Bronchoalveolar lavage and interstitial cells have different roles in radiation-induced lung injury

PURPOSE: To determine the contribution of intra-alveolar cells as opposed to cells fixed in the interstitium in the development of radiation-induced lung injury. MATERIALS AND METHODS: C3H/HeN mice were irradiated to the thorax with various doses of radiation. The cellular composition and cytokine production were assessed in the two sites by histological staining and RNase protection assay. RESULTS: Following thoracic irradiation, there was an initial decrease in the number of bronchial alveolar lavage (BAL) cells that was followed after 2 months by a dose-dependent increase up to 4 months. Foamy Mac-1 positive macrophages were present early in the BAL populations, which also expressed the pro-inflammatory cytokines TNF-alpha, IL-1alpha and IL-1beta, but this response subsided by the time of onset of pneumonitis (3 months). In contrast, in whole lung tissue there was a steady increase in Mac-1 positive cells and increased expression of TNF-alpha, IL-1alpha and IL-1beta mRNAs to maximum levels at 3-4 months. CONCLUSIONS: These data indicate distinct temporal and spatial changes in pro-inflammatory cytokine gene expression in different cellular compartments of the irradiated lung. BAL cells became inflammatory early on, but interstitial cells became involved later and were probably more involved in contributing to the pneumonitis.     

Rapid induction of cytokine gene expression in the lung after single and fractionated doses of radiation

Purpose: To investigate cytokine gene expression in the lung after single and fractionated doses of radiation, and to investigate the effect of steroids and the genetic background. Materials and methods: Expression of cytokine genes (mTNF-alpha, mIL-1alpha, mIL-1beta, mIL-2, mIL-3, mIL-4, mIL-5, mIL-6, mIFNgamma) in the lungs of C3H/HeJ and C57BL/6J mice was measured by RNase protection assay at different times after various doses of radiation. The effects of dexamethasone and fractionated radiation treatment on gene expression were also studied. Results: IL-1beta was the major cytokine induced in the lungs of C3H/HeJ mice within the first day after thoracic irradiation. Radiation doses as low as 1Gy were effective. Responses to 20Gy irradiation peaked within 4-8h and subsided by 24h. With the exception of IL-1alpha and TNF-alpha, the other cytokines that were investigated had undetectable pre-treatment mRNA levels and were not radiation inducible. Similar responses were seen in C57BL/6J mice, although TNF-alpha was induced and there were some quantitative differences. Pre-treatment of C3H/HeJ mice with dexamethasone reduced basal and induced IL-1 levels, but complete inhibition was not achieved. Dexamethasone was also effective if given immediately after irradiation. Fractionated daily doses of radiation (4Gy/day) helped to maintain cytokine gene expression for a longer period. Conclusions: Inflammatory genes are rapidly induced in the lung by irradiation. This response cannot be readily abolished by steroid pre-treatment. Fractionated treatment schedules help to perpetuate the response.     

Rapid induction of cytokine gene expression in the lung after single and fractionated doses of radiation

PURPOSE: To investigate cytokine gene expression in the lung after single and fractionated doses of radiation, and to investigate the effect of steroids and the genetic background. MATERIALS AND METHODS: Expression of cytokine genes (mTNF-alpha, mIL-1alpha, mIL-1beta, mIL-2, mIL-3, mIL-4, mIL-5, mIL-6, mIFN-gamma) in the lungs of C3H/HeJ and C57BL/6J mice was measured by RNase protection assay at different times after various doses of radiation. The effects of dexamethasone and fractionated radiation treatment on gene expression were also studied. RESULTS: IL-1beta was the major cytokine induced in the lungs of C3H/HeJ mice within the first day after thoracic irradiation. Radiation doses as low as 1 Gy were effective. Responses to 20 Gy irradiation peaked within 4-8h and subsided by 24 h. With the exception of IL-1alpha and TNF-alpha, the other cytokines that were investigated had undetectable pre-treatment mRNA levels and were not radiation inducible. Similar responses were seen in C57BL/6J mice, although TNF-alpha was induced and there were some quantitative differences. Pre-treatment of C3H/HeJ mice with dexamethasone reduced basal and induced IL-1 levels, but complete inhibition was not achieved. Dexamethasone was also effective if given immediately after irradiation. Fractionated daily doses of radiation (4 Gy/day) helped to maintain cytokine gene expression for a longer period. CONCLUSIONS: Inflammatory genes are rapidly induced in the lung by irradiation. This response cannot be readily abolished by steroid pre-treatment. Fractionated treatment schedules help to perpetuate the response.     

