Bleomycin-induced Lung Injury In Mice Research Articles

Effect of IL-2-Bax, a novel interleukin-2-receptor-targeted chimeric protein, on bleomycin lung injury.

The role of lymphocytes in the pathogenesis of lung fibrosis is not clear, but the weight of the evidence supports a pro-fibrotic effect for lymphocytes. The high-affinity interleukin-2 receptor (haIL-2R) is expressed on activated, but not quiescent, T lymphocytes. This selective expression of haIL-2R provides the basis for therapeutic strategies that target IL-2R-expressing cells. We hypothesized that elimination of activated lymphocytes by IL-2R-targeted chimeric proteins might ameliorate lung fibrosis. We investigated the effects of IL-2-Bax, a novel apoptosis-inducing IL-2R-targeted chimeric protein, on bleomycin-induced lung injury in mice. Treatment groups included (i) a single intratracheal instillation of bleomycin and twice-daily intraperitoneal injections of IL-2-Bax; (ii) intratracheal bleomycin and intraperitoneal IL-2-PE66(4Glu), an older-generation chimeric protein; (iii) intratracheal bleomycin/intraperitoneal PBS; (iv) intratracheal saline/intraperitoneal PBS. Lung injury was evaluated 14 days after intratracheal instillation by cell count in bronchoalveolar lavage (BAL) fluid, semi-quantitative and quantitative histomorphological measurements and by biochemical analysis of lung hydroxyproline. Bleomycin induced a BAL lymphocytosis that was significantly attenuated by IL-2-Bax and IL-2-PE66(4Glu). However, morphometric parameters and lung hydroxyproline were unaffected by the chimeric proteins. These results show that IL-2-Bax reduces the lymphocytic infiltration of the lungs in response to bleomycin, but this effect is not accompanied by a decrease in lung fibrosis.

Bone Marrow–Derived Mesenchymal Stem Cells in Repair of the Injured Lung

We sought to determine whether an intact bone marrow is essential to lung repair following bleomycin-induced lung injury in mice, and the mechanisms of any protective effects conferred by bone marrow-derived mesenchymal stem cell (BMDMSC) transfer. We found that myelosupression increased susceptibility to bleomycin injury and that BMDMSC transfer was protective. Protection was associated with the differentiation of engrafted BMDMSC into specific and distinct lung cell phenotypes, with an increase in circulating levels of G-CSF and GM-CSF (known for their ability to promote the mobilization of endogenous stem cells) and with a decrease in inflammatory cytokines. In vitro, cells from injured, but not from normal, mouse lung produced soluble factors that caused BMDMSC to proliferate and migrate toward the injured lung. We conclude that bone marrow stem cells are important in the repair of bleomycin-injured lung and that transfer of mesenchymal stem cells protects against the injury. BMDMSC localize to the injured lung and assume lung cell phenotypes, but protection from injury and fibrosis also involves suppression of inflammation and triggering production of reparative growth factors.

Adenovirus is Profibrotic in Bleomycin-Induced Lung Injury in Mice through Effects on Adaptive Immunity

Adenovirus is Profibrotic in Bleomycin-Induced Lung Injury in Mice through Effects on Adaptive Immunity

Adenovirus is Profibrotic in Bleomycin-Induced Lung Injury in Mice through Effects on Adaptive Immunity

Adenovirus is Profibrotic in Bleomycin-Induced Lung Injury in Mice through Effects on Adaptive Immunity

Increased basigin in bleomycin-induced lung injury.

Basigin is expressed in many tissues during development, including lung. It is also found on tumor cells and in wounds where it is thought to stimulate adjacent fibroblasts to produce matrix metalloproteinases. To investigate whether basigin might be expressed in fibro-inflammatory lung processes, we generated bleomycin-induced lung injury in mice. At 14 d after intratracheal bleomycin, we found basigin prominently in areas of fibrosis, alveolar macrophages, and bronchiolar epithelium, whereas it was only weakly present in bronchiolar epithelium in untreated mice. Western blots of radioimmunoprecipitation assay RIPA-insoluble fractions of bleomycin-treated lungs showed increased basigin compared with RIPA-insoluble fractions of lung from untreated mice. By quantitative reverse transcriptase-polymerase chain reaction, lung basigin mRNA was significantly increased 14 d after bleomycin, and by in situ hybridization, basigin mRNA was prominent in bronchiolar epithelium. Western blots of bronchoalveolar lavage fluid (BALF) showed various forms of basigin after bleomycin that were not present in BALF from untreated lung. These results demonstrate that bleomycin-induced lung injury is associated with increased basigin expression in bronchiolar epithelium, deposition of basigin in fibrotic sites, and increased basigin in BALF. Accordingly, basigin may play a role in diffuse alveolar injury.

COMBINING HISTOLOGY AND BIOCHEMICAL MEASUREMENTS OF CONNECTIVE TISSUE COMPONENTS IN SMALL SAMPLES OF LUNG: APPLICATION TO BLEOMYCIN-INDUCED FIBROSIS IN THE MOUSE

Bleomycin-induced lung injury in mice was used to illustrate the plausibility of quantitating changes in elastin or protein concentration in histologically defined regions of mouse lungs. Mice were instilled by either the intranasal or intratracheal route with varying levels of bleomycin and the lungs were removed and fixed under pressure for histology and analysis. Histological sections that bracketed a defined volume of tissue was used for biochemical analysis and was limited to the alveolar region of the lung. Our studies showed that the intranasal method of instillation was as effective as the intratracheal method for inducing fibrosis. Even though distribution to the 4 major lobes of the lung was fairly uniform, there was no consistency in the degree of fibrosis between the various lobes of the same animal. The most dramatic change in biochemical parameters was protein concentration, which increased over 10-fold in some fibrotic lungs compared to normal lungs, whereas desmosine concentration increased up to 5-fold. Fibrosis scores agreed well with desmosine concentration, except with moderate desmosine increases where histologically defined fibrosis was not observed. The experiments illustrate the feasibility and desirability of comparing histology and biochemical analysis on the same fixed lung sample. elastin desmosine lung fibrosis

Enalapril protects mice from pulmonary hypertension by inhibiting TNF-mediated activation of NF-kappaB and AP-1.

