Mouse breast regression protein 39 (BRP-39; Chi3l1) and its human homologue YKL-40 are chitinase-like proteins that lack chitinase activity. Although YKL-40 is expressed in exaggerated quantities and correlates with disease activity in asthma and many other disorders, the biological properties of BRP-39/YKL-40 have only been rudimentarily defined. We describe the generation and characterization of BRP-39−/− mice, YKL-40 transgenic mice, and mice that lack BRP-39 and produce YKL-40 only in their pulmonary epithelium. Studies of these mice demonstrated that BRP-39−/− animals have markedly diminished antigen-induced Th2 responses and that epithelial YKL-40 rescues the Th2 responses in these animals. The ability of interleukin13 to induce tissue inflammation and fibrosis was also markedly diminished in the absence of BRP-39. Mechanistic investigations demonstrated that BRP-39 and YKL-40 play an essential role in antigen sensitization and immunoglobulin E induction, stimulate dendritic cell accumulation and activation, and induce alternative macrophage activation. These proteins also inhibit inflammatory cell apoptosis/cell death while inhibiting Fas expression, activating protein kinase B/AKT, and inducing Faim 3. These studies establish novel regulatory roles for BRP-39/YKL-40 in the initiation and effector phases of Th2 inflammation and remodeling and suggest that these proteins are therapeutic targets in Th2- and macrophage-mediated disorders.
The exaggerated expression of chitinase-like protein YKL-40, the human homologue of breast regression protein–39 (BRP-39), was reported in a number of diseases, including chronic obstructive pulmonary disease (COPD). However, the in vivo roles of YKL-40 in normal physiology or in the pathogenesis of specific diseases such as COPD remain poorly understood. We hypothesized that BRP-39/YKL-40 plays an important role in the pathogenesis of cigarette smoke (CS)–induced emphysema. To test this hypothesis, 10-week-old wild-type and BRP-39 null mutant mice (BRP-39−/−) were exposed to room air (RA) and CS for up to 10 months. The expression of BRP-39 was significantly induced in macrophages, airway epithelial cells, and alveolar Type II cells in the lungs of CS-exposed mice compared with RA-exposed mice, at least in part via an IL-18 signaling–dependent pathway. The null mutation of BRP-39 significantly reduced CS-induced bronchoalveolar lavage and tissue inflammation. However, CS-induced epithelial cell apoptosis and alveolar destruction were further enhanced in the absence of BRP-39. Consistent with these findings in mice, the tissue expression of YKL-40 was significantly increased in the lungs of current smokers compared with the lungs of ex-smokers or nonsmokers. In addition, serum concentrations of YKL-40 were significantly higher in smokers with COPD than in nonsmokers or smokers without COPD. These studies demonstrate a novel regulatory role of BRP-39/YKL-40 in CS-induced inflammation and emphysematous destruction. These studies also underscore that maintaining physiologic concentrations of YKL-40 in the lung is therapeutically important in preventing excessive inflammatory responses or emphysematous alveolar destruction.
YKL-40/BRP-39; COPD; emphysema; cigarette smoke
We previously reported that YKL-40, the human analog of mouse breast regression protein-39 (BRP-39; chitinase 3-like 1), is elevated in the cerebrospinal fluid of patients with a variety of neuroinflammatory conditions, such as multiple sclerosis and traumatic brain injury. YKL-40 expression in the CNS was predominantly associated with reactive astrocytes in the vicinity of inflammatory lesions. Because previous studies have shown that reactive astrocytes play a critical role in limiting immune infiltration in the mouse model of experimental autoimmune encephalomyelitis (EAE), we explored the role of BRP-39 in regulating neuroinflammation in EAE. Using BRP-39-deficient mice (BRP-39−/−), we demonstrate the importance of BRP-39 in modulating the severity of clinical EAE and CNS neuroinflammation. At disease onset, absence of BRP-39 had little effect on clinical disease or lymphocytic infiltrate, but by 14 days post-immunization (dpi), differences in clinical scores were evident. By 28 dpi, BRP-39−/− mice showed more severe and persistent clinical disease than BRP-39+/+ controls. Histopathological evaluation showed that BRP-39−/− mice had more marked lymphocytic and macrophage infiltrates and gliosis vs. BRP-39+/+ mice. These findings support the role of BRP-39 expression in limiting immune cell infiltration into the CNS and offer a new target to modulate neuroinflammation.