Die Bedeutung von Zytokinen für die radiogene Lungenreaktion

BACKGROUND: The radiosensitivity of the lung tissue limits the dose of radiation which can be delivered to tumors in the thoracic region. Radiation-induced lung damage implies the induction of numerous cytokines which form the basis for the multicellular interactions of the inflammatory and fibrogenic processes associated with radiation injury. It is of special clinical significance, how far local radiation induced cytokine production in the lung tissue may be reflected in increased cytokine blood levels in patients during radiotherapy and may predict the later development of radiation-induced lung damage. Another potential cause of increased cytokine levels in the blood of oncologic patients is the secretion of cytokines in the blood circulation by tumor specimens. METHODS: Published data on radiation-induced cytokine expression from experimental and clinical studies are reviewed. RESULTS AND CONCLUSION: The major pro-inflammatory cytokines in the radiation response of the lung include tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), and interleukin-6 (IL-6). Transforming growth factor-beta (TGF-beta) appears to be of particular importance in the development of lung fibrosis. First approaches with radioprotective agents and gene therapy to modify radiation-induced cytokine expression have been investigated for prevention of late effects of irradiation lung damage in animal experiments. Preliminary data of clinical studies suggest that elevated plasma TGF-beta-levels during radiotherapy may predict the development of symptomatic radiation pneumonitis. The biological impacts of endogenous radiation-induced cytokine production by tumor cells in respect of tumor behavior, potential damage to normal tissue, and clinical status of the host still need to be determined more precisely.     

