Bleomycin Binding Sites on Alveolar Macrophages

Previous work has demonstrated that bleomycin can directly stimulate alveolar macrophage secretion of fibroblast growth factors and monocyte chemotactic factors. In this study, rat alveolar macrophages obtained by bronchoalveolar lavage were examined for the presence of bleomycin binding sites, which might mediate this response. The results indicated that alveolar macrophages have specific, saturable, and reversible binding sites. Both high‐ and low‐affinity binding sites were found; each macrophage possessed 6.7 × 104 high‐affinity sites, with a Kd, of 528 nM, and 2.2 × 106 low‐affinity sites, with a Kd of 65 μM. The Kd of the high‐affinity sites corresponds closely to the ED50 obtained from dose‐response curves of the bleomycin‐stimulated secretion of both fibroblast growth and monocyte chemotactic factors, suggesting that bleomycin stimulation of alveolar macrophage function responses may be mediated by bleomycin interaction with these sites.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141017/1/jlb0519.pd

Therapies for bleomycin induced lung fibrosis through regulation of TGF-Β1 induced collagen gene expression

This review describes normal and abnormal wound healing, the latter characterized by excessive fibrosis and scarring, which for lung can result in morbidity and sometimes mortality. The cells, the extracellular matrix (ECM) proteins, and the growth factors regulating the synthesis, degradation, and deposition of the ECM proteins will be discussed. Therapeutics with particular emphasis given to gene therapies and their effects on specific signaling pathways are described. Bleomycin (BM), a potent antineoplastic antibiotic increases TGF-Β1 transcription, TGF-Β1 gene expression, and TGF-Β protein. Like TGF-Β1, BM acts through the same distal promoter cis -element of the COL1A1 gene causing increased COL1 synthesis and lung fibrosis. Lung fibroblasts exist as subpopulations with one subset predominately responding to fibrogenic stimuli which could be a specific cell therapeutic target for the onset and development of pulmonary fibrosis. J. Cell. Physiol. 211: 585–589, 2007. © 2007 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/55994/1/20972_ftp.pd

3‐Deaza‐Adenosine Inhibition of Stimulus‐Response Coupling in Human Polymorphonuclear Leukocytes

In an effort to define better the functional role of S‐adenosyl‐methionine‐mediated methylation reactions in modulating polymorphonuclear (PMN) functional responses to chemotactic stimuli, we investigated the effects of 3‐deaza‐adenosine (3‐DZA), a known inhibitor of methylation reactions in phagocytic cells, on formyl methionyl‐leucyl‐phenylalanine (FMLP)‐induced responses in human PMN leukocytes. Using the fluorescent cyanine dye 3,3’‐dipropylthiocarbocyanine (di‐S‐C3‐(5)) as an optical probe of membrane potential we observed that 3‐DZA at concentrations that inhibit FMLP‐induced O2− production does not significantly alter FMLP‐induced changes in transmembrane potential. Additional studies showed an inhibitory effect of 3‐DZA on FMLP‐induced PMN pinocytosis and to a lesser degree on FMLP‐induced degranulation. However, pretreatment of PMNs with 3‐DZA did not alter FMLP‐induced changes in Quin‐2 fluorescence, an indicator of changes in intracellular calcium levels. These findings demonstrate a dissociation between chemotactic factor‐induced cell membrane depolarization, changes in intracellular calcium, and specific neutrophil functional responses and suggest that chemotactic factor‐induced changes in transmembrane potential and intracellular calcium are independent of chemotactic factor‐induced methylation reactions. Furthermore, 3‐DZA did not alter phorbol myristate acetate induced O2− production or fluid pinocytosis indicating a stimulus specificity for the inhibitory effects of this agent on O2− production.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141044/1/jlb0121.pd