BRP-39; Chitinase-like proteins; Experimental autoimmune encephalomyelitis; Multiple sclerosis; Neuroimmunology; YKL-40
The chitinase-like protein YKL-40 was found to be increased in patients with severe asthma and chronic obstructive pulmonary disease (COPD), two disease conditions featuring neutrophilic infiltrates. Based on these studies and a previous report indicating that neutrophils secrete YKL-40, we hypothesized that YKL-40 plays a key role in cystic fibrosis (CF) lung disease, a prototypic neutrophilic disease. The aim of this study was (i) to analyze YKL-40 levels in human and murine CF lung disease and (ii) to investigate whether YKL-40 single-nucleotide polymorphisms (SNPs) modulate CF lung disease severity. YKL-40 protein levels were quantified in serum and sputum supernatants from CF patients and control individuals. Levels of the murine homologue BRP-39 were analyzed in airway fluids from CF-like βENaC-Tg mice. YKL-40SNPs were analyzed in CF patients. YKL-40 levels were increased in sputum supernatants and in serum from CF patients compared to healthy control individuals. Within CF patients, YKL-40 levels were higher in sputum than in serum. BRP-39 levels were increased in airways fluids from βENaC-Tg mice compared to wild-type littermates. In both CF patients and βENaC-Tg mice, YKL-40/BRP-39 airway levels correlated with the severity of pulmonary obstruction. Two YKL-40 SNPs (rs871799 and rs880633) were found to modulate age-adjusted lung function in CF patients. YKL-40/BRP-39 levelsare increased in human and murine CF airway fluids, correlate with pulmonary function and modulate CF lung disease severity genetically. These findings suggest YKL-40 as a potential biomarker in CF lung disease.
BRP-39 and its human homolog YKL-40 have been regarded as a prototype of chitinase-like proteins (CLP) in mammals. Exaggerated levels of YKL-40 protein and/or mRNA have been noted in a number of diseases characterized by inflammation, tissue remodeling, and aberrant cell growth. Asthma is an inflammatory disease characterized by airway hyperresponsiveness and airway remodeling. Recently, the novel regulatory role of BRP-39/YKL-40 in the pathogenesis of asthma has been demonstrated both in human studies and allergic animal models. The levels of YKL-40 are increased in the circulation and lungs from asthmatics where they correlate with disease severity, and CHI3L1 polymorphisms correlate with serum YKL-40 levels, asthma and abnormal lung function. Animal studies using BRP-39 null mutant mice demonstrated that BRP-39 was required for optimal allergen sensitization and Th2 inflammation. These studies suggest the potential use of BRP-39 as a biomarker as well as a therapeutic target for asthma and other allergic diseases. Here, we present an overview of chitin/chitinase biology and summarize recent findings on the role of BRP-39 in the pathogenesis of asthma and allergic responses.
BRP-39; human CHI3L1 protein; asthma; hypersensitivity
While the presence of the chitinase-like molecule YKL40 has been reported in COPD and asthma, its relevance to inflammatory processes elicited by cigarette smoke and common environmental allergens, such as house dust mite (HDM), is not well understood. The objective of the current study was to assess expression and function of BRP-39, the murine equivalent of YKL40 in a murine model of cigarette smoke-induced inflammation and contrast expression and function to a model of HDM-induced allergic airway inflammation.
CD1, C57BL/6, and BALB/c mice were room air- or cigarette smoke-exposed for 4 days in a whole-body exposure system. In separate experiments, BALB/c mice were challenged with HDM extract once a day for 10 days. BRP-39 was assessed by ELISA and immunohistochemistry. IL-13, IL-1R1, IL-18, and BRP-39 knock out (KO) mice were utilized to assess the mechanism and relevance of BRP-39 in cigarette smoke- and HDM-induced airway inflammation.
Cigarette smoke exposure elicited a robust induction of BRP-39 but not the catalytically active chitinase, AMCase, in lung epithelial cells and alveolar macrophages of all mouse strains tested. Both BRP-39 and AMCase were increased in lung tissue after HDM exposure. Examining smoke-exposed IL-1R1, IL-18, and IL-13 deficient mice, BRP-39 induction was found to be IL-1 and not IL-18 or IL-13 dependent, while induction of BRP-39 by HDM was independent of IL-1 and IL-13. Despite the importance of BRP-39 in cellular inflammation in HDM-induced airway inflammation, BRP-39 was found to be redundant for cigarette smoke-induced airway inflammation and the adjuvant properties of cigarette smoke.
These data highlight the contrast between the importance of BRP-39 in HDM- and cigarette smoke-induced inflammation. While functionally important in HDM-induced inflammation, BRP-39 is a biomarker of cigarette smoke induced inflammation which is the byproduct of an IL-1 inflammatory pathway.
Hyperoxic acute lung injury (HALI) is characterized by a cell death response with features of apoptosis and necrosis that is inhibited by IL-11 and other interventions. We hypothesized that Bfl-1/A1, an antiapoptotic Bcl-2 protein, is a critical regulator of HALI and a mediator of IL-11–induced cytoprotection. To test this, we characterized the expression of A1 and the oxygen susceptibility of WT and IL-11 Tg(+) mice with normal and null A1 loci. In WT mice, 100% O2 caused TUNEL+ cell death, induction and activation of intrinsic and mitochondrial-death pathways, and alveolar protein leak. Bcl-2 and Bcl-xl were also induced as an apparent protective response. A1 was induced in hyperoxia, and in A1-null mice, the toxic effects of hyperoxia were exaggerated, Bcl-2 and Bcl-xl were not induced, and premature death was seen. In contrast, IL-11 stimulated A1, diminished the toxic effects of hyperoxia, stimulated Bcl-2 and Bcl-xl, and enhanced murine survival in 100% O2. In A1-null mice, IL-11–induced protection, survival advantage, and Bcl-2 and Bcl-xl induction were significantly decreased. VEGF also conferred protection via an A1-dependent mechanism. In vitro hyperoxia also stimulated A1, and A1 overexpression inhibited oxidant-induced epithelial cell apoptosis and necrosis. A1 is an important regulator of oxidant-induced lung injury, apoptosis, necrosis, and Bcl-2 and Bcl-xl gene expression and a critical mediator of IL-11– and VEGF-induced cytoprotection.
