Limb-girdle muscular dystrophy type 2B causes HDL-C abnormalities in patients and statin-resistant muscle wasting in dysferlin-deficient mice.

Limb-girdle muscular dystrophy (MD) type 2B (LGMD2B) and Duchenne MD (DMD) are caused by mutations to the Dysferlin and Dystrophin genes, respectively. We have recently demonstrated in typically mild dysferlin- and dystrophin-deficient mouse models that increased plasma cholesterol levels severely exacerbate muscle wasting, and that DMD patients display primary dyslipidemia characterized by elevated plasma cholesterol and triglycerides. Herein, we investigate lipoprotein abnormalities in LGMD2B and if statin therapy protects dysferlin-deficient mice (Dysf) from muscle damage. Herein, lipoproteins and liver enzymes from LGMD2B patients and dysferlin-null (Dysf) mice were analyzed. Simvastatin, which exhibits anti-muscle wasting effects in mouse models of DMD and corrects aberrant expression of key markers of lipid metabolism and endogenous cholesterol synthesis, was tested in Dysf mice. Muscle damage and fibrosis were assessed by immunohistochemistry and cholesterol signalling pathways via Western blot. LGMD2B patients show reduced serum high-density lipoprotein cholesterol (HDL-C) levels compared to healthy controls and exhibit a greater prevalence of abnormal total cholesterol (CHOL)/HDL-C ratios despite an absence of liver dysfunction. While Dysf mice presented with reduced CHOL and associated HDL-C and LDL-C-associated fractions, simvastatin treatment did not prevent muscle wasting in quadriceps and triceps muscle groups or correct aberrant low-density lipoprotein receptor (LDLR) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) protein expression. LGMD2B patients present with reduced serum concentrations of HDL-C, a major metabolic comorbidity, and as a result, statin therapy is unlikely to prevent muscle wasting in this population. We propose that like DMD, LGMD2B should be considered as a new type of genetic dyslipidemia.

Losartan metabolite EXP3179 is a unique blood pressure-lowering AT1R antagonist with direct, rapid endothelium-dependent vasoactive properties.

BACKGROUND AND PURPOSE: Losartan is an anti-hypertensive angiotensin II (ANGII) type 1 receptor (AT1R) blocker (ARB) with many unexpected therapeutic properties, even in non-blood pressure (BP)-related diseases. Administered as a prodrug, losartan undergoes serial metabolism into EXP3179, a metabolite alleged to lack AT1R-blocking properties, and EXP3174, the dominant AT1R antagonist. Having observed that losartan can decrease vascular tone in mice with low AT1R expression and inhibit Marfan aortic widening at very high doses, we investigated whether EXP3179 may have unique, AT1R-independent effects on vascular tone and endothelial function. EXPERIMENTAL APPROACH: We compared the AT1R blocking capabilities of EXP3179 and EXP3174 using AT1R-expressing cell lines. Their BP lowering and vasoactive properties were studied in normal, hypertensive and transgenic rodents, and ex vivo wire myography. KEY RESULTS: We observed that both EXP3179 and EXP3174 can fully block (100%) AT1R signaling in vitro and significantly decrease BP in normotensive and spontaneously hypertensive rats. Only EXP3179 prevented PE-induced contraction by up to 65% (p < 0.01) in L-NAME and endothelium removal-sensitive fashion. Use of transgenic mice revealed that these effects involve the eNOS/caveolin-1 axis and the endothelium-dependent hyperpolarization factor (EDHF). CONCLUSION AND IMPLICATIONS: We provide direct structure-activity evidence that EXP3179 is a BP-lowering AT1R blocker with unique endothelial function-enhancing properties not shared with losartan or EXP3174. The major pharmacological effects of losartan in patients are therefore likely more complex than simple blockade of AT1R by EXP3174, which helps rationalize its therapeutic and prophylactic properties, especially at very high doses. Reports relying on EXP3179 as an AT1R-independent losartan analogue may require careful re-evaluation.

Mitochondrial dysfunction contributes to the senescent phenotype of IPF lung fibroblasts.

