Cheryl L. Fattman

Interleukin 1 receptor antagonist mediates the antiinflammatory and antifibrotic effect of mesenchymal stem cells during lung injury

Mesenchymal stem cells (MSCs) have been exploited as cellular vectors to treat a wide array of diseases but the mechanisms responsible for their therapeutic effect remain indeterminate. Previously, we reported that MSCs inhibit bleomycin (BLM)-induced inflammation and fibrosis within the lungs of mice. Interrogation of the MSC transcriptome identified interleukin 1 receptor antagonist (IL1RN) as a potential mediator of this effect. Fractionation studies indicated that MSCs are the principal source of IL1RN in murine bone marrow and that its expression is restricted to a unique subpopulation of cells. Moreover, MSC-conditioned media was shown to block proliferation of an IL-1α-dependent T cell line and inhibit production of TNF-α by activated macrophages in vitro. Studies conducted in mice revealed that MSC administration was more effective than recombinant IL1RN delivered via adenoviral infection or osmotic pumps in inhibiting BLM-induced increases in TNF-α, IL-1α, and IL1RN mRNA in lung, IL1RN protein in bronchoalveolar lavage (BAL) fluid, and trafficking of lymphocytes and neutrophils into the lung. Therefore, MSCs protect lung tissue from BLM-induced injury by blocking TNF-α and IL-1, two fundamental proinflammatory cytokines in lung. Identification of IL1RN-expressing human MSC subpopulations may provide a novel cellular vector for treating chronic inflammatory diseases in humans.

Extracellular superoxide dismutase in biology and medicine

Accumulated evidence has shown that reactive oxygen species (ROS) are important mediators of cell signaling events such as inflammatory reactions (superoxide) and the maintenance of vascular tone (nitric oxide). However, overproduction of ROS such as superoxide has been associated with the pathogenesis of a variety of diseases including cardiovascular diseases, neurological disorders, and pulmonary diseases. Antioxidant enzymes are, in part, responsible for maintaining low levels of these oxygen metabolites in tissues and may play key roles in controlling or preventing these conditions. One key antioxidant enzyme implicated in the regulation of ROS-mediated tissue damage is extracellular superoxide dismutase (EC-SOD). EC-SOD is found in the extracellular matrix of tissues and is ideally situated to prevent cell and tissue damage initiated by extracellularly produced ROS. In addition, EC-SOD is likely to play an important role in mediating nitric oxide-induced signaling events, since the reaction of superoxide and nitric oxide can interfere with nitric oxide signaling. This review will discuss the regulation of EC-SOD and its role in a variety of oxidant-mediated diseases.

A Role for the Receptor for Advanced Glycation End Products in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a severely debilitating disease associated with a dismal prognosis. There are currently no effective therapies for IPF, thus the identification of novel therapeutic targets is greatly needed. The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface receptors whose activation has been linked to various pathologies. In healthy adult animals, RAGE is expressed at the highest levels in the lung compared to other tissues. To investigate the hypothesis that RAGE is involved in IPF pathogenesis, we have examined its expression in two mouse models of pulmonary fibrosis and in human tissue from IPF patients. In each instance we observed a depletion of membrane RAGE and its soluble (decoy) isoform, sRAGE, in fibrotic lungs. In contrast to other diseases in which RAGE signaling promotes pathology, immunohistochemical and hydroxyproline quantification studies on aged RAGE-null mice indicate that these mice spontaneously develop pulmonary fibrosis-like alterations. Furthermore, when subjected to a model of pulmonary fibrosis, RAGE-null mice developed more severe fibrosis, as measured by hydroxyproline assay and histological scoring, than wild-type controls. Combined with data from other studies on mouse models of pulmonary fibrosis and human IPF tissues indicate that loss of RAGE contributes to IPF pathogenesis.

Enhanced bleomycin-induced pulmonary damage in mice lacking extracellular superoxide dismutase

Extracellular superoxide dismutase (EC-SOD) is highly expressed in the extracellular matrix of lung and vascular tissue. Localization of EC-SOD to the matrix of the lung may protect against oxidative tissue damage that leads to pulmonary fibrosis. This study directly examines the protective role of EC-SOD in a bleomycin model of pulmonary fibrosis and the effect of this enzyme on oxidative protein fragmentation. Mice null for ec-sod display a marked increase in lung inflammation at 14 d post-bleomycin treatment as compared to their wild-type counterparts. Hydroxyproline analysis determined that both wild-type and ec-sod null mice display a marked increase in interstitial fibrosis at 14 d post-treatment, and the severity of fibrosis is significantly increased in ec-sod null mice compared to wild-type mice. To determine if the lack of EC-SOD promotes bleomycin-induced oxidative protein modification, 2-pyrrolidone content (as a measure of oxidative protein fragmentation at proline residues) was assessed in lung tissue from treated mice. 2-Pyrrolidone levels in the lung hydrolysates from ec-sod null mice were increased at both 7 and 14 d post-bleomycin treatment as compared to wild-type mice, indicating EC-SOD can inhibit oxidative fragmentation of proteins in this specific model of oxidative stress.

