circulatory mcp 1 near the end of radiation up to 8 weeks after treatments . data on e - selectin is similar to l - selectin in that there was some decline of levels near the end of radiation and after radiation ( p & lt ; 0 . 03 ) as well as a decrease of relative changes through most time points of the period investigated ( p & lt ; 0 . 01 ). there also was not a significant difference between the pneumonitis group and the non - pneumonitis group . radiation pneumonitis and fibrosis can be regarded as the consequences of a wound - healing inflammatory reaction to radiation damage of lung tissues . research in immunological regulation of inflammation has revealed the complex interaction between local tissues and immune cells mediated through chemokines , adhesion molecules , inflammatory cytokine , and fibrotic cytokines . we have shown that lung radiation is associated with a temporal expression of il - 1α , tgf - β1 , collagen i , collagen iii , and collagen iv gene expression in fibrosis - prone mice ( c57bl / 6 ). among the panel of cytokines potentially involved in the inflammatory response to radiation lung injury , il - 1α and il - 6 were the only two cytokines that correlated significantly with radiation pneumonitis ( fig4 ). in addition , pre - treatment levels of both il - 1α and il - 6 were significantly higher in patients who subsequently developed pneumonitis , supporting their role as predictors of radiation pneumonitis . our data showed some differences between il - 1α and il - 6 , however , in that when normalized to individual pre - treatment levels , il - 1α remains relatively constant during treatment course , but there is a trend toward elevation of il - 6 at 8 to 12 weeks post - radiation . the rise of il - 6 after completion of radiation was observed . it coincided with the period of clinical symptomatic pneumonitis and this deserves further investigation ( fig1 ). both il - 6 and il - 1α are important immunoregulatory moieties . although both are inflammatory cytokines , they differ somewhat in origin of cells and in some functional aspects . both cytokines mediate fever and regulate inflammation and fibrotic response through immune cells . the source of il - 1 is primarily from monocytes as well as alveolar macrophages . il - 6 is synthesized by a variety of cells in the lung parenchyma , including the alveolar macrophages , type it pneumocytes , t lymphocytes , and lung fibroblasts . in the in vitro system , when alveolar macrophages were exposed to clinically relevant dose of radiation ( 2 gy ), it was found that both il - 1α and il - 1β were released in increased amounts . it has been shown that il - 1 stimulates human lung fibroblast in the production of il - 6 and stabilizes il - 6 messenger rna production . in patients with higher pre - treatment levels of il - 1α , il - 1α may regulate a subsequent increase of il - 6 after radiation , was observed ( fig2 ). pro - fibrotic cytokines participate in radiation lung injury , especially during the development of lung fibrosis phase , which generally starts at 4 to 6 months after treatment and continues without a clear end point . lung fibrosis is equivalent to the scar after the initial inflammatory phase of lung reaction to radiation injury . although radiographic fibrosis in general is not observed until 4 to 6 months after completion of radiation , it has been reported that circulatory tgf - β1 changes may serve as an early predictor for radiation pneumonitis and its expression increases with radiation in animal research models . two pro - fibrotic cytokines , bfgf and tgf - β1 , and their changes in the association to radiation pneumonitis ( fig3 ) was investigated . as the incidence of radiation pneumonitis peaks at 6 to 13 weeks in our cohort of patients , we analyzed our data up to 12 weeks and did not find an association in predicting radiation pneumonitis with either bfgf or tgf - β1 . this finding may be attributed to the patient population and sample size differences . since cytokines are relatively fragile molecules , technical differences in specimen collection , processing , and laboratory assays may also result in the differences in laboratory measurements . we have discussed that circulatory measure of il - 1α and il - 6 turned are significantly associated with radiation pneumonitis . thus , patients with higher baseline levels of inflammatory cytokines are more vulnerable to radiation lung injury . fig1 . twenty - four patients were followed prospectively for clinical symptoms of radiation pneumonitis and radiographic changes . the scattered plot demonstrates the time of either symptomatic pneumonitis ( top line ) or only radiographic infiltrates without symptoms ( bottom line ). data showed that symptomatic pneumonitis was diagnosed