Oxygen metabolite detoxifying enzyme levels in bleomycin-induced fibrotic lungs

The activities of three enzymes cytosolic superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSHP), and malonylialdehyde (MDA), a by-product of lipid peroxidation, were determined in whole lungs of normal and bleomycin-treated rats. Two days after bleomycin treatment total lung SOD, CAT, and GSHP activities were significantly (p p p 2 metabolites may play an important role in the development of bleomycin-induced pulmonary fibrosis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27559/1/0000603.pd

Regulation of Macrophage‐Derived Fibroblast Growth Factor Release by Arachidonate Metabolites

The macrophage is a source of many mediators with direct and indirect fibrogenic potential. In this study, release of macrophage‐derived fibroblast growth factor (MDGF) activity by murine peritoneal macrophages is examined with regard to its regulation by arachidonate metabolites. Upon stimulation with 10 μg/ml lipopolysaccharide (LPS), resident peritoneal macrophages from CBA/J mice released MDGF activity into media rapidly, reaching maximal levels in approximately 1 h. Lysates of these stimulated cells also revealed significantly increased cell‐associated MDGF activity, composing 45% of the total assayable activity. This activity, as assayed by radioactive thymidine incorporation by primary cultures of rat lung fibroblasts, was separable from interleukin‐1 (IL‐1) activity by reverse phase high performance liquid chromatography (HPLC). Furthermore, purified murine IL‐1 had no MDGF activity in this assay system. This stimulated MDGF release was enhanced by the cylooxygenase inhibitors indomethacin, Ibuprofen, and aspirin at micromolar concentrations, but inhibited in a dose‐dependent manner by prostaglandin E2 (PGE2). On the other hand, nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor was inhibitory at 0.1 and 0.4 μM but not at 2.5 μM. Zymosan‐stimulated macrophages also markedly increased MDGF release, albeit with a different time course which was characterized by a delay of approximately 7 h before peak levels were attained. Such stimulation, which is known to cause increased lipoxygenase activity, was also inhibited by 0.5 μM NDGA. In contrast, the lipoxygenase pathway products leukotrienes B4 (LTB4) and C4 (LTC4) stimulated MDGF release in a dose‐dependent (10‐10‐10‐8 M) manner, with LTC4 being more potent on a per unit dose basis. Stimulation by LTC4 was inhibited by the putative leukotriene receptor antagonist, FPL55712, while LTD4 and LTE4 did not stimulate MDGF release, thus suggesting the mediation of this effect by specific LTC4 receptors. These data suggest also that products of the cyclooxygenase and lipoxygenase pathways are potentially important both as exogenous (ie, derived from cells other than the macrophage itself) and auto‐ or self‐regulators of macrophage MDGF release. This, in turn, implies that cyclooxygenase products are antifibrogenic and important in maintaining or returning to the quiescent or normal state, whereas the lipoxygenase products are profibrogenic and important in induction of fibrosis or wound‐healing and tissue repair. Any alteration in the balance between these two pathways may result in either a desirable or a harmful outcome.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141690/1/jlb0106.pd

Tackling MARCKS-PIP3 circuit attenuates fibroblast activation and fibrosis progression.