Treg分化对放射性肺损伤的影响

目的 探讨调节性T细胞（Treg）的分化对放射性肺损伤的影响及其作用机制。 方法 建立Treg抑制小鼠模型,按随机数字表法将C57BL/6小鼠分成4组:空白对照组、单纯照射组、照射+免疫球蛋白G（IgG）组和照射+CD25组,每组12只,除空白对照组外其余3组小鼠给予单次20 Gy X射线全胸照射,照射+IgG组和照射+CD25组小鼠每周腹腔注射IgG抗体和CD25抗体。分别于照射后第4周和第8周各处死小鼠6只,采用流式细胞术检测小鼠肺组织内CD25+Foxp3+Treg（Foxp3:叉头样转录因子3）的百分比以鉴定模型是否建立成功;采用Western blot法检测单纯照射组小鼠肺组织内神经纤毛蛋白1（NRP1）的表达;采用免疫荧光法检测每组小鼠肺组织内CD25+NRP1+Treg的百分比;拍照并观察每组小鼠皮肤的损伤情况,采用苏木精-伊红染色法检测小鼠肺组织的病理学改变;采用酶联免疫吸附测定法检测每组小鼠肺组织内转化生长因子β1（TGF-β1）、白细胞介素（IL）-17A、干扰素γ（IFN-γ）、IL-2和IL-4的水平变化。两组间比较采用独立样本t检验。 结果 流式细胞术检测结果显示,照射后第4周和第8周,单纯照射组小鼠肺组织内CD25+Foxp3+Treg百分比[（1.73±0.04）%、（2.13±0.15）%]均较空白对照组[（1.14±0.02）%、（1.70±0.06）%] 明显升高,差异均有统计学意义（t=?26.680、?4.545,P=0.000、0.010）,抑制Treg后,第4周和第8周时照射+CD25组小鼠肺组织内CD25+Foxp3+Treg百分比[（0.72±0.14）%、（0.27±0.02）%]均较单纯照射组明显降低,差异均有统计学意义（t=5.296、37.538,均P=0.000）。Western blot结果显示,照射后第4周和第8周,单纯照射组小鼠肺组织内NRP1蛋白表达水平均较空白对照组升高,差异均有统计学意义（t=?7.341、?9.127,均P=0.000）。免疫荧光法检测结果显示,照射后第4周和第8周,单纯照射组小鼠肺组织内CD25+NRP1+Treg的百分比均较空白对照组升高,而照射+CD25组CD25+NRP1+Treg百分比均较单纯照射组降低,且差异均有统计学意义（t=8.926、14.457,P=0.001、0.000）。观察小鼠皮肤损伤程度后发现,照射后第4周和第8周,单纯照射组小鼠皮肤损伤严重,而照射+CD25组小鼠照射后第4周时皮肤基本完好,第8周时出现脱毛脱皮。病理学结果显示,照射后第4周和第8周,与空白对照组相比,单纯照射组小鼠的肺组织结构破坏,肺泡壁增厚,细胞外基质增多,而照射+CD25组小鼠的肺组织结构完整,肺泡壁纤细。酶联免疫吸附测定结果显示,与空白对照组相比,照射后第4周,单纯照射组小鼠肺组织内IL-17A和IL-4的水平均升高,差异均有统计学意义（t=?8.492、?15.796,P=0.001、0.000）,照射后第8周,TGF-β1和IL-17A水平升高,差异均有统计学意义（t=?11.072、?7.167,P=0.000、0.002）,IL-2水平在第4周和第8周时均降低,IFN-γ水平在第4周时升高,差异有统计学意义（t=?27.393,P=0.000）,第8周时下降;与单纯照射组相比,照射+CD25组小鼠TGF-β1和IL-17A水平在第4周和第8周时均降低（t=6.037、4.524、5.496、4.772,均P=0.000）,IFN-γ水平升高（t=?7.006、?12.565,P=0.002、0.000）,差异均有统计学意义,而IL-2和IL-4水平在第4周时均降低,第8周时均明显升高,差异均有统计学意义（t=2.866、?9.090、8.833、?7.191,均P=0.000）。 结论 放射性肺损伤小鼠的肺组织中出现Treg分化,并增强分泌TGF-β1促炎因子,同时干扰辅助T细胞（Th1、Th2型）细胞因子的平衡来促进放射性肺损伤的发生。     

Irradiation induces increased production of haemopoietic and proinflammatory cytokines in the mouse lung

PURPOSE: To investigate cytokine expression following irradiation of mice, predominantly in lung tissue but also in selected other tissues. MATERIALS AND METHODS: Mice of strain ICR were whole-body (unilaterally) exposed to 3-20 Gy of (60)Co gamma-rays. Colony-stimulating activity (CSA) of lung-conditioned media (LCM), and also other non-haemopoietic and haemopoietic organs, and blood serum of mice was assayed using a GM-CFC bioassay. The production of GM-CSF, IL-6 and TNF-alpha protein in LCM and sera was determined by an ELISA method. RESULTS: Greatest CSA was detected in conditioned media from the lungs and was induced in a dose- and time-dependent fashion, peaking at 3-9 days after irradiation with a lethal dose of 9 Gy. Conditioned medium prepared from lungs that had been irradiated with a dose of 9 Gy in vitro did not exhibit an increase in CSA. However, whereas the lung-conditioned medium from irradiated mice was found to produce CSA, sera from normal or irradiated mice did not lead to this effect. A significant increase in CSA in sera was observed in the presence of a suboptimal concentration of IL-3, implying that they comprise the co-stimulatory activity (CoSA). The results showed that radiation exposure increased GM-CSF and TNF-alpha protein levels but did not affect IL-6 production in LCM. In contrast, IL-6 and TNF-alpha protein levels in serum were increased after irradiation but no GM-CSF production could be detected. CONCLUSION: Whole-body irradiation enhances CSA in lungs as well as in other haemopoietic and non-haemopoietic organs. The increase of CSA correlates with increased levels of haemopoietic and proinflammatory cytokines in lung.     