Increasing evidence highlights that senescence plays an important role in idiopathic pulmonary fibrosis (IPF). This study delineates the specific contribution of mitochondria and the superoxide they form to the senescent phenotype of lung fibroblasts from IPF patients (IPF-LFs). Primary cultures of IPF-LFs exhibited an intensified DNA damage response (DDR) and were more senescent than age-matched fibroblasts from control donors (Ctrl-LFs). Furthermore, IPF-LFs exhibited mitochondrial dysfunction, exemplified by increases in mitochondrial superoxide, DNA, stress and activation of mTORC1. The DNA damaging agent etoposide elicited a DDR and augmented senescence in Ctrl-LFs, which were accompanied by disturbances in mitochondrial homoeostasis including heightened superoxide production. However, etoposide had no effect on IPF-LFs. Mitochondrial perturbation by rotenone involving sharp increases in superoxide production also evoked a DDR and senescence in Ctrl-LFs, but not IPF-LFs. Inhibition of mTORC1, antioxidant treatment and a mitochondrial targeting antioxidant decelerated IPF-LF senescence and/or attenuated pharmacologically induced Ctrl-LF senescence. In conclusion, increased superoxide production by dysfunctional mitochondria reinforces lung fibroblast senescence via prolongation of the DDR. As part of an auto-amplifying loop, mTORC1 is activated, altering mitochondrial homoeostasis and increasing superoxide production. Deeper understanding the mechanisms by which mitochondria contribute to fibroblast senescence in IPF has potentially important therapeutic implications.

Functional Toll-Like Receptor 9 Expression and CXCR3 Ligand Release in Pulmonary Sarcoidosis.

Sarcoidosis is a granulomatous disease characterized by a T-helper type 1 (Th1) cell-dominated alveolitis. As a role of bacteria in the pathogenesis of sarcoidosis has been discussed, Toll-like receptors (TLRs) may be involved in the initiation of a first immune reaction. We analyzed expression and functional relevance of several TLRs in bronchoalveolar lavage (BAL) cells from patients with pulmonary sarcoidosis. In parallel, we determined the release of C-X-C motif chemokine 9 (CXCL9), CXCL10, and CXCL11 by BAL cells from patients with pulmonary sarcoidosis. Nucleotide-binding oligomerization domain-containing protein (NOD) 1 and 2, TLR2, TLR6, and TLR9 expression by BAL cells was analyzed by real-time RT-PCR and cell surface expression by flow cytometry. Chemokine release was measured in BAL cell culture supernatants by ELISA. We found increased TLR9 mRNA expression in patients with sarcoidosis with chest X-ray type I and II and TLR9 protein expression in BAL cells from patients with chest X-ray type II and III. Stimulation with CpG nucleotides increased CXCL10 release by BAL cells from patients with sarcoidosis type II significantly compared with control subjects or other patients with sarcoidosis. In contrast, no increase in TNF, IL-12p40, or CXCL8 was detected. Spontaneous release of CXCL10, but not CXCL9 or CXCL11, by cultured BAL cells was also highest in cells from patients with chest X-ray type II. We found a significant association between TLR9 expression and CD4+ lymphocytes in BAL. Our data demonstrate that TLR9 ligands may contribute to the immunopathogenesis of sarcoidosis via induction of CXCL10 release in the alveolar macrophages.

miR-638 regulates gene expression networks associated with emphysematous lung destruction.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease characterized by varying degrees of emphysematous lung destruction and small airway disease, each with distinct effects on clinical outcomes. There is little known about how microRNAs contribute specifically to the emphysema phenotype. We examined how genome-wide microRNA expression is altered with regional emphysema severity and how these microRNAs regulate disease-associated gene expression networks. METHODS: We profiled microRNAs in different regions of the lung with varying degrees of emphysema from 6 smokers with COPD and 2 controls (8 regions × 8 lungs = 64 samples). Regional emphysema severity was quantified by mean linear intercept. Whole genome microRNA and gene expression data were integrated in the same samples to build co-expression networks. Candidate microRNAs were perturbed in human lung fibroblasts in order to validate these networks. RESULTS: The expression levels of 63 microRNAs (P < 0.05) were altered with regional emphysema. A subset, including miR-638, miR-30c, and miR-181d, had expression levels that were associated with those of their predicted mRNA targets. Genes correlated with these microRNAs were enriched in pathways associated with emphysema pathophysiology (for example, oxidative stress and accelerated aging). Inhibition of miR-638 expression in lung fibroblasts led to modulation of these same emphysema-related pathways. Gene targets of miR-638 in these pathways were amongst those negatively correlated with miR-638 expression in emphysema. CONCLUSIONS: Our findings demonstrate that microRNAs are altered with regional emphysema severity and modulate disease-associated gene expression networks. Furthermore, miR-638 may regulate gene expression pathways related to the oxidative stress response and aging in emphysematous lung tissue and lung fibroblasts.