Carbon Monoxide Suppresses Bleomycin-Induced Lung Fibrosis

Idiopathic pulmonary fibrosis is an incurable fibrosing disorder that progresses relentlessly to respiratory failure. We hypothesized that a product of heme oxygenase activity, carbon monoxide (CO), may have anti-fibrotic effects. To test this hypothesis, mice treated with intratracheal bleomycin were exposed to low-concentration inhaled CO or ambient air. Lungs of mice treated with CO had significantly lower hydroxyproline accumulation than controls. Fibroblast proliferation, thought to play a central role in the progression of fibrosis, was suppressed by in vitro exposure to CO. CO caused increased cellular levels of p21(Cip1) and decreased levels of cyclins A and D. This effect was independent of the observed suppression of MAPKs phosphorylation by CO but was dependent on increased cGMP levels. Further, CO-exposed cells elaborated significantly less fibronectin and collagen-1 than control cells. This same effect was seen in vivo. Suppression of collagen-1 production did not depend on MAPK or guanylate cyclase signaling pathways but did depend on the transcriptional regulator Id1. Taken together, these data suggest that CO exerts an anti-fibrotic effect in the lung, and this effect may be due to suppression of fibroblast proliferation and/or suppression of matrix deposition by fibroblasts.

IL-22 Is Essential for Lung Epithelial Repair following Influenza Infection

Influenza infection is widespread in the United States and the world. Despite low mortality rates due to infection, morbidity is common and little is known about the molecular events involved in recovery. Influenza infection results in persistent distal lung remodeling, and the mechanism(s) involved are poorly understood. Recently IL-22 has been found to mediate epithelial repair. We propose that IL-22 is critical for recovery of normal lung function and architecture after influenza infection. Wild-type and IL-22(-/-) mice were infected with influenza A PR8/34 H1N1 and were followed up for up to 21 days post infection. IL-22 receptor was localized to the airway epithelium in naive mice but was expressed at the sites of parenchymal lung remodeling induced by influenza infection. IL-22(-/-) mice displayed exacerbated lung injury compared with wild-type mice, which correlated with decreased lung function 21 days post infection. Epithelial metaplasia was observed in wild-type mice but was not evident in IL-22(-/-) animals that were characterized with an increased fibrotic phenotype. Gene expression analysis revealed aberrant expression of epithelial genes involved in repair processes, among changes in several other biological processes. These data indicate that IL-22 is required for normal lung repair after influenza infection. IL-22 represents a novel pathway involved in interstitial lung disease.

Altered expression of extracellular superoxide dismutase in mouse lung after bleomycin treatment

The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) is highly expressed in the extracellular matrix of lung tissue and is believed to protect the lung from oxidative damage that results in diseases such as pulmonary fibrosis. This study tests the hypothesis that proteolytic removal of the heparin-binding domain of EC-SOD results in clearance of the enzyme from the extracellular matrix of pulmonary tissues and leads to a loss of antioxidant protection. Using a polyclonal antibody to mouse EC-SOD, the immunodistribution of EC-SOD in normal and bleomycin-injured lungs was examined. EC-SOD labeling was strong in the matrix of vessels, airways, and alveolar surfaces and septa in control lungs. At 2 d post-treatment, a slight increase in EC-SOD staining was evident. In contrast, lungs examined 4 or 7 d post-treatment, showed an apparent loss of EC-SOD from the matrix and surface of alveolar septa. Notably, at 7 d post-treatment, the truncated form of EC-SOD was found in the bronchoalveolar lavage fluid of bleomycin-treated mice, suggesting that EC-SOD is being removed from the extracellular matrix through proteolysis. However, loss of EC-SOD through proteolysis did not correlate with a decrease in overall pulmonary EC-SOD activity. The negligible effect on EC-SOD activity may reflect the large influx of intensely staining inflammatory cells at day 7. These results indicate that injuries leading to pulmonary fibrosis have a significant effect on EC-SOD distribution due to proteolytic removal of the heparin-binding domain and may be important in enhancing pulmonary injuries by altering the oxidant/antioxidant balance in alveolar interstitial spaces.

