Carl J. Johnston

A perpetual cascade of cytokines postirradiation leads to pulmonary fibrosis

PURPOSE Radiation-induced pulmonary reactions have classically been viewed as distinct phases--acute pneumonitis and, later, fibrosis--occurring at different times after irradiation and attributed to different target cell populations. We prefer to view these events as a continuum, with no clear distinction between the temporal sequence of the different pulmonary reactions; the progression is the result of an early activation of an inflammatory reaction, leading to the expression and maintenance of a cytokine cascade. In the current study, we have examined the temporal and spatial expression of cytokine and extracellular matrix messenger ribonucleic acid (mRNA) abundance in fibrosis-sensitive mice after thoracic irradiation. METHODS AND MATERIALS Radiation fibrosis-prone (C57BL/6) mice received thoracic irradiation of 5 and 12.5 Gy. At Day 1, and 1, 2, 8, 16, and 24 weeks after treatment, animals were killed and lung tissue processed for light microscopy and isolation of RNA. Expression of cytokine and extracellular matrix mRNA abundance was evaluated by slot-blot analysis and cellular localization by in situ hybridization and immunochemistry. RESULTS One of the cytokines responsible for the inflammatory phase (IL-1 alpha) is elevated at 2 weeks, returns to normal baseline values, then increases at 8 weeks, remaining elevated until 26 weeks when lung fibrosis appears. Transforming growth factor-beta (TGF beta), a proliferative cytokine, is elevated at 2 weeks, persists until 8 weeks, and then returns to baseline values. In parallel with the cytokine cascade, the fibrogenic markers for CI/CIII/IV (collagen genes) correlate by showing a similar early and then later elevation of activity. For instance, the collagen gene expression of CI/CIII is a biphasic response with an initial increase at 1-2 weeks that remits at 8 weeks, remains inactive from 8 to 16 weeks, and then becomes elevated at 6 months when collagen deposition is recognized histopathologically. CONCLUSION These studies clearly demonstrate the early and persistent elevation of cytokine production following pulmonary irradiation. The temporal relationship between the elevation of specific cytokines and the histological and biochemical evidence of fibrosis serves to illustrate the continuum of response, which, we believe, underlies pulmonary radiation reactions and supports the concept of a perpetual cascade of cytokines produced immediately after irradiation, prompting collagen genes to turn on, and persisting until the expression of late effects becomes apparent pathologically and clinically.

Early alterations in extracellular matrix and transforming growth factor β gene expression in mouse lung indicative of late radiation fibrosis

PURPOSE Fibrosis, characterized by the accumulation of collagen, is a late result of thoracic irradiation. The expression of late radiation injury can be found immediately after irradiation by measuring messenger RNA (mRNA) abundance. METHODS AND MATERIALS To determine if extracellular matrix mRNA and transforming growth factor beta abundance was affected acutely after irradiation, we measured mRNA levels of collagen I (CI), collagen III (CIII), collagen IV (CIV), fibronectin (FN), and transforming growth factor beta (TGF beta 1,2&3) in mouse lungs on day 1 and day 14 after graded doses of radiation. C57BL/6 female mice were irradiated with a single dose to the thorax of 5 or 12.5 Gy. Total lung RNA was prepared and immobilized by Northern and slot blotting and hybridized with radiolabelled cDNA probes for CI, CIII, CIV, FN, TGF beta 1,2&3 and a control probe encoding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Autoradiographic data were quantified by video densitometry and results normalized to GAPDH. RESULTS Changes in the expression of CI, CIII, CIV, FN and TGF beta 1,2&3 were observed as early as 1 day after exposure. Through 14 days, changes in mRNA up to 5-fold were seen for any one dose. Dose related changes as high as 10-fold were also evident. The CI:CIII ratio increased gradually for the 5 Gy dose at 14 days postirradiation while the CI:CII ratio for the 12.5 Gy dose decreased by approximately 4-fold as compared to the control. CONCLUSION These studies suggest that alterations in expression of extracellular matrix and TGF beta mRNA occur very early after radiation injury even at low doses and may play a role in the development of chronic fibrosis.