Bronchoalveolar lavage and interstitial cells have different roles in radiation-induced lung injury

Purpose : To determine the contribution of intra-alveolar cells as opposed to cells fixed in the interstitium in the development of radiation-induced lung injury. Materials and methods : C3H/HeN mice were irradiated to the thorax with various doses of radiation. The cellular composition and cytokine production were assessed in the two sites by histological staining and RNase protection assay. Results : Following thoracic irradiation, there was an initial decrease in the number of bronchial alveolar lavage (BAL) cells that was followed after 2 months by a dose-dependent increase up to 4 months. Foamy Mac-1 positive macrophages were present early in the BAL populations, which also expressed the pro-inflammatory cytokines TNF- !, IL-1 ! and IL-1 #, but this response subsided by the time of onset of pneumonitis (3 months). In contrast, in whole lung tissue there was a steady increase in Mac-1 positive cells and increased expression of TNF- !, IL-1 ! and IL-1 # mRNAs to maximum levels at 3-4 months. Conclusions : These data indicate distinct temporal and spatial changes in pro-inflammatory cytokine gene expression in different cellular compartments of the irradiated lung. BAL cells became inflammatory early on, but interstitial cells became involved later and were probably more involved in contributing to the pneumonitis.     

GATA-3在放射性肺纤维化中的作用机制

目的 通过建立小鼠放射性肺纤维化模型,了解转录因子GATA-3及白细胞介素-13(IL-13)在放射性肺纤维化形成过程中的表达情况及其意义,为放射性肺纤维化的防治提供实验和理论依据。方法 成年雌性C57BL/6小鼠63只,随机分作2组:对照组21只, 不做任何处理;照射组42只,全肺单次照射12 Gy。于照射后1 h、1、2、4、8、16和24周,取照射组及相同鼠龄对照组小鼠肺组织和血清,分别用HE和Masson染色在光镜下观察组织病理改变和胶原纤维沉积;用碱水解法测定羟脯氨酸含量;用实时定量RT-PCR法检测GATA-3mRNA相对含量;用ELISA法测定血清中IL-13的含量;用Western blot法检测肺组织中GATA-3的蛋白表达变化情况。结果 光镜下,照射组小鼠肺组织较对照组组织学改变和胶原纤维沉积明显;测定羟脯氨酸含量显示,照射组小鼠肺组织中的羟脯氨酸含量较对照组明显增高(Z=3.142,P＜0.05);蛋白和mRNA检测结果显示,较之对照组,GATA-3和IL-13的表达在照射组中明显升高,特别是在肺组织尚未发生明显的纤维化组织学改变时(1~2周),照射组GATA-3表达明显升高(t=6.50,6.33,P＜0.01),IL-13受GATA-3调节,在照射后第16周表达最为明显(t=32.21,P＜0.01)。结论 转录因子GATA-3可以通过诱导Th2免疫细胞因子IL-13的表达,参与放射性肺纤维化的形成。     

Effects of NF-kappaB1 (p50) targeted gene disruption on ionizing radiation-induced NF-kappaB activation and TNFalpha, IL-1alpha, IL-1beta and IL-6 mRNA expression in vivo.