The 18 glycosyl hydrolase family of chitinases is an ancient gene family that is widely expressed from prokaryotes to eukaryotes. In mammals, despite the absence of endogenous chitin, a number of chitinases and chitinase-like proteins (C/CLPs) have been identified. However, their roles have only recently begun to be elucidated. Acidic mammalian chitinase (AMCase) inhibits chitin-induced innate inflammation; augments chitin-free, allergen-induced Th2 inflammation; and mediates effector functions of IL-13. The CLPs BRP-39/YKL-40 (also termed chitinase 3-like 1) inhibit oxidant-induced lung injury, augments adaptive Th2 immunity, regulates apoptosis, stimulates alternative macrophage activation, and contributes to fibrosis and wound healing. In accord with these findings, levels of YKL-40 in the lung and serum are increased in asthma and other inflammatory and remodeling disorders and often correlate with disease severity. Our understanding of the roles of C/CLPs in inflammation, tissue remodeling, and tissue injury in health and disease is reviewed below.
asthma; fibrosis; BRP-39/YKL-40; AMCase; chitotriosidase
Rationale. Hyperoxia exposure to developing lungs—critical in the pathogenesis of bronchopulmonary dysplasia—may augment lung inflammation by inhibiting anti-inflammatory mediators in alveolar macrophages. Objective. We sought to determine the O2-induced effects on the polarization of macrophages and the role of anti-inflammatory BRP-39 in macrophage phenotype and neonatal lung injury. Methods. We used RAW264.7, peritoneal, and bone marrow derived macrophages for polarization (M1/M2) studies. For in vivo studies, wild-type (WT) and BRP-39−/− mice received continuous exposure to 21% O2 (control mice) or 100% O2 from postnatal (PN) 1 to PN7 days, along with intranasal lipopolysaccharide (LPS) administered on alternate days (PN2, -4, and -6). Lung histology, bronchoalveolar lavage (BAL) cell counts, BAL protein, and cytokines measurements were performed. Measurements and Main Results. Hyperoxia differentially contributed to macrophage polarization by enhancing LPS induced M1 and inhibiting interleukin-4 induced M2 phenotype. BRP-39 absence led to further enhancement of the hyperoxia and LPS induced M1 phenotype. In addition, BRP-39−/− mice were significantly more sensitive to LPS plus hyperoxia induced lung injury and mortality compared to WT mice. Conclusions. These findings collectively indicate that BRP-39 is involved in repressing the M1 proinflammatory phenotype in hyperoxia, thereby deactivating inflammatory responses in macrophages and preventing neonatal lung injury.
Supplemental oxygen is frequently prescribed. However, prolonged exposure to high concentrations of oxygen causes hyperoxic acute lung injury (HALI), which manifests as acute respiratory distress syndrome in adults and leads to bronchopulmonary dysplasia in newborns (NBs). Nitric oxide (NO), NO synthases (NOSs), and angiopoietin (Ang) 2 have been implicated in the pathogenesis of HALI. However, the mechanisms of the contributions of NOS/NO and the relationship(s) between NOS/NO and Ang2 have not been addressed. In addition, the relevance of these moieties in adults and NBs has not been evaluated. To address these issues, we compared the responses in hyperoxia of wild-type (NOS [+/+]) and NOS null (−/−) young adult and NB mice. When compared with NOS2+/+ adult controls, NOS2−/− animals manifest exaggerated alveolar–capillary protein leak and premature death. These responses were associated with enhanced levels of structural cell death, enhanced expression of proapoptotic regulatory proteins, and Ang2. Importantly, silencing RNA knockdown of Ang2 decreased the levels of cell death and the expression of proapoptotic mediators. These effects were at least partially NOS2 specific, and were development dependent, because survival was similar in adult NOS3+/+ and NOS3−/− mice and NB NOS2+/+ and NOS2−/− mice, respectively. These studies demonstrate that NOS2 plays an important protective role in HALI in adult animals. They also demonstrate that this response is mediated, at least in part, by the ability of NOS2 to inhibit hyperoxia-induced Ang2 production and thereby decrease Ang2-induced tissue injury.
cytokines; hyperoxia; lung
Mice and humans produce chitinase-like proteins (CLPs), which are highly homologous to chitinases but lack chitinolytic activity. Mice express primarily three CLPs, including breast regression protein-39 (BRP-39) [chitinase 3-like-1 (Chi3l1) or 38-kDa glycoprotein (gp38k)], Ym1 (Chi3l3) and Ym2 (Chi3l4). Recently, CLPs have attracted considerable attention due to their increased expression in a number of pathological conditions, including asthma, allergies, rheumatoid arthritis and malignant tumors. Although the exact functions of CLPs are largely unknown, the significance of their increased expression levels during pathophysiological states needs to be determined. The quantification of BRP-39, Ym1 and Ym2 is an important step in gaining insight into the in vivo regulation of the CLPs.