CLARA CELL SECRETORY PROTEIN DEFICIENCY INCREASES OXIDANT STRESS RESPONSE IN CONDUCTING AIRWAYS

Little is known about the molecular basis for differential pulmonary oxidant sensitivity observed between genetically disparate members of the same species. We have generated mice that are deficient in Clara cell secretory protein (CCSP -/-) and that exhibit an oxidant-sensitive phenotype. We characterized the kinetics and distribution of altered stress-response [interleukin-6 (IL-6) and metallothionein (MT)] and epithelial cell-specific [cytochrome P-450 2F2 (CYP2F2)] gene expression to further understand the cellular and molecular basis for altered oxidant sensitivity in 129 strain CCSP -/- mice. Increases in IL-6 and MT mRNA abundance were detected by 2 h of exposure to 1 part/million ozone and preceded reductions in Clara cell CYP2F2 mRNA expression. Despite being qualitatively similar, increases in IL-6 and MT mRNA expression were enhanced in CCSP -/- mice with respect to coexposed 129 strain wild-type mice. Increased MT mRNA expression, indicative of the stress response, localized to the airway epithelium, surrounding mesenchyme, and endothelium of blood vessels. These results demonstrate a protective role for Clara cells and their secretions and indicate potential genetic mechanisms that may influence susceptibility to oxidant stress.

Differences in correlation of mRNA gene expression in mice sensitive and resistant to radiation-induced pulmonary fibrosis.

Fibrosis, characterized by the accumulation of collagen, is a late result of thoracic irradiation. The purpose of this study was to determine if extracellular matrix protein and transforming growth factor beta mRNA expression are altered late in the course of pulmonary fibrosis after irradiation, and then to determine if these changes differ between two strains of mice which vary in their sensitivity to radiation. Radiation-sensitive (C57BL/6) and radiation-resistant (C3H/HeJ) mice were irradiated with a single dose of 5 or 12.5 Gy to the thorax. Total lung RNA was prepared and immobilized by Northern and slot blotting and hybridized with radiolabeled cDNA probes for collagens I, III and IV, fibronectin, and transforming growth factor beta 1 and beta 3. Autoradiographic data were quantified by video densitometry and results normalized to a control probe encoding for glyceraldehyde-3-phosphate dehydrogenase. Alterations in mRNA abundance were observed in the sensitive mice at all times, while levels in the resistant mice were unaffected until 26 weeks after irradiation. The relationship between extracellular matrix protein per se and increased mRNA abundance suggests that late matrix protein accumulation may be a function of gene expression. Differences in levels of transforming growth factor beta mRNA may lead to strain-dependent variation in fibrotic response and may also contribute to the radiation-induced component of pulmonary fibrosis.

Effect of Administration of Lovastatin on the Development of Late Pulmonary Effects after Whole-Lung Irradiation in a Murine Model

Abstract Williams, J. P., Hernady, E., Johnston, C. J., Reed, C. M., Fenton, B., Okunieff, P. and Finkelstein, J. N. Effect of Administration of Lovastatin on the Development of Late Pulmonary Effects after Whole-Lung Irradiation in a Murine Model. Radiat. Res. 161, 560–567 (2004). Our groups work on late radiation effects has been governed by the hypothesis that the effects observed in normal tissues are a consequence of multicellular interactions through a network of mediators. Further, we believe that inflammation is a necessary component of this process. We therefore investigated whether the recruitment of mononuclear cells, observed during the pneumonitic period in the irradiated normal lung, is dependent on the expression of chemokines, notably Mcp1. Since statins have been shown to reduce chemokine expression and inflammatory cell recruitment, we specifically examined whether statins could be used to reduce monocyte recruitment. Mice received 15 Gy whole-lung irradiation; treated groups were administered lovastatin three times weekly starting either immediately or 8 weeks postirradiation. At subsequent intervals, animals were killed humanely, and cellular, mRNA and protein analyses were undertaken. Statin-treated animals demonstrated a statistically significant reduction in both macrophage and lymphocyte populations in the lung compared to radiation alone as well as improved rates of survival and decreased collagen content. In addition, ELISA measurements showed that radiation-induced increases in Mcp1 protein were reduced by statin treatment. Additional experiments are needed to assess whether statins offer a potential treatment for the amelioration of late effects in breast and lung cancer patients undergoing radiation therapy.