The present study was undertaken to investigate the effects of treatment with the angiotensin-converting enzyme (ACE) inhibitor enalapril in a mouse model of pulmonary hypertension induced by bleomycin. Bleomycin-induced lung injury in mice is mediated by enhanced tumor necrosis factor-alpha (TNF) expression in the lung, which determines the murine strain sensitivity to bleomycin, and murine strains are sensitive (C57BL/6) or resistant (BALB/c). Bleomycin induced significant pulmonary hypertension in C57BL/6, but not in BALB/c, mice; average pulmonary arterial pressure (PAP) was 26.4 +/- 2.5 mmHg (P < 0.05) vs. 15.2 +/- 3 mmHg, respectively. Bleomycin treatment induced activation of nuclear factor (NF)-kappaB and activator protein (AP)-1 and enhanced collagen and TNF mRNA expression in the lung of C57BL/6 but not in BALB/c mice. Double TNF receptor-deficient mice (in a C57BL/6 background) that do not activate NF-kappaB or AP-1 in response to bleomycin did not develop bleomycin-induced pulmonary hypertension (PAP 14 +/- 3 mmHg). Treatment of C57BL/6 mice with enalapril significantly (P < 0.05) inhibited the development of pulmonary hypertension after bleomycin exposure. Enalapril treatment inhibited NF-kappaB and AP-1 activation, the enhanced TNF and collagen mRNA expression, and the deposition of collagen in bleomycin-exposed C57BL/6 mice. These results suggest that ACE inhibitor treatment decreases lung injury and the development of pulmonary hypertension in bleomycin-treated mice.

Exacerbation of bleomycin-induced lung injury in mice by amifostine.

Bleomycin (BLM) induces lung injury and fibrosis in the murine lung and enhances tumor necrosis factor (TNF)-alpha and collagen mRNA expression in the murine lung. Amifostine is a cytoprotective agent that protects normal tissues from the cytotoxic effects of chemo- and radiation therapy. We investigated the effect of amifostine in BLM-induced lung injury in mice. Mice received intraperitoneal amifostine (200 mg/kg) 30 min before and/or 1, 3, and 7 days after an intratracheal injection of saline or BLM (4 U/kg). The animals were killed 14 days after BLM exposure, and their lungs were studied for TNF-alpha and collagen mRNA expression, hydroxyproline content, and histopathology. Light microscopy demonstrated that amifostine exacerbated the BLM-induced lung injury in mice. Increased TNF-alpha mRNA expression as a result of BLM exposure was not modulated by amifostine treatment. In contrast, amifostine treatment enhanced the BLM-induced expression of alpha(1)(I) procollagen mRNA in the lung. Similarly, mice treated with amifostine before BLM exposure accumulated significantly higher amounts of hydroxyproline (111 +/- 5 microg/lung) than BLM-treated animals (90 +/- 6 microg/lung). These data suggest that amifostine treatment exacerbates BLM-induced lung injury in mice.

Expression of TNF and the Necessity of TNF Receptors in Bleomycin-Induced Lung Injury in Mice

Bleomycin (BLM) induction of lung fibrosis in mice is an established model to study the mechanism of pulmonary fibrosis. Cytokine secretion has been implicated as a fundamental component of the lung fibrotic process observed in response to BLM. Among the cytokines implicated in lung fibrosis, Tumor necrosis factor (TNF) alpha has been considered to play a fundamental role. In the present study, we characterized the cellular sources of TNF during BLM-induced lung injury and examined the importance of TNF receptors in this process. To characterize the expression of TNF, we utilized two strains of mice, one sensitive (C57BL/6) and one resistant (BALB/c) to BLM-induced lung injury. Mice received BLM (120 mg/kg total) or saline, as control, by multiple subcutaneous injections. BLM induced the development of inflammation in subpleural areas only in the lungs of BLM-sensitive mice. These subpleural areas were characterized by infiltration of CD68-positive macrophages and increased collagen deposition. BLM enhanced the expression of TNF mRNA in BLM-sensitive, but not in BLM-resistant, mice. In situ hybridization studies localized the expression of TNF in the areas of BLM-induced inflammation in 6% and 27% of macrophages at 14 and 21 days post BLM treatment. In addition to TNF, BLM exposure resulted in the upregulated expression of transforming growth factor (TGF)-β1, but not interleukin (IL)-1, mRNA in the lungs of both murine strains at 14 and 21 days. This upregulated expression of TGF-β1 mRNA was greater in the lungs of BLM-sensitive mice. In separate experiments, double TNF receptor knockout mice were exposed to BLM. These animals demonstrated an increased expression of TNF, but not TGF-β1, mRNA in response to BLM and did not exhibit histologic evidence of lung injury following BLM exposure. In summary, the upregulation of TNF mRNA in macrophages correlated with the appearance of inflammation following BLM exposure and was limited to the BLM-sensitive strain. Furthermore, in addition to the release of the TNF ligand, it appears that the presence of TNF receptors is necessary for the development of BLM-induced lung injury, and signaling through these receptors may contribute to the regulation of the TGF-β1 mRNA expression observed in response to bleomycin. These results provide further support for a role of macrophages and TNF in the induction of lung inflammation