CHARACTERIZATION OF INTERIOR CLEAVAGE OF RETINOBLASTOMA PROTEIN IN APOPTOSIS

Previously we reported that at the onset of apoptotic execution, retinoblastoma protein (RB) was cleaved in its interior region, resulting in production of two major fragments, p48 and p68, and that the RB interior cleavage was mediated by a caspase‐like activity. Here, we further characterized the RB interior cleavage process in human leukemia cells treated with the anticancer agent etoposide. We found that the RB interior cleavage activity was much more sensitive to two specific tetrapeptide caspase inhibitors, YVAD‐CMK and DEVD‐FMK, than the poly(ADP‐ribose) polymerase cleavage activity, suggesting that two distinct caspases are involved in these processes. Several Asp residues are located in amino acids 341–421 of RB protein, and cleavage of any one of these sites by a caspase would generate a p48, which contains the amino terminus, and a p68 fragment, which contains the A/B pocket and the carboxyl terminus. This hypothesis was supported by the fact that the p48 and p68 fragments had selective binding affinity to different RB antibodies and that the p48 was found only in the low‐salt‐extracted cytoplasmic fraction, while the p68 was only in the nuclear fraction, of the apoptotic cells. However, the nuclear binding partner of the p68 RB fragment is not the transcription factor E2F‐1 since a specific E2F‐1 antibody coimmunoprecipitated only the unphosphorylated form of RB, but not the p68 fragment. Lastly, we confirmed that RB also underwent dephosphorylation and carboxyl terminal cleavage during apoptosis, as we and others reported previously. J. Cell. Biochem. 67:399–408, 1997.

Sequential two-step cleavage of the retinoblastoma protein by caspase-3/-7 during etoposide-induced apoptosis

During cellular apoptosis, retinoblastoma protein (RB) is subjected to cleavage near the carboxyl terminus by a caspase-3-like protease. In addition, an heretofore unidentified protease cleaves RB internally, generating fragments of 68 and 48 kDa. Internal cleavage abrogates the ability of RB to associate with E2F. To investigate the mechanism of RB internal cleavage, we developed and employed an in vitro cleavage assay. Incubation of in vitro translated 35S-RB with apoptotic cell extracts led to RB cleavage at the C-terminus, followed by internal cleavage. The caspase peptide inhibitors z-VAD-FMK or z-DEVD-FMK blocked both cleavage events. Rapid C-terminal and internal cleavage were also observed when recombinant caspase-3 was added to 35S-RB. Moreover, when caspase-3 was added to nonapoptotic cell extract, efficient internal cleavage of cellular RB was observed. Caspase-mediated internal cleavage occurred following RB residue aspartate349 in the sequence DSID349. This sequence is consistent with a DXXD recognition motif for caspase-3-like enzymes. Interestingly, we also observed RB internal cleavage in caspase-3-deficient MCF-7 cells, indicating that other caspases are capable of cleaving RB internally. Indeed, caspase-7, a member of the caspase-3 subfamily, was found to cleave 35S-RB at both the carboxyl terminus, and following aspartate349. By contrast, caspases that are not members of the caspase-3 subfamily failed to cleave RB. Taken together, our findings demonstrate that during apoptosis, a caspase-3-like protease is responsible for degradation and functional inactivation of RB by cleaving the protein internally following aspartate349.

Lung extracellular superoxide dismutase overexpression lessens bleomycin-induced pulmonary hypertension and vascular remodeling.

Interstitial lung disease is a devastating disease in humans that can be further complicated by the development of secondary pulmonary hypertension. Accumulating evidence indicates that the oxidant superoxide can contribute to the pathogenesis of both interstitial lung disease and pulmonary hypertension. We used a model of pulmonary hypertension secondary to bleomycin-induced pulmonary fibrosis to test the hypothesis that an imbalance in extracellular superoxide and its antioxidant defense, extracellular superoxide dismutase, will promote pulmonary vascular remodeling and pulmonary hypertension. We exposed transgenic mice overexpressing lung extracellular superoxide dismutase and wild-type littermates to a single dose of intratracheal bleomycin, and evaluated the mice weekly for up to 35 days. We assessed pulmonary vascular remodeling and the expression of several genes critical to lung fibrosis, as well as pulmonary hypertension and mortality. The overexpression of extracellular superoxide dismutase protected against late remodeling within the medial, adventitial, and intimal layers of the vessel wall after the administration of bleomycin, and attenuated pulmonary hypertension at the same late time point. The overexpression of extracellular superoxide dismutase also blocked the early up-regulation of two key genes in the lung known to be critical in pulmonary fibrosis and vascular remodeling, the transcription factor early growth response-1 and transforming growth factor-β. The overexpression of extracellular superoxide dismutase attenuated late pulmonary hypertension and significantly improved survival after exposure to bleomycin. These data indicate an important role for an extracellular oxidant/antioxidant imbalance in the pathogenesis of pulmonary vascular remodeling associated with secondary pulmonary hypertension attributable to bleomycin-induced lung fibrosis.