primarily between 6 weeks and 13 weeks after completion of radiation with rare outliers occurring prior to 6 weeks and between 6 months to 9 months . fig2 . il - 1α absolute levels ( a1 ), relative changes normalized to individual pretreatment levels ( b1 ), and the comparison of levels between patients with grade 1 to 3 pneumonitis ( solid bar ) and no pneumonitis ( hatched bar ) are presented for pre - treatment baseline level , weekly during radiation , and up to 12 weeks after radiation . fig2 a 2 , b2 , and c2 demonstrate the il - 6 absolute levels , and relative changes and the comparison between the two groups of patients , respectively . fig3 . basic fgf a absolute levels ( a1 ), relative changes normalized to individual pretreatment levels ( b1 ), and the comparison of levels between patients with grade 1 to 3 pneumonitis ( solid bar ) and no pneumonitis ( hatched bar ) are presented for pre - treatment baseline level , weekly during radiation and up to 12 weeks after radiation . fig3 a 2 , b2 , and c2 demonstrate the tgf - β1 absolute levels , relative changes , and the comparison between the two groups of patients , respectively . fig4 . basic mcp1 absolute levels ( a1 ), relative changes normalized to individual pretreatment levels ( b1 ), and the comparison of levels between patients with grade 1 to 3 pneumonitis ( solid bar ) and no pneumonitis ( hatched bar ) are presented for pre - treatment baseline level , weekly during radiation and up to 12 weeks after radiation . fig4 a 2 , b2 , and c2 demonstrate the l - selectin absolute levels , relative changes , and the comparison between the two groups of patients , respectively . in fig4 , a3 , b3 , and c3 demonstrate the e - selectin absolute levels , relative changes , and the comparison between the two groups of patients , respectively . six to 7 week - old female c3h / hen , balb / c and c57bl / 6 mice were used ( jackson laboratories , bar harbor , me .). the right hind leg ( 10 mice per group ) was given 10 , 20 , 30 , 40 , 60 , or 80 gy in a single radiation dose with a shephered irradiator , a 6000 ci cs source , together with collimating equipment . the left , non - irradiated hind leg was used as the non - irradiated control . mice were sacrificed at different time points after radiation ( 0 . 5 , 1 , 2 , 4 , 8 , 12hrs , day 1 , day 7 , and day 14 ). at least 10 mice were used at each time point . tissues from 3 mice were used for histology , and the remaining animals were used for mrna analysis . skin and muscle tissues from control and irradiated legs were dissected , and total rna was isolated . guidelines for the humane treatment of animals were followed as approved by the university of rochester committee on animal resources . skin and muscle tissues from each treatment group ( 7 - 10 mice ) were pooled and total rna was isolated by pulverizing the frozen tissue and dissolving it in tri reagent ( molecular research center , ohio ) according to the manufacturer &# 39 ; s specifications . to determine the integrity of isolated rna , 2 μg of rna from each sample was fractionated on a formaldehyde gel and visualized by staining in ethidium bromide . rnase protection was performed using established multi - probes template sets ( pharmingen , sandiago , calif .) as described previously . the interleukin ( il ) sets include : il - 1α :, il - 1β , il - 1rα , il - 6 , il - 10 and il - 12 . two internal controls , l32 and gapdh , were used as loading controls . the cocktail constructs were used to prepare p - utp labeled antisense crna probes using the pharmingen in vitro transcription kits ( pharmingen , sandiago , calif .). probes were hybridized with 30 μg of total rna at 50 ° c . for 16 hr . rnase a ( 1 mg / ml ) and rnase t1 ( 2000 u / ml ) were then added to digest single - stranded rna . after digestion , the rna was precipitated and resuspended in gel loading buffer , heated at 95 ° c . for 5 min , and run in 7 % denaturing polyacrylamide gel ( national diagnostics , ga .). the gel was run for 2 - 3 hr at 60v , dried on whatman filter paper , and placed on a phosphorimager screen for quantitative analysis using a cyclone phosphorimager device ( hp company , conn .). area integration of each mrna - protected fragment was normalized against the protected internal control band ( gapdh ) in the corresponding lane to calculate the ratio of targeted / gapdh mrna . in order to compare the basal levels with radiation - induced levels for each interleukine mrna tested , relative mrna levels ( folds ) were plotted . some gels are shown with over - exposure of the control lanes to highlight differences in il - 1α / β expression . blood samples were collected from 3 mice strains at various time points after radiation . after centrifugation for 30 minutes at 4 ° c ., plasmas were aliquated and stored at − 70 ° c . until analysis . immunoenzymetric assays for murine il - 1β ( endogen inc , cambridge , mass .) were performed according to the manufacturer &# 39 ; s instructions . a standard curve with cytokine - positive control was run in each assay and the lower limit of detection was determined to be 3 . 