INFLAMMATORY CELL RECRUITMENT FOLLOWING THORACIC IRRADIATION

Ionizing radiation leads to a progressive injury in which a monocyte/macrophage-rich pneumonitis is followed by a chronic progressive fibrosis. In the present study, the role of macrophage/monocyte recruitment in the genesis of radiation-induced pulmonary fibrosis was examined. The objectives were threefold: (i) characterize the inflammatory cells recruited into the lung during the development of radiation-induced fibrosis; (ii) investigate changes in lung response following depletion of resident alveolar macrophages in vivo prior to radiation treatment; (iii) assess if inhalation of low levels of endotoxin would potentiate the radiation-initiated injury. One group of fibrosis-sensitive C57BL/6 mice was irradiated with a single dose of 15 Gy to the thorax. In a second group, resident inflammatory cells were depleted using clodronate, encapsulated into liposomes, 48 hours prior to irradiation with a single dose of 15 Gy to the thorax. Control animals were sham irradiated. All groups of animals then were examined 8, 16, or 24 weeks post irradiation. No difference in total cell numbers or cell differentials was observed between irradiated mice or those that were both liposome treated and irradiated at any time point. At 16 weeks, mice that received radiation showed a 5- to 6-fold increase in lymphocytes regardless of treatment as compared to control animals. At 24 weeks post irradiation, select groups were exposed to lipopolysaccharide (LPS) and examined 24 hours post inhalation. Lavageable protein was increased several fold in mice that received both radiation and LPS exposure as compared to 15 Gy or LPS exposure alone. These results demonstrate: (i) macrophages and lymphocytes are the predominately recruited cell types through 24 weeks post irradiation; (ii) recovery of inflammatory cells, regardless of prior macrophage depletion, were similar, suggesting that early responses are primarily driven by parenchymal cell injury; (iii) thoracic irradiation-induced injury can cause sensitization to a secondary stimulus that may result in injuries/responses not predicted by evaluating exposures individually.

Alterations in the expression of chemokine mRNA levels in fibrosis-resistant and -sensitive mice after thoracic irradiation.

Fibrosis, characterized by the accumulation of collagen, is a consequence of a chronic inflammatory response. The purpose of this study was to determine if the mRNA expression of the chemokines, lymphotactin (Ltn), RANTES, eotaxin, macrophage inflammatory protein (MIP)-1 alpha, -1 beta, and -2, interferon-inducible protein 10 (IP-10), and monocyte chemotactic protein-1 (MCP-1), are altered during the development of radiation-induced pneumonitis and fibrosis. Further, we wished to determine if these changes differ between two strains of mice that vary in their sensitivity to radiation fibrosis. Fibrosis-sensitive (C57BL/6) and fibrosis-resistant (C3H/HeJ) mice were irradiated with a single dose of 12.5 Gy to the thorax. Total lung RNA was prepared and hybridized utilizing RNase protection assays. Data were quantified by phosphorimaging and results normalized to a constituitively expressed mRNA L32. 8 weeks post-irradiation most chemokines measured were elevated to varying degrees. The degree of elevation of each chemokine was identical in both strains. This suggested that chemotactic activity for neutrophils, macrophages, and lymphocytes were occurring during pneumonitis. By 26 weeks post-irradiation, messages encoding Ltn, RANTES, IP-10, and MCP-1 were elevated only in fibrosis sensitive (C57BL/6) mice. In situ hybridization demonstrated that MCP-1 and RANTES transcripts were produced predominantly from macrophages and lymphocytes. These studies suggest that lymphocytic recruitment and activation are key components of radiation-induced fibrosis.

Inflammatory and Epithelial Responses in Mouse Strains That Differ in Sensitivity to Hyperoxic Injury

The pulmonary response to various toxicants including bleomycin, ozone, ionizing radiation, and hyperoxia is highly variable among mouse strains. The current study tests the hypothesis that at a similar stage of injury, regardless of strain, expression of inflammatory cytokine and epithelial marker genes would be similar, indicating a common pathway of injury progression. Three strains of mice, C57B1/6J, 129/J, and C3H/HeJ, ranging from sensitive to resistant, were exposed to > 95% O2 for varying times. Ribonuclease protection was used to quantify changes in cytokine mRNA. Despite differences in the kinetics, each strain demonstrated similar hyperoxia-induced changes in the abundance of interleukin (IL)-6, IL-1 beta, IL-3, and tumor neucrosis factor (TNF)-alpha. For each strain, death was accompanied by similar increases in cytokine mRNAs above steady-state control levels. Other inflammatory cytokines, including IL-1 alpha, IL-4, and interferon (IFN)-gamma, were unaltered in all strains at all times. In situ hybridization analysis of the epithelial markers, surfactant protein B (SPB), and clara cell secretory protein (CCSP) at the time of proinflammatory induction showed a similar pattern of expression in all strains. Increased SPB was detected in bronchiolar epithelium, while the number of type II cells expressing this message declined. Both the number of cells expressing CCSP as well as abundance per cell declined. These results suggest that although differences in acute sensitivity to hyperoxia exist between mouse strains, once initiated, acute epithelial cell injury and associated inflammatory changes follow the same pattern in all strains.