PURPOSE: To investigate the role of the NF-kappaB1 (p50) gene in ionizing radiation (IR)-induced NF-kappaB activation and TNFalpha, IL-1alpha, IL-1beta and IL-6 mRNA expression in vivo. MATERIALS AND METHODS: NF-kappaB activation was analysed by the gel shift/supershift assay and the levels of TNFalpha, IL-1alpha, IL-1beta and IL-6 mRNA were measured using RNase protection assay (RPA). Various tissues from BALB/c, B6,129P-Nfkb1 (NF-kappaB1 or p50 gene knockout, p50(-/-)) and B6,129PF2 (wild-type, p50(+/+)) mice were analysed before or after exposure to a lethal dose (8.5 Gy) of total-body gamma-irradiation. RESULTS: Exposure of BALB/c mice to total-body IR selectively activated NF-kappaB in the spleen, mesenteric lymph nodes (LN) and bone marrow (BM). Gel supershift assay using polyclonal antibodies against NF-kappaB p50, p65 or c-Rel protein revealed that the NF-kappaB p50 subunit is a critical component of the NF-kappaB complexes activated by IR in vivo. Discretely augmented TNFalpha, IL-1alpha, IL-1beta and IL-6 mRNA expression was found in the spleen, LN and BM after BALB/c mice received IR. However, mice lacking the p50 gene (p50(-/-)) showed a significant reduction in IR-induced activation of NF-kappaB and increases in TNFalpha, IL-1alpha, IL-1beta and IL-6 mRNA expression, as compared with that of wild-type mice (p50(+/+)). CONCLUSIONS: The NF-kappaB p50 subunit is a critical component of the NF-kappaB complexes activated by IR and it plays an important role in mediating IR-induced TNFalpha, IL-1alpha, IL-1beta and IL-6 mRNA expression in vivo.     

乌司他丁对大鼠放射性肺损伤中TNF-α及IL-6的影响

目的 观察乌司他丁(天普洛安)对大鼠放射性肺损伤过程中TNF-α及IL-6的影响,试图寻找一种预防或治疗放射性肺损伤的有效途径。方法 72只雌性SD大鼠随机分为3组:健康对照组、单纯照射组和乌司他丁治疗组,每组24只。照射组及治疗组动物麻醉后,行直线加速器全胸部照射一次,剂量为25 Gy。照射组照射后通过尾静脉注射乌司他丁(100?000 U·kg^－1·d^－1),共计7 d。对照组和照射组注射同等体积的生理盐水。于照射后2 h、4、8和24周处死动物,取部分肺组织行HE染色及Masson染色,观察组织学变化,另提取组织蛋白用免疫印迹杂交方法检测肺组织中TNF-α水平,使用酶联免疫吸附分析法检测血清中IL-6水平。所有数据采用SPSS统计软件进行方差分析。结果 照射组大鼠肺组织中TNF-α及血清中IL-6水平与对照组相比,明显升高,并在第4周时达到顶峰(q=5.63、6.21,P<0.01);治疗组TNF-α和IL-6水平与对照组相比,第4周时也增加明显,但是与照射组相比明显下降(q=4.97、7.42,P<0.01)。结论 大鼠放射性肺损伤时TNF-α及IL-6水平明显升高,在放射性肺损伤发展中起到重要作用,乌司他丁能显著降低其水平,并抑制炎性反应,从而能有效地防治放射性肺损伤。     

Irradiation induces increased production of haemopoietic and proinflammatory cytokines in the mouse lung

Purpose : To investigate cytokine expression following irradiation of mice, predominantly in lung tissue but also in selected other tissues. Materials and methods : Mice of strain ICR were whole-body (unilaterally) exposed to 3-20 Gy of 60 Co γ-rays. Colony-stimulating activity (CSA) of lung-conditioned media (LCM), and also other non-haemopoietic and haemopoietic organs, and blood serum of mice was assayed using a GM-CFC bioassay. The production of GM-CSF, IL-6 and TNF- α protein in LCM and sera was determined by an ELISA method. Results : Greatest CSA was detected in conditioned media from the lungs and was induced in a dose- and time-dependent fashion, peaking at 3-9 days after irradiation with a lethal dose of 9 Gy. Conditioned medium prepared from lungs that had been irradiated with a dose of 9 Gy in vitro did not exhibit an increase in CSA. However, whereas the lung-conditioned medium from irradiated mice was found to produce CSA, sera from normal or irradiated mice did not lead to this effect. A significant increase in CSA in sera was observed in the presence of a suboptimal concentration of IL-3, implying that they comprise the co-stimulatory activity (CoSA). The results showed that radiation exposure increased GM-CSF and TNF- α protein levels but did not affect IL-6 production in LCM. In contrast, IL-6 and TNF- α protein levels in serum were increased after irradiation but no GM-CSF production could be detected. Conclusion : Whole-body irradiation enhances CSA in lungs as well as in other haemopoietic and non-haemopoietic organs. The increase of CSA correlates with increased levels of haemopoietic and proinflammatory cytokines in lung.     