A gene expression signature of emphysema-related lung destruction and its reversal by the tripeptide GHK.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease consisting of emphysema, small airway obstruction, and/or chronic bronchitis that results in significant loss of lung function over time. METHODS: In order to gain insights into the molecular pathways underlying progression of emphysema and explore computational strategies for identifying COPD therapeutics, we profiled gene expression in lung tissue samples obtained from regions within the same lung with varying amounts of emphysematous destruction from smokers with COPD (8 regions × 8 lungs = 64 samples). Regional emphysema severity was quantified in each tissue sample using the mean linear intercept (Lm) between alveolar walls from micro-CT scans. RESULTS: We identified 127 genes whose expression levels were significantly associated with regional emphysema severity while controlling for gene expression differences between individuals. Genes increasing in expression with increasing emphysematous destruction included those involved in inflammation, such as the B-cell receptor signaling pathway, while genes decreasing in expression were enriched in tissue repair processes, including the transforming growth factor beta (TGFß) pathway, actin organization, and integrin signaling. We found concordant differential expression of these emphysema severity-associated genes in four cross-sectional studies of COPD. Using the Connectivity Map, we identified GHK as a compound that can reverse the gene-expression signature associated with emphysematous destruction and induce expression patterns consistent with TGFß pathway activation. Treatment of human fibroblasts with GHK recapitulated TGFß-induced gene-expression patterns, led to the organization of the actin cytoskeleton, and elevated the expression of integrin ß1. Furthermore, addition of GHK or TGFß restored collagen I contraction and remodeling by fibroblasts derived from COPD lungs compared to fibroblasts from former smokers without COPD. CONCLUSIONS: These results demonstrate that gene-expression changes associated with regional emphysema severity within an individual's lung can provide insights into emphysema pathogenesis and identify novel therapeutic opportunities for this deadly disease. They also suggest the need for additional studies to examine the mechanisms by which TGFß and GHK each reverse the gene-expression signature of emphysematous destruction and the effects of this reversal on disease progression.

STAT3-mediated signaling dysregulates lung fibroblast-myofibroblast activation and differentiation in UIP/IPF.

STAT3 is a latent transcription factor that plays a role in regulating fibroblast function in fibrotic lung diseases. To further understand the role of STAT3 in the phenotypic divergence and function of human lung fibroblasts (LFs), we investigated the effect of basal and cytokine-induced STAT3 activity on indices of LF differentiation and activation, including expression of α-smooth muscle actin (α-SMA), collagen, and adhesion molecules Thy-1/CD90 and α(v) ß(3) and ß(5) integrins. We identified a population of fibroblasts from usual interstitial pneumonia (UIP)/idiopathic pulmonary fibrosis (IPF) lungs characterized by constitutively phosphorylated STAT3, lower proliferation rates, and diminished expression of α-SMA, Thy-1/CD90, and ß(3) integrins compared with control LFs. Staining of UIP lung biopsy specimens demonstrated that phosphorylated STAT3 was not present in α-SMA-positive fibroblastic foci but was observed in the nuclei of cells located in the areas of dense fibrosis. STAT3 activation in LFs did not significantly influence basal or transforming growth factor ß(1)-induced collagen I expression but inhibited expression of α-SMA, Thy-1/CD90, and αv ß(3) integrins. Suppression of STAT3 signaling diminished resistance of IPF LFs to staurosporine-induced apoptosis and responsiveness to transforming growth factor ß(1) but increased basal α-SMA and restored ß(3) integrin expression in LFs via an ALK-5-dependent, SMAD3/7-independent mechanism. These data suggest that STAT3 activation regulates several pathways in human LFs associated with normal wound healing, whereas aberrant STAT3 signaling plays a critical role in UIP/IPF pathogenesis.

Granzyme K activates protease-activated receptor-1.

Granzyme K (GrK) is a trypsin-like serine protease that is elevated in patients with sepsis and acute lung inflammation. While GrK was originally believed to function exclusively as a pro-apoptotic protease, recent studies now suggest that GrK may possess other non-cytotoxic functions. In the context of acute lung inflammation, we hypothesized that GrK induces pro-inflammatory cytokine release through the activation of protease-activated receptors. The direct effect of extracellular GrK on PAR activation, intracellular signaling and cytokine was assessed using cultured human lung fibroblasts. Extracellular GrK induced secretion of IL-6, IL-8 and MCP-1 in a dose- and time-dependent manner in lung fibroblasts. Heat-inactivated GrK did not induce cytokine release indicating that protease activity is required. Furthermore, GrK induced activation of both the ERK1/2 and p38 MAP kinase signaling pathways, and significantly increased fibroblast proliferation. Inhibition of ERK1/2 abrogated the GrK-mediated cytokine release. Through the use of PAR-1 and PAR-2 neutralizing antibodies, it was determined that PAR-1 is essential for GrK-induced IL-6, IL-8 and MCP-1 release. In summary, extracellular GrK is capable of activating PAR-1 and inducing fibroblast cytokine secretion and proliferation.