INFLAMMATORY AND ANTIOXIDANT GENE EXPRESSION IN C57BL/6J MICE AFTER LETHAL AND SUBLETHAL OZONE EXPOSURES

Ozone (O3) is a highly reactive and toxic oxidant pollutant. The objective of this study is to compare cytokine, chemokine, and metallothionein (Mt) changes elicited by lethal and sublethal exposure to ozone in a genetically sensitive strain of mice. Eight-week-old C57BL/6J mice were exposed to 0.3 ppm ozone for 0, 24, or 96 hours; 1.0 ppm ozone for 0, 1, 2, or 4 hours; or 2.5 ppm ozone for 0, 2, 4, or 24 hours. After 24 hours of exposure to 0.3 ppm ozone, increases in mRNA abundance were detected for messages encoding eotaxin, macrophage inflammatory protein (MIP)-1 alpha, and MIP-2. These increases persisted through 96 hours of exposure. At this time point messages encoding lymphotactin (Ltn) and metallothionein were also increased. After 4 hours of 1.0 ppm ozone exposure, increases in mRNA abundance were detected for messages encoding eotaxin, MIP-1 alpha, MIP-2, and interleukin (IL)-6. Mt mRNA abundance was increased after 1 hour of exposure and persisted through 4 hours, although the magnitude of the alterations increased. After 2 hours of 2.5 ppm ozone exposure, increases were detected for messages encoding eotaxin, MIP-1 alpha, MIP-2, IL-6, and Mt. These increases persisted through 4 hours of exposure. Lung weights of mice exposed to 2.5 ppm ozone for 24 hours were approximately 2 times greater than air-exposed mice. At this dose lethality occurred by 36 hours. Increased mRNAs for eotaxin, MIP-1 alpha, MIP-2, and Mt were to a higher magnitude than were detected after 2 and 4 hours of exposure. Messages encoding IL-12, IL-10, interferon (IFN)-gamma, IL-1 alpha, IL-1 beta, and IL-1Ra were unaltered at all time points and doses examined. Our results demonstrate dose- and time-dependent changes in chemokine, cytokine, and Mt mRNA abundance and that early acute changes may be predictive of subacute and chronic responses to ozone.

Pneumocystis carinii Activates the NF-κB Signaling Pathway in Alveolar Epithelial Cells

ABSTRACT Pneumocystis carinii pneumonia (PcP) is a clinically important infection of immunocompromised patients. Although the interaction of Pneumocystis with the alveolar epithelium has been well documented, very little information regarding the epithelial response to Pneumocystis is currently available. In order to study Pneumocystis-epithelium interactions, a murine cell line derived specifically from an alveolar epithelial cell (AEC) was utilized. The coculture of murine AECs with mouse Pneumocystis induced a dose- and time-dependent release of the CXC chemokine MIP-2. Importantly, the specific removal of Pneumocystis from the preparation, or the pretreatment of AECs with sulfasalazine, a potent and specific inhibitor of NF-κB, nearly completely abrogated the chemokine response to Pneumocystis. Since the murine MIP-2 promoter contains consensus κB binding sequences, the ability of Pneumocystis to stimulate NF-κB signaling in AECs was examined. Pneumocystis stimulation of an AEC line stably transfected with a κB-dependent reporter construct triggered the NF-κB signaling pathway and reporter production. These data were confirmed in gel shift assays, providing direct evidence that Pneumocystis induced the nuclear translocation of the p50/p65 heterodimeric form of NF-κB. Maximal NF-κB activation was dependent upon direct contact with viable Pneumocystis organisms. These data demonstrate that Pneumocystis activates NF-κB signaling in AECs and establish a reporter cell line for studying NF-κB activation in AECs. Given the global regulatory functions of the NF-κB family, these findings suggest that Pneumocystis directly alters AEC gene expression in a manner that promotes pulmonary immune and inflammatory responses.