尾吊对空间诱变肺炎克雷伯菌感染小鼠炎症反应的增强作用

目的 探索空间诱变肺炎克雷伯菌(T16-169菌)感染尾吊模拟失重小鼠后炎症反应变化.方法 尾吊小鼠模拟失重生理效应;C57BL/6小鼠随机分为对照、对照染菌、尾吊及尾吊染菌4组,采用RT-qPCR法和xMAP技术分别检测小鼠肺组织中炎症因子TNF-α、IL-6、IL-1β 的mRNA表达量及血浆中炎症因子浓度,HE染色光镜下观察肺组织病理变化.结果 肺组织RT-qPCR及血浆炎症因子xMAP检测结果显示,与对照组相比,对照染菌及尾吊染菌组小鼠肺组织及血浆中炎症因子TNF-α、IL-6、IL-1β 的表达均显著升高,且尾吊染菌组最为显著(P<0.01或P<0.001);肺组织病理结果显示,对照染菌组和尾吊染菌组小鼠肺组织均出现不同程度的损伤,以尾吊染菌组的损伤最为严重.结论 空间诱变肺炎克雷伯菌感染尾吊小鼠可显著升高血浆及肺组织中炎症因子表达,导致更严重的肺组织受损,提示尾吊模拟失重感染空间诱变肺炎克雷伯菌可致机体的炎症反应增强.     

乳铁蛋白通过调节HMGB1/TLR4炎症反应改善放射性肺损伤

目的:研究乳铁蛋白对放射性肺损伤的防护作用。方法:将15只C57BL/6 J小鼠按随机数表法分为健康对照组、15 Gy照射组(单纯照射组)和乳铁蛋白联合15 Gy照射组(联合组),每组5只。联合组全程饮用10 mg/ml乳铁蛋白水溶液,3 d后单纯照射组和联合组给予单次15 Gy X射线全胸照射。于照后14 d处死,苏...     

Effects of IL-4 on pulmonary fibrosis and the accumulation and phenotype of macrophage subpopulations following thoracic irradiation

Purpose: Thoracic irradiation injures lung parenchyma, triggering inflammation and immune cell activation, leading to pneumonitis and fibrosis. Macrophage polarization contributes to these processes. Since IL-4 promotes pro-fibrotic macrophage activation, its role in radiation-induced lung injury was investigated.

Materials and methods: Lung macrophage subpopulations were characterized from 3–26 weeks following exposure of WT and IL-4?/? mice to 0 or 12.5 Gray single dose thoracic irradiation.

Results: Loss of IL-4 did not prevent fibrosis, but blunted macrophage accumulation within the parenchyma. At 3 weeks following exposure, cell numbers and expression of F4/80 and CD206, an alternative activation marker, decreased in alveolar macrophages but increased in infiltrating macrophages in WT mice. Loss of IL-4 impaired recovery of these markers in alveolar macrophages and blunted expansion of these populations in infiltrating macrophages. CD206+ cells were evident in fibrotic regions of WT mice only, however Arg-1+ cells increased in fibrotic regions in IL-4?/? mice only. Radiation-induced proinflammatory Ly6C expression was more apparent in alveolar and interstitial macrophages from IL-4?/? mice.

Conclusions: IL-4 loss did not prevent alternative macrophage activation and fibrosis in irradiated mice. Instead, a role is indicated for IL-4 in maintenance of macrophage populations in the lung following high single dose thoracic irradiation.