Minimally invasive imaging method based on second harmonic generation and multiphoton excitation fluorescence in translational respiratory research.

For translational respiratory research including in the development of clinical diagnostic tools, a minimally invasive imaging method, which can provide both cellular and extracellular structural details with sufficient specificity, sensitivity and spatial resolution, is particularly useful. Multiphoton microscopy causes excitation of endogenously fluorescent macromolecular systems and induces highly specific second harmonic generation signals from non-centrosymmetric macromolecules such as fibrillar collagens. Both these signals can be captured simultaneously to provide spatially resolved 3D structural organization of extracellular matrix as well as the cellular morphologies in their native states. Besides briefly discussing the fundamentals of multiphoton excitation fluorescence and harmonic generation signals and the instrumentation details, this review focuses on the specific applications of these imaging modalities in lung structural imaging, particularly morphological features of alveolar structures, visualizing and quantifying extracellular matrix remodelling accompanying emphysematous destructions as well as the IPF, detecting lung cancers and the potential use in the tissue engineering applications.

Alternatively activated alveolar macrophages in pulmonary fibrosis-mediator production and intracellular signal transduction.

Activated macrophages have been characterized as M1 and M2 according to their inflammatory response pattern. Here we analyzed the M2 marker expression and intracellular signal transduction in the course of cytokine-driven differentiation. We found elevated spontaneous production of the chemokines CCL17, CCL18 and CCL22 and increased expression of CD206 by alveolar macrophages from patients with lung fibrosis. Stimulation of normal human AM with Th2 cytokines IL-4 and/or IL-10 in vitro revealed IL-4 as the most powerful inducer of M2-phenotype in AM and monocytes. Importantly, IL-10 enhanced IL-4-induced expression of CCL18 and IL-1RA in a synergistic fashion. IL-4/IL-10 stimulation induces a strong activation of STAT3 in AM from fibrosis patients. These results suggest an important role for M2 polarized AM in the pathogenesis of pulmonary fibrosis and indicate that both IL-4 and IL-10 account for human AM phenotype shift to M2, as seen in patients with fibrotic interstitial lung diseases.

Human lung parenchyma but not proximal bronchi produces fibroblasts with enhanced TGF-beta signaling and alpha-SMA expression.

Given the contribution various fibroblast subsets make to wound healing and tissue remodeling, the concept of lung fibroblast heterogeneity is of great interest. However, the mechanisms contributing to this heterogeneity are unknown. To this aim, we compared molecular and biophysical characteristics of fibroblasts concurrently isolated from normal human proximal bronchi (B-FBR) and distal lung parenchyma (P-FBR). Using quantitative RT-PCR, spontaneous expression of more than 30 genes related to repair and remodeling was analyzed. All P-FBR lines demonstrated significantly increased basal α-smooth muscle actin (α-SMA) mRNA and protein expression levels when compared with donor-matched B-FBR. These differences were not associated with sex, age, or disease history of lung tissue donors. In contrast to B-FBR, P-FBR displayed enhanced transforming growth factor (TGF)-ß/Smad signaling at baseline, and inhibition of either ALK-5 or neutralization of endogenously produced and activated TGF-ß substantially decreased basal α-SMA protein in P-FBR. Both B-FBR and P-FBR up-regulated α-SMA after stimulation with TGF-ß1, and basal expression levels of TGF-ß1, TGF-ßRI, and TGF-ßRII were not significantly different between fibroblast pairs. Blockade of metalloproteinase-dependent activation of endogenous TGF-ß did not significantly modify α-SMA expression in P-FBR. However, resistance to mechanical tension of these cells was significantly higher in comparison with B-FBR, and added TGF-ß1 significantly increased stiffness of both cell monolayers. Our data suggest that in contrast with human normal bronchial tissue explants, lung parenchyma produces mesenchymal cells with a myofibroblastic phenotype by intrinsic mechanisms of TGF-ß activation in feed-forward manner. These results also offer a new insight into mechanisms of human fibroblast heterogeneity and their function in the airway and lung tissue repair and remodeling.