Targeting activated fibroblasts, including myofibroblast differentiation, has emerged as a key therapeutic strategy in patients with idiopathic pulmonary fibrosis (IPF). However, there is no available therapy capable of selectively eradicating myofibroblasts or limiting their genesis. Through an integrative analysis of the regulator genes that are responsible for the activation of IPF fibroblasts, we noticed the phosphatidylinositol 4,5-bisphosphate (PIP2)-binding protein, myristoylated alanine-rich C-kinase substrate (MARCKS), as a potential target molecule for IPF. Herein, we have employed a 25-mer novel peptide, MARCKS phosphorylation site domain sequence (MPS), to determine if MARCKS inhibition reduces pulmonary fibrosis through the inactivation of PI3K/protein kinase B (AKT) signaling in fibroblast cells. We first observed that higher levels of MARCKS phosphorylation and the myofibroblast marker α-smooth muscle actin (α-SMA) were notably overexpressed in all tested IPF lung tissues and fibroblast cells. Treatment with the MPS peptide suppressed levels of MARCKS phosphorylation in primary IPF fibroblasts. A kinetic assay confirmed that this peptide binds to phospholipids, particularly PIP2, with a dissociation constant of 17.64 nM. As expected, a decrease of phosphatidylinositol (3,4,5)-trisphosphate pools and AKT activity occurred in MPS-treated IPF fibroblast cells. MPS peptide was demonstrated to impair cell proliferation, invasion, and migration in multiple IPF fibroblast cells in vitro as well as to reduce pulmonary fibrosis in bleomycin-treated mice in vivo. Surprisingly, we found that MPS peptide decreases α-SMA expression and synergistically interacts with nintedanib treatment in IPF fibroblasts. Our data suggest MARCKS as a druggable target in pulmonary fibrosis and also provide a promising antifibrotic agent that may lead to effective IPF treatments.-Yang, D. C., Li, J.-M., Xu, J., Oldham, J., Phan, S. H., Last, J. A., Wu, R., Chen, C.-H. Tackling MARCKS-PIP3 circuit attenuates fibroblast activation and fibrosis progression

An ST2‐dependent role of bone marrow‐derived group 2 innate lymphoid cells in pulmonary fibrosis

Recent evidence supports that bone marrow (BM)‐derived hematopoietic progenitor cells play an important role in lung injury and fibrosis. While these cells give rise to multiple cell types, the ST2 (Il1rl1)‐expressing group 2 innate lymphoid cells (ILC2s) derived from BM progenitors have been implicated in tissue repair and remodeling, including in lung fibrosis. To further investigate the precise role of BM‐derived ILC2s in the pathogenesis of fibrotic lung disease, their importance in the bleomycin‐induced lung fibrosis model was evaluated by analyzing the effects of selective ST2 deficiency in the BM compartment. The results showed that while ST2‐sufficient control mice exhibited activation of lung IL‐33/ST2 signaling, ILC2 recruitment, IL‐13 induction, and fibrosis, these responses were significantly diminished in ST2‐deficient‐BM chimera mice, with selective loss of ST2 expression only in the BM. This diminished response to bleomycin was similar to that seen in ST2 global knockout mice, suggesting the predominant importance of ST2 from the BM compartment. In wild‐type mice, ILC2 recruitment to the lung was accompanied by a concomitant decrease in ST2+ BM cells. ST2‐deficient BM cells were unresponsive to IL‐33‐induced ILC2 maturation. Finally, lineage‐negative wild‐type, but not ST2‐deficient BM cells from bleomycin‐treated mice stimulated lung fibroblast type I collagen expression, which was associated with elevated TGFβ expression in the BM cells. Taken together, these findings suggested that the BM‐derived ILC2s were recruited to fibrotic lung through the IL‐33/ST2 pathway, and contributed to fibroblast activation to promote lung fibrosis. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/145267/1/path5092.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/145267/2/path5092_am.pd