5 pg / ml . most of non - irradiated mice had circulating il - 1β protein levels near the limit of detection . localization of the il - 1β gene in soft tissue was determined by in situ localization and was performed as previously published . briefly , leg tissues were fixed in 10 % formalin and 2 % paraformadhyde by cutting the whole leg into 3 - 5 pieces . tissue sections were then placed on specially prepared slides ( acid washed and t3 - aminopropyl trietlioxysilane coated ) and were deparaffinized and rebydrated . proteinase k - digested sections were hybridized with appropriate amounts of il - 1β riboprobe . sections to be examined were hybridized with anti - sense rna under conditions of probe excess , and , after washing , they were prepared for autoradiography using nbtii emulsion ( kodak , rochester , n . y .). after autoradiography and staining , the slides were analyzed by bright and dark field microscopy . backgrounds for these studies were determined using the sense stand rna probe . as positive controls for hybridization , some sections were hybridized with constitutively expressed mrna ( gapdh ) and were analyzed for cell specific expression of the molecule of interest . cell types and locations of il - 1β over - expression were identified histologically . cytokine mrna expression levels from skin and muscle in non - irradiated versus irradiated tissues were compared using the unpaired student &# 39 ; s t - test , or mann - whitney rank sum test as appropriate . differences were considered significant for p & lt ; 0 . 05 . at early time points after irradiation of the skin , the gross appearance was only mildly different from one strain of mice to another . fig5 shows typical changes seen after 30 gy . during this acute process , which occurs over the 14 days following limb irradiation , the c3h / hen mice ( least fibrosis sensitive strain ) had some hair loss and leg swelling ( fig5 b ). the balb / c mice , which have intermediate fibrosis sensitivity , had the most edema and hair loss ( fig5 f ), while the fibrosis sensitive c57bl / 6 mice had only a thinning of the fur with minimal edema over the first 14 days ( fig5 d ). local hair loss was noted during the first 14 days in all 3 mice strains , in a dose dependent manner . ulceration was seen only in the high dose groups ( 60 and 80 gy ) at 14 days , and it was less common in the c57bl / 6 mice . however , the acute inflammation that occurred in these animals over the 2 week observation period did not correspond to the degree of fibrosis that is expected 2 months after therapy . the histological changes mirrored the clinical examinations , with some qualitative differences . as an example , 30 gy irradiated tissues at various times after treatment are shown in fig6 . c3h / hen and balb / c mice had lower basal densities of hair follicles compared to c57bl / 6 mice ( fig6 a , d , and g ). three days after 30 gy irradiation , all 3 strains had similar follicle densities ; however , the sub - epidermal matrix accumulation was more pronounced in balb / c and c3h / hen mice ( fig6 c and f ). at 14 days , inflammatory cells and fibroblasts in the dermis were more pronounced in c57bl / 6 mice ( fig6 ). in order to determine the molecular correlation of radiation - induced soft tissue damage , we examined mrna expression of interleukins , il - 1α , il - 1β , and il - 1ra by rnase protection assay in skin and muscle tissues from the 3 mice strains before and after different doses of radiation . as shown in fig7 and fig8 , and summarized in table 2 , all 3 mice strains had detectable basal levels of il - β mrna in their skin ( c3h / hen mice had the highest ), but only very low levels of il - 1β mrna in muscle . skin il - 1β mrna expression was substantially increased within a few hours following 30 gy leg irradiation ( fig3 ). there were two phases of il - 1β mrna elevations : first from 0 . 