Induction of epithelial-mesenchymal transition in primary airway epithelial cells from patients with asthma by transforming growth factor-beta1.

RATIONALE: Airway remodeling in asthma is associated with the accumulation of fibroblasts, the primary cell responsible for synthesis and secretion of extracellular matrix proteins. The process by which the number of fibroblasts increases in asthma is poorly understood, but epithelial-mesenchymal transition (EMT) may play a significant role. OBJECTIVES: To evaluate whether EMT occurs in primary airway epithelial cells (AECs), the mechanisms involved, and if this process is altered in asthmatic AECs. METHODS: AECs were obtained from subjects with asthma (n = 8) and normal subjects without asthma (n = 10). Monolayer and air-liquid interface-AEC (ALI-AEC) cultures were treated with transforming growth factor (TGF)-beta1 (10 ng/ml) for 72 hours and assayed for mesenchymal and epithelial markers using quantitative polymerase chain reaction, confocal microscopy, and immunoblot. The involvement of BMP-7, Smad3, and MAPK-mediated signaling were also evaluated. MEASUREMENTS AND MAIN RESULTS: TGF-beta1-induced EMT in AEC monolayers derived from subjects with asthma and normal donors. EMT was characterized by changes in cell morphology, increased expression of mesenchymal markers EDA-fibronectin, vimentin, alpha-smooth muscle actin, and collagen-1, and loss of epithelial markers E-cadherin and zonular occludin-1. Inhibition of TGF-beta1-induced signaling with Smad3-inhibiting siRNA or TGF-beta1-neutralizing antibodies prevented and reversed EMT, respectively, whereas BMP-7 had no effect. In ALI-AEC cultures derived from normal subjects, EMT was confined to basally situated cells, whereas in asthmatic ALI-AEC cultures EMT was widespread throughout the epithelium. CONCLUSIONS: TGF-beta1 induces EMT in a Smad3-dependent manner in primary AECs. However, in asthmatic-derived ALI-AEC cultures, the number of cells undergoing EMT is greater. These findings support the hypothesis that epithelial repair in asthmatic airways is dysregulated.

Characterization of side population cells from human airway epithelium.

The airway epithelium is the first line of contact with the inhaled external environment and is continuously exposed to and injured by pollutants, allergens, and viruses. However, little is known about epithelial repair and in particular the identity and role of tissue resident stem/progenitor cells that may contribute to epithelial regeneration. The aims of the present study were to identify, isolate, and characterize side population (SP) cells in human tracheobronchial epithelium. Epithelial cells were obtained from seven nontransplantable healthy lungs and four asthmatic lungs by pronase digestion. SP cells were identified by verapamil-sensitive efflux of the DNA-binding dye Hoechst 33342. Using flow cytometry, CD45(-) SP, CD45(+) SP, and non-SP cells were isolated and sorted. CD45(-) SP cells made up 0.12% +/- 0.01% of the total epithelial cell population in normal airway but 4.1% +/- 0.06% of the epithelium in asthmatic airways. All CD45(-) SP cells showed positive staining for epithelial-specific markers cytokeratin-5, E-cadherin, ZO-1, and p63. CD45(-) SP cells exhibited stable telomere length and increased colony-forming and proliferative potential, undergoing population expansion for at least 16 consecutive passages. In contrast with non-SP cells, fewer than 100 CD45(-) SP cells were able to generate a multilayered and differentiated epithelium in air-liquid interface culture. SP cells are present in human tracheobronchial epithelium, exhibit both short- and long-term proliferative potential, and are capable of generation of differentiated epithelium in vitro. The number of SP cells is significantly greater in asthmatic airways, providing evidence of dysregulated resident SP cells in the asthmatic epithelium. Disclosure of potential conflicts of interest is found at the end of this article.

Transforming growth factor beta1 induces alphavbeta3 integrin expression in human lung fibroblasts via a beta3 integrin-, c-Src-, and p38 MAPK-dependent pathway.