We constructed a standard DNA for quantitative real-time PCR (qPCR) by containing three CLPs target fragments and five reference genes cDNA in a one-to-one ratio. We evaluated this system by analyzing the eight target cDNA sequences. Tissue cDNAs obtained by reverse transcription from total RNA from four embryonic stages and eight adult tissues were analyzed using the qPCR system with the standard DNA.
We established a qPCR system detecting CLPs and comparing their expression levels with those of five reference genes using the same scale in mouse tissues. We found that BRP-39 and Ym1 were abundant in the mouse lung, whereas Ym2 mRNA was abundant in the stomach, followed by lung. The expression levels of BRP-39 and Ym1 in the mouse lung were higher than those of two active chitinases and were comparable to glyceraldehyde-3-phosphate dehydrogenase, a housekeeping gene which is constitutively expressed in all tissues.
Our results indicate that catalytically inactive BRP-39 and Ym1 are constitutive genes in normal mouse lung.
BRP-39; Chitinase; Chitinase-like protein; Gene expression analysis; Quantitative real-time PCR system; Ym1; Ym2
To determine the roles of breast regression protein-39 (BRP-39) in regulating dendritic cell maturation and in pathology of acute asthma.
Mouse bone marrow-derived dendritic cells (BMDCs) were prepared, and infected with adenovirus over-expressing BRP-39. Ovalbumin (OVA)-induced murine model of acute asthma was made in female BALB/c mice by sensitizing and challenging with chicken OVA and Imject Alum. The transfected BMDCs were adoptively transferred into OVA-treated mice via intravenous injection. Airway hyperresponsiveness (AHR), inflammation and pulmonary histopathology were characterized.
The expression of BRP-39 mRNA and protein was significantly increased in lung tissues of OVA-treated mice. The BMDCs infected with adenovirus BRP-39 exhibited greater maturation and higher activity in vitro. Adoptive transfer of the cells into OVA-treated mice significantly augmented OVA-induced AHR and eosinophilic inflammation. Meanwhile, BRP-39 further enhanced the production of OVA-induced Th2 cytokines IL-4, IL-5 and IL-13, but significantly attenuated OVA-induced IFN-γ production in bronchoalveolar lavage fluid.
In OVA-induced murine model of acute asthma, BRP-39 is over-expressed in lung tissue and augments Th2 inflammatory response and AHR. BRP-39 promotes dendritic cell maturation in vitro. Therefore, BRP-39 may be a potential therapeutic target of asthma.
asthma; ovalbumin; bone marrow-derived dendritic cells (BMDCs); breast regression protein-39 (BRP-39); YKL-40; Th2 inflammation; airway hyperresponsiveness; bronchoalveolar lavage fluid
Cystic fibrosis (CF) lung disease begins in early life and is progressive with the major risk factor being an exaggerated inflammatory response. Currently, assessment of neutrophilic inflammation in early cystic fibrosis (CF) lung disease relies on bronchoalveolar lavage (BAL). The chitinase-like protein YKL-40 is raised in sputum and serum of adults with CF. We investigated YKL-40 in BAL, serum and urine to determine whether this reflected inflammation and infection in young children with CF.
YKL-40 was measured in matched samples of BAL, serum and urine obtained from 36 infants and young children with CF participating in an early surveillance program. Levels were compared to clinical data and markers of inflammation detected in the lung.
YKL-40 in BAL correlated with pulmonary infection [β=1.30 (SE 0.34), p < 0.001] and BAL markers of inflammation [macrophage number: r2 = 0.34, p < 0.001; neutrophil number: r2 = 0.74, p < 0.001; neutrophil elastase: r2 = 0.47, p < 0.001; CXCL8: r2 = 0.45, p < 0.001; IL-β: r2 = 0.62, p < 0.001]. YKL-40 was detectable in serum but levels did not correlate with BAL levels in the same individuals (r2 = 0.04, p = 0.14) or with inflammatory markers. YKL-40 was below the limit of detection in urine (30 pg/ml).
This study demonstrates that levels of the chitinase-like protein YKL-40 reflect airway inflammation and infection in early CF lung disease. The lack of increased YKL-40 in serum in the absence of systemic inflammation limits the benefit of this potential biomarker in early disease.