5 to 4 hrs and then from 7 - 14 days following irradiation ( fig7 a , b , and c ). c3h / hen and balb / c mice had similar patterns of il - β mrna expression after irradiation ( fig7 ). in contrast , the fibrosis - sensitive c57bl / 6 mice had little if any il - 1β mrna induction . neither of the bimodal peaks were seen in irradiated muscle of c57bl / 6 mice . elevation of skin and muscle il - 1β mrna in c3h / hen and balb / c mice was radiation dose - dependent . very high dose ( 80 gy ) radiation significantly increased mrna expression of skin and muscle il - β ( 6 and 9 fold , respectively ) in c3h / hen mice at 4 hrs ( fig8 a , b , and c ). after 14 days , the 80 gy dose significantly increased il - 1β mrna levels in both c57bl / 6 and c3h / hen mice , which were over 15 - fold higher than those of non - irradiated controls . local leg radiation not only increased local tissue il - 1β mrna expression , but it also increased circulating il - 1β measured by elisa . circulating il - 1β was associated with tissue mrna expression with bimodal elevations at 4 - 8 hr and again at 7 to 14 days in both balb / c and c3h / hen mice ( fig9 b ). there appeared to be a dose response , with higher local radiation doses leading to chronically higher circulating il - 1β levels . in order to define the cell types producing il - 1β mrna , in situ hybridization was performed on irradiated soft tissues . increased il - 1β mrna was mainly localized in keratinocytes and stroma cells in the dermis of non - irradiated skin . as shown in fig7 and table 2 , undetectable or very low levels of il - 1α mrna were measured in the skin of c3h / hen mice . a 2 to 6 fold induction of skin il - 1α mrna was detected in both c57bl / 6 ( fig7 a ) and balb / c mice after high doses of radiation ( fig1 a and 10 b ). this increased skin il - 1α mrna expression was radiation dose - dependent , progressed with time , and was minimal at the sub - fibrogenic radiation doses (≦ 30gy ). radiation did not appear to induce il - 1α mrna expression in muscle of any of the 3 mice strains ( fig1 c ). like il - 1β mrna , il - 1ra mrna was highly expressed in skin tissue , and no substantial difference in the basal levels of il - 1ra mrna was seen among the three strains ( fig1 ). skin il - 1ra , however , was dramatically induced by radiation in c57bl / 6 mice , but not in c3h / hen or balb / c mice . induction of il - 1ra mrna in c57bu6 mice was radiation dose dependent . the effects of radiation on il - 1ra mrna expression in muscle of any strain was minimal ( fig1 d ). murine models were used to simulate the situation that occurs in human skin after irradiation . this enabled us to examine the molecular characteristics of soft tissue fibrosis . doses that caused little or no fibrosis (& lt ; 30gy ), as well as highly fibrogenic doses ( 60 - 80gy ) were used in the 3 mice strains . we expected that , if radiation - induced cytokine mrna expression is a causal event , then high doses would induce higher levels of cytokine mrna , explaining strain variation in fibrosis sensitivity . two key questions were asked in this study : 1 ) is there a difference in basal mrna expression of certain cytokines in skin or muscle tissues among 3 mouse strains with different fibrosis sensitivities ? 2 ) does this difference in mrna expression contribute to the various radiation - related fibrosis responses in the three mouse strains ? we demonstrated that : 1 ) skin tissues express higher levels of several interleukins than muscle tissues , independent of mouse strain . this is consistent with prominent initial fibrosis occurring in the subepidermal regions , with less and later development of fibrosis in muscle tissue . 2 ) c3h / hen mice have the lowest predisposition for developing fibrosis and did not express il - 1α mrna in their skin . the most fibrosis sensitive strain , c57bl / 6 , had high basal and radiation - induced levels of this cytokine . muscle , which is more fibrosis resistant than skin , also had lower or undetectable il - 1α expression compared to skin . 3 ) radiation induced elevation of il - 1β mrna was biphasic with an early peak ( 1 to 4 hr ) and another at a later time ( 3 to 14 day ). the first phase was absent in the fibrosis sensitive strain , and it was intermediate in the strain with intermediate fibrosis sensitivity . 4 ) cytokine responses in muscle were more blunted , compared to those in skin , and required higher radiation doses . 5 ) cytokine responses after local radiation could be large enough to be detected in the circulation . 6 ) the cells synthesizing the greatest quantities of il - 1β appear to be the keratinocytes and stromal cells of the epidermis and dermis . taken together we propose that these patterns suggest that brisk il - 1α responses to radiation and high basal il - 1α mrna levels are associated with a higher risk for late radiation fibrosis . an early pulse of il - 1β expression after irradiation appears to correlate with a lower risk for developing radiation soft tissue fibrosis . the data also provided evidence that circulating levels of cytokines might be a useful marker of local cytokine production following radiation . it has been demonstrated both experimentally and clinically that high basal levels of fibrogenic cytokines and / or growth factors are related