TNF and IL‐6 mediate MIP‐1α expression in bleomycin‐induced lung injury

Previously, macrophage inflammatory protein‐1α (MIP‐1α), a member of the C‐C chemokine family, has been implicated in bleomycin‐induced pulmonary fibrosis, a model of the human disease idiopathic pulmonary fibrosis. Neutralization of MIP‐1α protein with anti‐MIP‐1α antibodies significantly attenuated both mononuclear phagocyte recruitment and pulmonary fibrosis in bleomycin‐challenged CBA/J mice. However, the specific stimuli for MIP‐1α expression in the bleomycin‐induced lesion have not been characterized. In this report, two mediators of the inflammatory response to bleomycin, tumor necrosis factor (TNF) and interleukin‐6 (IL‐6), were evaluated as putative stimuli for MIP‐1α expression after bleomycin challenge in CBA/J mice. Elevated levels of bioactive TNF and IL‐6 were detected in bronchoalveolar lavage (BAL) fluid and lung homogenates from bleomycin‐treated CBA/J mice at time points post‐bleomycin challenge, which precede MIP‐1α protein expression. Treatment of bleomycin‐challenged mice with soluble TNF receptor (sTNFr) or anti‐IL‐6 antibodies significantly decreased MIP‐1α protein expression in the lungs. Furthermore, normal alveolar macrophages secreted elevated levels of MIP‐1α protein in response to treatment with TNF plus IL‐6 or bleomycin plus IL‐6, but not TNF, bleomycin, or IL‐6 alone. Finally, leukocytes recovered from the BAL fluid of bleomycin‐challenged mice secreted higher levels of MIP‐1α protein, compared to controls, when treated with TNF alone. Based on the data presented here, we propose that TNF and IL‐6 are part of a cytokine network that modulates MIP‐1α protein expression in the profibrotic inflammatory lesion during the response to intratracheal bleomycin challenge. J. Leukoc. Biol. 64: 528–536; 1998.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141711/1/jlb0528.pd

Prostaglandin modulation of N-formylmethionylleucylphenylalanine-induced transmembrane potential changes in rat neutrophils

Prostaglandins of the E-series (PGEs) and PGI2 will inhibit formylmethionylleucylphenylalanine- (f-Met-Leu-Phe) induced lysosomal enzyme release and superoxide-anion (O-2) production by neutrophils. The inhibitory effects of PGEs and PGI2 on neutrophil functional responses have been correlated with their ability to increase intracellular cAMP. In this study we have examined the effects of PGEs and PGI2 on f-Met-Leu-Phe- and phorbol-myristate-acetate-induced rat neutrophil membrane potential changes using an optical probe of membrane potential 3,3-dipropylthiodicarbocyanine iodide. 15-(S)-15-methyl-PGE1 (15-methyl-PGE1), a stable analogue of PGE1 and PGI2 inhibited f-Met-Leu-Phe-induced transmembrane potential changes in a dose-dependent manner. This inhibition was correlated with the ability of these agents to increase intracellular cAMP levels and inhibit O-2 production and degranulation. In contrast, 15-methyl-PGE1 and PGI2, did not inhibit phorbol-myristate-acetate-induced transmembrane potential changes and O-2 production. These results suggest independent mechanisms of activation of neutrophils by phorbol myristate acetate and f-Met-Leu-Phe, and they also suggest that the inhibitory effects of prostaglandins and cAMP on f-Met-Leu-Phe-stimulated cells is at a step or steps prior to activation of those processes involved in effecting changes in transmembrane potential, which are common to both stimuli.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24755/1/0000177.pd

The role of ILâ 5 in bleomycinâ induced pulmonary fibrosis

Eosinophils are known to express cytokines capable of promoting fibrosis. Interleukinâ 5 (ILâ 5) is important in regulating eosinophilopoiesis, eosinophil recruitment and activation. Lung ILâ 5 expression is elevated in pulmonary fibrosis, wherein the eosinophil is a primary source of fibrogenic cytokines. To determine the role of ILâ 5 in pulmonary fibrosis, the effects of antiâ ILâ 5 antibody were investigated in a model of bleomycinâ induced pulmonary fibrosis. Fibrosis was induced in mice by endotracheal bleomycin treatment. Animals were also treated with either antiâ ILâ 5 antibody or control IgG. Lungs were then analyzed for fibrosis, eosinophil influx, chemotactic activity, and cytokine expression. The results show that a primary chemotactic activity at the height of eosinophil recruitment is ILâ 5. Furthermore, antiâ ILâ 5 antibody caused significant reduction in lung eosinophilia, cytokine expression, and fibrosis. These findings taken together suggest an important role for ILâ 5 in pulmonary fibrosis via its ability to regulate eosinophilic inflammation, and thus eosinophilâ dependent fibrogenic cytokine production. J. Leukoc. Biol. 64: 657â 666; 1998.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141191/1/jlb0657.pd