Cystic fibrosis; YKL-40; Biomarker; Lung disease
YKL-40 (chitinase 3-like protein 1) is expressed in a broad spectrum of inflammatory conditions and cancers. We have previously reported that YKL-40 levels are elevated in the cerebrospinal fluid (CSF) of macaques and humans with lentiviral encephalitis, as well as multiple sclerosis (MS). The current study assessed temporal CSF YKL-40 levels in subjects with severe traumatic brain injury (TBI; Glasgow Coma Scale [GCS] score ≤8). We also evaluated temporal expression of YKL-40 after parasagittal controlled cortical impact (CCI) injury over the parietal cortex (2.8 mm deep, 4 m/sec). We demonstrate that CSF YKL-40 levels are elevated after acute TBI, and that YKL-40 levels are higher in patients who died following injury than in patients who survived. YKL-40 levels significantly correlate with CSF levels of inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), as well as the inflammatory marker C-reactive protein (CRP). After CCI, in situ hybridization (ISH) showed that YKL-40 transcription is primarily associated with reactive astrocytes in pericontusional cortex. Tissue YKL-40 transcription time course analysis after CCI showed that YKL40 transcription in astrocytes began 1 day after injury, remained elevated for several days, and then declined by day 12. Similarly to our temporal CSF measurements in humans, YKL-40 induction after CCI is coincident with IL-1β expression. Taken together these findings demonstrate that YKL-40 is induced in astrocytes during acute neuroinflammation, is temporally related to inflammatory mediator expression, and may be a useful biomarker for understanding secondary injury and for patient prognosis.
chitinase; controlled cortical impact; cytokine; gliosis; neuroinflammation; traumatic brain injury; YKL-40
Chitinase 3-like 1 (CHI3L1 or YKL40) is a secreted glycoprotein highly expressed in tumours from patients with advanced stage cancers, including prostate cancer (PCa). The exact function of YKL40 is poorly understood, but it has been shown to play an important role in promoting tumour angiogenesis and metastasis. The therapeutic value and biological function of YKL40 are unknown in PCa. The objective of this study was to examine the expression and function of YKL40 in PCa. Gene expression analysis demonstrated that YKL40 was highly expressed in metastatic PCa cells when compared with less invasive and normal prostate epithelial cell lines. In addition, the expression was primarily limited to androgen receptor-positive cell lines. Evaluation of YKL40 tissue expression in PCa patients showed a progressive increase in patients with aggressive disease when compared with those with less aggressive cancers and normal controls. Treatment of LNCaP and C4-2B cells with androgens increased YKL40 expression, whereas treatment with an anti-androgen agent decreased the gene expression of YKL40 in androgen-sensitive LNCaP cells. Furthermore, knockdown of YKL40 significantly decreased invasion and migration of PCa cells, whereas overexpression rendered them more invasive and migratory, which was commensurate with an enhancement in the anchorage-independent growth of cells. To our knowledge, this study characterises the role of YKL40 for the first time in PCa. Together, these results suggest that YKL40 plays an important role in PCa progression and thus inhibition of YKL40 may be a potential therapeutic strategy for the treatment of PCa.
YKL40; prostate cancer; cell migration; cell invasion; metastasis; targeted therapy
Prolonged exposure to hyperoxia in neonates can cause hyperoxic acute lung injury (HALI), which is characterized by increased pulmonary permeability and diffuse infiltration of various inflammatory cells. Disruption of the epithelial barrier may lead to altered pulmonary permeability and maintenance of barrier properties requires intact epithelial tight junctions (TJs). However, in neonatal animals, relatively little is known about how the TJ proteins are expressed in the pulmonary epithelium, including whether expression of TJ proteins is regulated in response to hyperoxia exposure. This study determines whether changes in tight junctions play an important role in disruption of the pulmonary epithelial barrier during hyperoxic acute lung injury.
Newborn rats, randomly divided into two groups, were exposed to hyperoxia (95% oxygen) or normoxia for 1–7 days, and the severity of lung injury was assessed; location and expression of key tight junction protein occludin and ZO-1 were examined by immunofluorescence staining and immunobloting; messenger RNA in lung tissue was studied by RT-PCR; transmission electron microscopy study was performed for the detection of tight junction morphology.
We found that different durations of hyperoxia exposure caused different degrees of lung injury in newborn rats. Treatment with hyperoxia for prolonged duration contributed to more serious lung injury, which was characterized by increased wet-to-dry ratio, extravascular lung water content, and bronchoalveolar lavage fluid (BALF):serum FD4 ratio. Transmission electron microscopy study demonstrated that hyperoxia destroyed the structure of tight junctions and prolonged hyperoxia exposure, enhancing the structure destruction. The results were compatible with pathohistologic findings. We found that hyperoxia markedly disrupted the membrane localization and downregulated the cytoplasm expression of the key tight junction proteins occludin and ZO-1 in the alveolar epithelium by immunofluorescence. The changes of messenger RNA and protein expression of occludin and ZO-1 in lung tissue detected by RT-PCR and immunoblotting were consistent with the degree of lung injury.
These data suggest that the disruption of the pulmonary epithelial barrier induced by hyperoxia is, at least in part, due to massive deterioration in the expression and localization of key TJ proteins.