to a higher incidence of radiation - or chemotherapy - induced late tissue damage . our recent animal studies also suggest that high blood tgf - β levels are associated with a high risk for radiation - induced tissue fibrosis . we measured local and circulating levels of interleukin mrna in our 3 mice strains with different fibrosis sensitivities because higher basal mrna levels of these cytokines may also be related to a higher risk of radiation - mediated normal tissue fibrosis . it is apparent from our data that c3h / hen skin does not have detectable il - 1α mrna . low or undetectable skin il - 1α mrna in c3h / hen mice , a fibrosis resistance mouse strain , may be responsible for its resistant phenotype . in our radiation - induced lung fibrosis models , similar results were also observed . the correlation of low mrna levels of skin and lung il - 1α with increased resistance of radiation - induced fibrosis warrants further investigation . radiation - induced expression of interleukin mrna is organ - dependent . all interleukin responses were more pronounced in the skin than in muscle . inducible levels of each cytokine , however , varied between skin and muscle tissues . for example , radiation induced an elevation of skin il - 1α mrna , not muscle il - 1α mrna , in c57bl / 6 mice . our previous data in cultured cell lines ( keratinocytes , skin fibroblast , and squamous cell carcinoma cells ) also demonstrated that different cell types not only express different levels of each cytokine , but also respond to radiation differently . our data here may also provide some guidance for clinical radiation therapy . for example , avoidance of cutaneous radiation might prevent cytokine cascades that could result in late tissue fibrosis . this is because soft tissue fibrosis begins in the subepidermis , later extends through the dermis , and eventually involves the superficial and the deeper muscle layers . clinically , the efficacy of megavoltage radiation is in large part due to the lower epidermal dosimetry . it is an intriguing notion that patients with elevated basal il - 1α mrna might be treated prophylactically with anti - cytokine therapy to prevent fibrosis . while radiation - induced alteration of interleukin mrna in lung and other organs have been reported in several strains of mice , altered mrna levels of cytokines in soft tissues from different strains of mice have not yet been reported . in this study , we collected and processed rna samples of three strains in the same rnase protection gel , and we also compared the il - 1 mrna expression difference between skin and muscle . we found that the patterns of cytokine mrna expression were consistent with the degree of fibrotic response . in contrast , macroscopic and microscopic acute alterations were weak predictors of fibrosis sensitivity . the lack of correlation between acute reactions and late effects has been studied for decades , and the role that cytokines and growth factors play appears to finally help explain the phenomenon . radiation increased il - 1 mrna expression in two waves , the first at approximately 4 hours after therapy and another 3 to 14 days post - radiation . examination of corresponding skin tissue morphology at each time point suggested that acute tissue response in preexisting cellular components may be responsible for the first peak of cytokine production . in situ hybridization studies suggest that keratinocytes , endothelial cells , and skin fibroblasts are the source of the early il - 1β mrna expression . infiltrating inflammatory cells and activated fibroblasts are probably responsible for the second peak in cytokine mrna production . several studies have demonstrated that pulses of il - 1 , given within 24 hours of radiation , are radioprotective . endogenous pulsing of il - 1β in c3h / hen mice after radiation may therefore partly explain this strain &# 39 ; s higher resistance to fibrosis compared to c57bl / 6 mice . in conclusion , we have shown that skin tissues produce more interleukin mrna compared with muscle tissues . skin il - 1α and il - 1ra mrna are upregulated in c57bl / 6 mice , while il - 1β mrna is increased in c3h / hen and balb / c mice within a few hours of local leg radiation . these results show that radiation - induced differential mrna expression for interleukin and varied basal levels of interleukin mrna participate in radiation - induced normal tissue damage . fig5 . typical gross observation of radiation changes seen in control ( a , c and e ) and 14 days following 30 gy irradiation ( b , d and f ) of the right hind limb in 3 mice strains . edema was similar in all three strains , and hair loss was similar in c3h / hen and c57bl / 6 mice , with slightly greater hair loss in balb / c mice ( f ). fig6 . the characteristic histological observation of progressive pathological changes of radiation fibrosis are shown in panels a through i . normal mouse skin for c3h / hen ( a ), balb / c ( d ), and c57bl / 6 ( g ). note the thin epidermis with underlying papillary dermis , hair follicles containing multiple hairs . leg muscle is free of significant inflammation . day 3 ( b , e , and h ) and day 14 ( c , f , and i ) after 30 gy radiation are shown . early soft tissue reaction includes progressive loss of dermal papilla , reduced hair follicle number , increased empty hair follicles , and a superficial filling of the dermis with matrix and inflammatory cells . there is little inflammation of muscle , and the dermal inflammatory cell infiltrates were grossly similar in all strains . fig7 . il - 1β mrna expression in irradiated limbs in 3 mice strains by rnase protection assay ( a ). il - 1β mrna expression was quantitatively determined using a cyclone phosphorimager ( hp co , mi ). il - 1β mrna values are pooled from seven mice per measurement for irradiated skin ( b ) and muscle ( c ). lanes are shown over - exposed to demonstrate the absence of il - 1α in the skin of c3h / hen mice , and the brisk il - 1β response to radiation in c3h / hen and balb / c but not in c57bl / 6 . the early phase of il - 1β mrna expression was seen in muscle , while the later increase at 1 to 2 weeks was less evident in muscle . 30 gray is sufficient to cause a high frequency of severe acute reactions in all strains , but , at 2 months following radiation , 30 gy is sub - fibrogenic for most c3h / hen and balb / c mice . fig8 . determination of il - 1β mrna expression in high dose ( 80 gy ) irradiated limbs from c3h / hen and c57bl / 6 mice by rnase protection assay ( a and b ). mrna from seven mice was pooled . 80 gy radiation induced elevated il - 1β mrna expression in both skin and muscle tissues . 80 gy is sufficient to cause substantial fibrosis and acute reaction in all strains . fig9 . plasma il - 1β levels in c3h / hen and balb / c mice after limb irradiation . circulating levels of il - 1β in platelet depleted plasma were significantly increased after 30 gy radiation in balb / c mice ( left ). the difference from baseline was not significant at any time after 10 gy , which is a sub - fibrogenic dose . in a separate experiment ( right ), 30 gy radiation significantly increased blood il - 1β in both c3h / hen and balb / c mice . the results suggest that circulating il - 1β is a surrogate for protein locally produced in the hind limb . fig1 . determination of il - 1α mrna expression in 30 , 40 , or 60 gy irradiated limbs from 3 mice strains by rnase protection assay . each value was normalized to its internal control gapdh and represents the pooled expression from seven mice per measurement . radiation elevated il - 1α mrna in skin ( a and b ) but not in muscle tissue ( c ). the effect was greater with increased radiation dose . c3h / hen mice express no detectable il - 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1 by human alveolar macrophages after in vitro irradiation . radiat res 136 : 37 - 41 , 1993 . 50 . elias j a , lentz v : il - 1 and tumor necrosis factor synergistically stimulate fibroblasts il - 6 production and stabilize il - 6 messenger rna . j immunol 145 : 161 - 166 , 1990 . 51 . helle m , brakenhoff j p j , de groot e r et a :. interleukin 6 is involved in interleukin 1 - induced activities . eur j immunol 18 : 957 - 959 , 1988 . 52 . roach m iii , gandara d r , yuo h s , et al : radiation pneumonitis following combined modality therapy for lung cancer : analysis of prognostic factors . j clin oncol . 13 : 2606 - 2612 , 1995 . 53 . kimsey f c , mendenliall n p , ewald l m , et al : is radiation treatment volume a predictor for acute or late effect on pulmonary function ? cancer 73 : 2549 - 2555 , 1994 . 54 . byhardt r w , martin l , pajak t f et al : the influence of field size and other treatment factors on pulmonary toxicity following hyperfractionated irradiation for inoperable non - small cell lung cancer ( nsclc )- analysis of a radiation therapy oncology group ( rtog ) protocol . int j radiat oncol biol phys 27 : 537 - 544 , 1993 . 55 . kwa s l s , lebesque j v , theuws j c m et al :, radiation pneumonitis as a function of mean lung dose : an analysis of pooled data of 540 patients . int j radiat oncol biol phys 42 : 1 - 9 , 1998 . isotransplantable murine mca - 35 mammary tumor cells was inoculated i . m . into right hind thighs of 6 - 7 week - old female c3h / hen mice ( nci , fredrick , md .). right hind thigh tumors were given 60 gy ( single dose using a cs irradiator ) when tumors reached 8 - 9 mm in diameter . mice were sacrificed 20 days after radiation . tumors and the overlaying skin tissues were removed for histology and rna preparation . irradiated tissues ( tumor and skin ) were also collected for making paraffin blocks for immunohistochemical staining . guidelines for the humane treatment of animals were followed as approved by the university of rochester committee on animal resources . celebrex ( pfizer inc .) powder was dissolved in pbs . due to partial dissolution , the agent was mixed very well every time before gavaging . 