Acute lung injury; Hyperoxia; Newborn; Permeability; Tight Junction
Large-tidal volume (VT) mechanical ventilation and hyperoxia used in patients with acute respiratory distress syndrome can damage pulmonary epithelial cells through lung inflammation and apoptotic cell death. Hyperoxia has been shown to increase ventilator-induced lung injury, but the mechanisms regulating interaction between large VT and hyperoxia are unclear. We hypothesized that the addition of hyperoxia to large-VT ventilation would increase neutrophil infiltration by upregulation of the cytokine macrophage inflammatory protein-2 (MIP-2) and would increase apoptosis via the mitogen-activated protein kinase pathways.
C57BL/6 mice were exposed to high-VT (30 ml/kg) mechanical ventilation with room air or hyperoxia for one to five hours.
The addition of hyperoxia to high-VT ventilation augmented lung injury, as demonstrated by increased apoptotic cell death, neutrophil migration into the lung, MIP-2 production, MIP-2 mRNA expression, increased DNA binding activity of activator protein-1, increased microvascular permeability, and c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2 activation. Hyperoxia-induced augmentation of high-VT-induced lung injury was attenuated in JNK-deficient mice and in mice with pharmacologic inhibition of ERK activity by PD98059. However, only JNK-deficient mice, and not mice with ERK activity inhibition by PD98059, were protected from high-VT-induced lung injury without hyperoxia.
We conclude that hyperoxia increased high-VT-induced cytokine production, neutrophil influx, and apoptotic cell death through activation of the JNK and ERK1/2 pathways.
Earlier studies have reported that transforming growth factor beta 1(TGFβ1) is a critical mediator of hyperoxia-induced acute lung injury (HALI) in developing lungs, leading to impaired alveolarization and a pulmonary phenotype of bronchopulmonary dysplasia (BPD). However, the mechanisms responsible for the TGFβ1-induced inflammatory signals that lead to cell death and abnormal alveolarization are poorly understood. We hypothesized that TGFβ1 signaling via TGFβR2 is necessary for the pathogenesis of the BPD pulmonary phenotype resulting from HALI.
We utilized lung epithelial cell-specific TGFβ1 overexpressing transgenic and TGFβR2 null mutant mice to evaluate the effects on neonatal mortality as well as pulmonary inflammation and apoptosis in developing lungs. Lung morphometry was performed to determine the impaired alveolarization and multicolor flow cytometry studies were performed to detect inflammatory macrophages and monocytes in lungs. Apoptotic cell death was measured with TUNEL assay, immunohistochemistry and western blotting and protein expression of angiogenic mediators were also analyzed.
Our data reveals that increased TGFβ1 expression in newborn mice lungs leads to increased mortality, macrophage and immature monocyte infiltration, apoptotic cell death specifically in Type II alveolar epithelial cells (AECs), impaired alveolarization, and dysregulated angiogenic molecular markers.
Our study has demonstrated the potential role of inhibition of TGFβ1 signaling via TGFβR2 for improved survival, reduced inflammation and apoptosis that may provide insights for the development of potential therapeutic strategies targeted against HALI and BPD.
Transforming growth factor; Oxygen; Inflammation; Cell death; Angiopoietin; Newborn; Pulmonary; Bronchopulmonary dysplasia
The aim of this study was to investigate the hepatoprotective effect of BRP, a polysaccharide fraction isolated from Boschniakia rossica, against galactosamine and lipopolysaccharide induced fulminant hepatic failure. Mice were injected with a single dose of galactosamine/lipopolysaccharide with or without pretreatment of BRP. Results showed marked reduction of hepatic necrosis, serum marker enzymes and levels of tumor necrosis factor-α and interleukin-6 in BRP pretreated mice when compared with galactosamine/lipopolysaccharide-challenged mice. Mice pretreated with BRP decreased the activation of caspases-3 and caspase-8, and showed a reduced level of DNA fragmentation of liver cells. BRP also reduced hepatic lipid peroxidation, increased potential of hepatic antioxidative defense system, and reduced hepatic nitric oxide level which was elevated by galactosamine/lipopolysaccharide injection. Immunoblot analysis showed down-regulation of inducible nitric oxide synthase and cyclooxygenase-2 proteins of liver tissues in BRP pretreated group when compared with galactosamine/lipopolysaccharide-challenged group. Furthermore, treatment with galactosamine/lipopolysaccharide markedly increased toll-like receptor 4, nuclear level of nuclear factor-κB, and phosphorylation of both extracellular signal-regulated kinase and c-Jun N-terminal kinase in liver tissues. However, these increases were attenuated by pretreatment with BRP. The results suggest that BRP alleviates galactosamine/lipopolysaccharide-induced liver injury by enhancing antioxidative defense system, suppressing inflammatory responses and reducing apoptotic signaling.
Boschniakia rossica; polysaccharide; hepatic failure; mice
Rationale: Acute lung injury and the acute respiratory distress syndrome are characterized by increased lung oxidant stress and apoptotic cell death. The contribution of epithelial cell apoptosis to the development of lung injury is unknown.
Objectives: To determine whether oxidant-mediated activation of the intrinsic or extrinsic apoptotic pathway contributes to the development of acute lung injury.