50 mg / kg ( 0 . 2 ml ) celebrex was given daily , and five days per week for constitutive three weeks . four experiment groups were used . all mice were treated with single 60 gy radiation in tumor - bearing leg . group 1 was radiation alone ; mice in group 2 were given 50 mg / kg celebrex 2 hours before radiation ( 2 hr pre - radiation ); mice in group 3 and 4 were received the same amount of celebrex at day 2 or day 7 post - radiation . mice in the group 4 were received total 10 doses , and rest treated mice were given total 15 doses . all mice were sacrificed 20 day after radiation . radiation induce skin damage was assessed using 5 - scales skin scoring system . 20 days after single 60 gy radiation , mice from each treatment group were determined blindly for the degree of skin damage by three investigators . grade 1 : normal skin ; grade 2 : slight hair loss in irradiated area ; grade 3 : radish and swollen tissue ; grad 4 : small area erosion ; grade 5 : small ulceration . grades 2 - 3 is referred as mild , and grades 4 - 5 is considered as severe skin damage . total rna was isolated from tumors and skin overlying tumors , respectively , with 9 - 10 mice in each treatment group by pulverizing the frozen tissue , and dissolved in tri reagent ( molecular research center , ohio ) according to the manufacturer &# 39 ; s specifications . to determine the integrity of isolated rna , 2 μg of rna from each sample was fractionated on a formaldehyde gel and visualized by staining in ethidium bromide . rnase protection was performed using established multi - probe template sets ( pharmingen , san diego , calif .) as described previously [ okunieff , 1998 # 4388 ]. the chemokine multiple templet includes : mcp - 1 , mip - 1α , mip - 1β , mip - 2 , rantes , eotaxin and ip - 10 . the c - c chemokine receptor multiple templete includes : ccr1 , ccr2 , ccr3 , ccr4 and ccr5 . the c - x - c chemokine receptor multiple templets includes : cxcr2 and cxcr4 . two internal standards , l32 and gapdh , were used as loading controls . the cocktail constructs were used to prepare 32 p - utp labeled antisense cdna probes using pharmingen in vitro transcription kits ( pharmingen , san diego , calif .). probes were hybridized with 30 μg of total rna at 50 ° c . for 16 hrs rnase a ( 1 mg / ml ), and rnase t1 ( 2000 u / ml ) was then added to digest single - stranded rna . after digestion , the rna was precipitated and resuspended in gel loading buffer , heated at 95 ° c . for 5 min , and run on a 6m urea , 7 % denaturing polyacrylamide gel ( national diagnostics , ga .). the gel was dried on filter paper and placed on a phosphorimager screen for quantitative analysis of mrna expression levels for each cytokine / chemokine . area integration of each mrna - protected fragment probe was normalized against the protected band for gapdh or l32 mrna in each corresponding lane to calculate the ratio of targeted mrna / gapdh mrna expression . in order to compare the basal levels of each gene tested , relative levels ( ratios ) were plotted . immunohistochemistry methods have previously been described in detail . immediately following cryostat sectioning , tissue slices ( normal muscle and tumor ) were stained with cd31 antibody ( pharmingen calif .) for determination of total vasculature . the stained sections were imaged using an epi - fluorescence equipped microscope , digitized ( 3 - ccd camera ), background - corrected , and image - analyzed using image pro software ( media cybernetics , mass .) and a 450 mhz pentium computer . color images from individual microscope fields were automatically acquired and digitally combined to form four montages of the tumor cross - section ( total area = 15 . 5 mm2 ) using a motorized stage and controller . the image montages were processed to enhance the contrast between background and cd31 staining . from the enhanced images , locations of cd31 - stained vessels were recorded . the quantitative vascular information was analyzed using custom fortran programs to perform a “ closest individual ” analysis as previously described . briefly , the distances from computer - superimposed sampling points to the nearest blood vessel were determined . the cumulative frequency distribution of these distances provided the probability of encountering vessels within any specified distance from the tumor cells . median distances ( μm ) to the nearest vessel were used for statistical comparisons . mrna levels ( ratios ) of tumors and skin from irradiated or non - irradiated mice were evaluated using the unpaired students t - test or mann - whitney rank sum test as appropriate . differences were considered significant for p & lt ; 0 . 