Methods: Exposure of tissue-specific or global knockout mice or cells lacking critical components of the apoptotic pathway to hyperoxia, a well-established mouse model of oxidant-induced lung injury, for measurement of cell death, lung injury, and survival.
Measurements and Main Results: We found that the overexpression of SOD2 prevents hyperoxia-induced BAX activation and cell death in primary alveolar epithelial cells and prolongs the survival of mice exposed to hyperoxia. The conditional loss of BAX and BAK in the lung epithelium prevented hyperoxia-induced cell death in alveolar epithelial cells, ameliorated hyperoxia-induced lung injury, and prolonged survival in mice. By contrast, Cyclophilin D–deficient mice were not protected from hyperoxia, indicating that opening of the mitochondrial permeability transition pore is dispensable for hyperoxia-induced lung injury. Mice globally deficient in the BH3-only proteins BIM, BID, PUMA, or NOXA, which are proximal upstream regulators of BAX and BAK, were not protected against hyperoxia-induced lung injury suggesting redundancy of these proteins in the activation of BAX or BAK.
Conclusions: Mitochondrial oxidant generation initiates BAX- or BAK-dependent alveolar epithelial cell death, which contributes to hyperoxia-induced lung injury.
cell death; epithelium; Bcl-2 proteins; acute respiratory distress syndrome
Human Glyco_18 domain-containing proteins constitute a family of chitinases and chitinase-like proteins. Chitotriosidase and AMCase are true enzymes which hydrolyse chitin and have a C-terminal chitin-binding domain. YKL-40, YKL-39, SI-CLP and murine YM1/2 proteins possess solely Glyco_18 domain and do not have the hydrolytic activity. The major sources of Glyco_18 containing proteins are macrophages, neutrophils, epithelial cells, chondrocytes, synovial cells, and cancer cells. Both macrophages and neutrophils use the regulated secretory mechanism for the release of Glyco_18 containing proteins. Glyco_18 containing proteins are established biomarkers for human diseases. Chitotriosidase is overproduced by lipid-laden macrophages and is a major marker for the inherited lysosomal storage Gaucher disease. AMCase and murine lectin YM1 are upregulated in Th2-environment, and enzymatic activity of AMCase contributes to asthma pathogenesis. YKL proteins act as soluble mediators for the cell proliferation and migration, and are also involved in rheumatoid arthritis, inflammatory bowel disease, hepatic fibrosis and cirrhosis. Chitotriosidase and YKL-40 reflect the macrophage activation in atherosclerotic plaques. Serum level of YKL-40 is a diagnostic and prognostic marker for numerous types of solid tumors. YKL-39 is a marker for the activation of chondrocytes and the progression of the osteoarthritis in human. Recently identified SI-CLP is upregulated by Th2 cytokine IL-4 as well as by glucocorticoids. This unique feature of SI-CLP makes it an attractive candidate for the examination of individual sensitivity of patients to glucocorticoid treatment and prediction of side effects of glucocorticoid therapy. Human chitinases and chitinase-like proteins are found in tissues and circulation, and can be detected by non-invasive technologies.
Glyco_18 Domain; Macrophage; Tumor; Arthritis; Gaucher Disease; Asthma
YKL-39 is a Glyco_18 domain containing chitinase-like protein which is currently recognized as a biomarker for the activation of chondrocytes and the progress of the osteoarthritis in human. YKL-39 was identified as an abundantly secreted protein in primary culture of human articular chondrocytes. Two biological activities of YKL-39 might contribute to the disease progression. One is the induction of autoimmune response and second is the participation in tissue remodeling. Other mammalian chitinase-like proteins including chitotriosidase, SI-CLP, YKL-40 and YM1 are expressed by macrophages in various pathological conditions. In contrast, YKL-39 was never reported to be produced by macrophages. We used in vitro model of human monocyte-derived macrophage differentiation to analyse regulation of YKL-39 expression. Expression of YKL-39 was examined by real-time RT-PCR. CD14+ MACS sorted human monocytes differentiated for 6 days under different stimulations including IFNγ, IL-4, dexamethasone and TGF-β. We found that both IL-4 and TGF-β have weak stimulatory effect on YKL-39 expression in all donors tested (3.2 ± 1.7 fold, p = 0.006 and 6.3 ± 3.1 fold, p = 0.014 respectively). However the combination of IL-4 and TGF-β had strong stimulatory effect on the expression of YKL-39 in all analysed individual macrophage cultures (34 ± 36 fold, p = 0.05). IFN-γ did not show statistically significant effect of YKL-39 mRNA expression. Presence of dexamethasone almost completely abolished the stimulatory effects of IL-4 and TGF-β. In summary, we show here for the first time, that human cells of monocyte origin are able to produce YKL-39. Maturation of monocyte derived macrophages in the presence of Th2 cytokine IL-4 and TGF-β leads to the strong activation of YKL-39 expression. Thus elevated levels of YKL-39 observed during chronic inflammations can not be attributed solely to the activity of chondrocytes. In perspective, YKL-39 might serve as a useful biomarker to detect macrophage-specific response in pathologies like tumour, atherosclerosis and Alzheimer disease.
osteoarthritis; chitinase; YKL-39; macrophage; TGF-beta; IL-4
YKL-40 (chitinase-3-like-1) is a member of "mammalian chitinase-like proteins". The protein is expressed in many types of cancer cells and the highest plasma YKL-40 levels have been found in patients with metastatic disease, short recurrence/progression-free intervals, and short overall survival. The aim of the study was to determine the expression of YKL-40 in tumor tissue and plasma in patients with borderline ovarian tumor or epithelial ovarian cancer (OC), and investigate prognostic value of this marker.