05 . 20 days after single 60 gy irradiated mca - 35 tumor skin had varied lesions including edema , erosion and superficial necrosis in most of saline - treated control mice 20 days after radiation ( fig1 a ). however , celebrex - treated tumors had less radiation - induced skin damage compared with saline - treated controls ( fig1 b - d ). the most of celebrex treated mice , regardless pre - ( 2 hr before radiation ) or post - radiation ( day 2 or day 7 after radiation ) had less inflammation and cellular component infiltration in the dermis ( fig1 b , c and d ) compared with saline - treated controls ( fig1 a ). 23 . 8 % ( 5 / 21 ) of mice in 60 gy alone treated group developed severe skin damage , but only 17 . 6 % of mice in the pre - 2hr celebrex treated group , 5 . 3 % of mice in the post - day 2 celebrex treated group , and 11 . 1 % of mice in post - day 7 celebrex - treated group , appeared as the severe skin damage 20 days after radiation . oral administration of celebrex also caused the reduction of blood vessels in mca - 35 tumor ( fig1 f ), focal necrosis ( fig1 g ) and even massive tumor necrosis ( fig1 h ) in some areas of tumors , compared with saline - treated controls ( fig1 e ). because radiation inducing soft tissue damages has been reported to associate with the persistent overproduction of cytokine or chemokine in irradiated normal or tumor cells , we next examined the effects of celebrex on the radiation - induced mrna expression of chemokines including five c - c family members ( rantes , eotactin , mip - 1α , mip - β and mcp - 1 ), one c - x - c family ( mip - 2 ) and one c family member ( lymphotactin ), as well as c - c receptors ( ccr1 , ccr2 and ccr5 ) and c - x - c receptors ( cxcr2 and cxcr4 ) in tumor skin and tumor tissues by rnase protection assay . as shown in fig1 and summarized in table 3 and 4 , celebrex treatment caused the significant reduction of rantes ( 2 . 3 ± 1 . 1 vs 7 . 4 ± 1 . 6 , p & lt ; 0 . 05 ) and mcp - 1 ( 10 . 2 ± 1 . 1 vs 18 . 8 ± 3 . 2 , p & lt ; 0 . 05 ) mrna expression in irradiated skin tissues , but not in tumor tissues ( table 3 ), although radiation induced higher levels of skin mip - 2 mrna expression in 37 . 5 % ( ⅜ ) of mice , only 14 . 3 %- 28 . 6 % of tumor skin had high mip - 2 mrna expression after celebrex treatment . similarly , celebrex - treatment did not significantly alter the tumor mip - 2 mrna ( table 4 ). celebrex not only reduced c - c chemokines , it also caused the decrease mrna expression of both c - c and c - x - c chemokine receptors in tumor skin ( fig1 b and d ), not in tumor tissues ( fig1 c ). all quantitative measurement are shown in tables 3 and 4 . due to each individual mouse variation , there was 15 - 30 % of celebrex - treated mice still developed the moderate or severe skin damage after radiation . radiation - induced skin damage was quantitatively determined by the skin scores from each individual mouse . in order to find out the relationship between overexpression of chemokines or their receptors mrna and radiation - induced skin damages , the correlation of skin scores and skin tissue chemokine and chemokine receptor mrna expression levels from each individual mouse were plotted and shown in fig1 . significant positive correlation between skin damages ( skin scores ) and overexpression of chemokine and its receptor mrna expression were observed in 60 gy radiation - treated mice . however , the correlation of celebrex - mediated the reduction of chemokine and chemokine receptor mrna expression with skin damages only occurred in rantes ( fig1 a ) and it receptor ccr5 ( fig1 d ), mcp - 1 ( fig1 b ) and its receptor ccr2 ( fig1 d ). although celebrex - mediated reduction of mip - 2 mrna expression did not correlate with less skin damage , related cxcr4 mrna expression was significantly reduced in celebrex - treated mice , which had less radiation - induced skin damage . as shown in fig1 , celebrex - treated mice had less infiltration of inflammatory cells in the dermas ( fig1 c ) compared with saline controls ( fig1 a ). however , the infiltration of inflammatory cells in tumor tissue was not obviously altered by celebrex treatment ( fig1 b and d ). thus we have discussed that : 1 ) radiation induced rantes / ccr5 and mcp - 1 / ccr2 mrna expression was decreased by celebrex ; and 2 ) celebrex - mediated reduction of chemokine and their receptor mrna expression was correlated with ameliorated skin damage . 2