YKL-40 protein expression was determined by immunohistochemistry in tissue arrays from 181 borderline tumors and 473 OC. Plasma YKL-40 was determined by ELISA in preoperative samples from 19 patients with borderline tumor and 76 OC patients.
YKL-40 protein expression was found in cancer cells, tumor associated macrophages, neutrophils and mast cells. The tumor cell expression was higher in OC than in borderline tumors (p = 0.001), and associated with FIGO stage (p < 0.0001) and histological subtype (p = 0.0009). Positive YKL-40 expression (≥ 5% staining) was not associated with reduced survival. Plasma YKL-40 was also higher in patients with OC than in patients with borderline tumors (p < 0.0001), and it was positively correlated to serum CA-125 (p < 0.0001) and FIGO stage (p = 0.0001). Univariate Cox analysis of plasma YKL-40 showed association with overall survival (p < 0.0001). Multivariate Cox analysis, including plasma YKL-40, serum CA125, FIGO stage, age and radicality after primary surgery as variables, showed that elevated plasma YKL-40 was associated with a shorter survival (HR = 2.13, 95% CI: 1.40–3.25, p = 0.0004).
YKL-40 in OC tissue and plasma are related to stage and histology, but only plasma YKL-40 is a prognostic biomarker in patients with OC.
A translational study in renal transplantation suggested YKL-40, a chitinase 3-like-1 gene product, plays an important role in acute kidney injury (AKI) and repair, but data are lacking about this protein in urine from native human kidneys.
This is an ancillary study to a single-center, prospective observational cohort of patients with clinically-defined AKI according to AKI Network serum creatinine criteria. We determined the association of YKL -40 ≥ 5 ng/ml, alone or combined with neutrophil gelatinase-associated lipocalin (NGAL), in urine collected on the first day of AKI with a clinically important composite outcome (progression to higher AKI stage and/or in-hospital death).
YKL-40 was detectable in all 249 patients, but urinary concentrations were considerably lower than in previously measured deceased-donor kidney transplant recipients. Seventy-two patients (29%) progressed or died in-hospital, and YKL-40 ≥ 5 ng/ml had an adjusted odds ratio (95% confidence interval) for the outcome of 3.4 (1.5-7.7). The addition of YKL-40 to a clinical model for predicting the outcome resulted in a continuous net reclassification improvement of 29% (P = 0.04). In patients at high risk for the outcome based on NGAL concentrations in the upper quartile, YKL-40 further partitioned the cohort into moderate-risk and very high-risk groups.
Urine YKL-40 is associated with AKI progression and/or death in hospitalized patients and improves clinically determined risk reclassification. Combining YKL-40 with other AKI biomarkers like NGAL may further delineate progression risk, though additional studies are needed to determine whether YKL-40 has general applicability and to define its association with longer-term outcomes in AKI.
Acute kidney injury; Biomarker; BRP-39; Chitinase 3-like-1; Net reclassification improvement; YKL-40
Epithelial cell death plays a critical role in hyperoxia-induced lung injury. We investigated the involvement of the autophagic marker microtubule-associated protein-1 light chain-3B (LC3B) in epithelial cell apoptosis after hyperoxia. Prolonged hyperoxia (>95% O2), which causes characteristic lung injury in mice, activated morphological and biochemical markers of autophagy. Hyperoxia induced the time-dependent expression and conversion of LC3B-I to LC3B-II in mouse lung in vivo and in cultured epithelial cells (Beas-2B, human bronchial epithelial cells) in vitro. Hyperoxia increased autophagosome formation in Beas-2B cells, as evidenced by electron microscopy and increased GFP-LC3 puncta. The augmented LC3B level after hyperoxia was transcriptionally regulated and dependent in part on the c-Jun N-terminal kinase pathway. We hypothesized that LC3B plays a regulatory role in hyperoxia-induced epithelial apoptosis. LC3B siRNA promoted hyperoxia-induced cell death in epithelial cells, whereas overexpression of LC3B conferred cytoprotection after hyperoxia. The autophagic protein LC3B cross-regulated the Fas apoptotic pathway by physically interacting with the components of death-inducing signaling complex. This interaction was mediated by caveolin-1 tyrosine 14, which is a known target of phosphorylation induced by hyperoxia. Taken together, hyperoxia-induced LC3B activation regulates the Fas apoptotic pathway and thus confers cytoprotection in lung epithelial cells. The interaction of LC3B and Fas pathways requires cav-1.
apoptosis; autophagy; hyperoxia; lung injury; caveolin-1