In response to transforming growth factor beta1 (TGFbeta) stimulation, fibroblasts modify their integrin repertoire and adhesive capabilities to certain extracellular matrix proteins. Although TGFbeta has been shown to increase the expression of specific alphav integrins, the mechanisms underlying this are unknown. In this study we demonstrate that TGFbeta1 increased both beta3 integrin subunit mRNA and protein levels as well as surface expression of alphavbeta3 in human lung fibroblasts. TGFbeta1-induced alphavbeta3 expression was strongly adhesion-dependent and associated with increased focal adhesion kinase and c-Src kinase phosphorylation. Inhibition of beta3 integrin activation by the Arg-Gly-Asp tripeptide motif-specific disintegrin echistatin or alphavbeta3 blocking antibody prevented the increase in beta3 but not beta5 integrin expression. In addition, echistatin inhibited TGFbeta1-induced p38 MAPK but not Smad3 activation. Furthermore, inhibition of the Src family kinases, but not focal adhesion kinase, completely abrogated TGFbeta1-induced expression of alphavbeta3 and p38 MAPK phosphorylation but not beta5 integrin expression and Smad3 activation. The TGFbeta1-induced alphavbeta3 expression was blocked by pharmacologic and genetic inhibition of p38 MAPK- but not Smad2/3-, Sp1-, ERK-, phosphatidylinositol 3-kinase, and NF-kappaB-dependent pathways. Our results demonstrate that TGFbeta1 induces alphavbeta3 integrin expression via a beta3 integrin-, c-Src-, and p38 MAPK-dependent pathway. These data identify a novel mechanism for TGFbeta1 signaling in human lung fibroblasts by which they may contribute to normal and pathological wound healing.

CCL18 as an indicator of pulmonary fibrotic activity in idiopathic interstitial pneumonias and systemic sclerosis.

OBJECTIVE: In diffuse parenchymal lung diseases, the evolution of pulmonary fibrosis is often devastating and may result in death. In this study the role of CCL18 as a biomarker of disease activity in idiopathic interstitial pneumonias (IIPs) and systemic sclerosis (SSc) with lung involvement was evaluated. METHODS: CCL18 was assessed in supernatants of cultured bronchoalveolar lavage (BAL) cells as well as BAL fluid and serum samples from 43 patients with IIPs, 12 patients with SSc, and 23 healthy control subjects. Concentrations of CCL18 were measured by enzyme-linked immunosorbent assay, and expression of CCL18 was assessed by flow cytometry. RESULTS: CCL18 concentrations were statistically significantly increased in all patients with fibrotic lung diseases. Spontaneous CCL18 production by BAL cells was negatively correlated with total lung capacity and the diffusion capacity for carbon monoxide, whereas there was a positive correlation of CCL18 concentrations with BAL neutrophil and eosinophil cell counts. Flow cytometry revealed an increase in the percentage of CCL18-positive alveolar macrophages and an increase in the CCL18 fluorescence intensity per cell in patients with fibrotic lung diseases. In a cohort of patients who were followed up for at least 6 months (n = 40), a close negative correlation was observed between changes in the predicted total lung capacity and changes in CCL18 serum concentrations. CONCLUSION: These findings suggest that CCL18 production by BAL cells and serum CCL18 concentrations reflect pulmonary fibrotic activity in patients with IIPs and those with SSc. Monitoring changes in CCL18 production might be an extraordinarily useful tool in clinical practice and in studies aimed at evaluating new approaches for treatment of fibrotic lung diseases.

IL-10-producing monocytes differentiate to alternatively activated macrophages and are increased in atopic patients.

BACKGROUND: Recently the immune regulatory role of T cell-derived IL-10 in allergic disease has been extensively studied. In contrast, there is mounting evidence that IL-10 might also have a role in the perpetuation of allergic inflammation and fibrotic remodeling. It has been reported that alternatively (IL-4) activated macrophages (aaMPhi) produce large quantities of IL-10 and lack IL-12 production. OBJECTIVE: Bearing this in mind, we hypothesized whether functionally different properties of IL-10-producing monocytes could be identified. METHODS: Intracellular cytokine expression of IL-10, IL-12, and IL-6 in peripheral blood CD14(+) monocytes was measured in 19 atopic patients and 18 healthy control subjects by means of flow cytometry. In addition, IL-10-secreting monocytes were sorted by means of flow cytometry. Capabilities of these cells regarding further differentiation, accessory cell capacity, and surface molecule expression were analyzed. RESULTS: Our data show a dichotomous expression pattern of either IL-10 or IL-12p40/p70 in peripheral blood monocytes after LPS stimulation. Compared with healthy control subjects, the percentage of IL-10-producing monocytes was significantly increased in atopic patients. IL-10-secreting monocytes were isolated by using an IL-10 secretion assay, and functional analysis of these sorted cells revealed that IL-10-secreting monocytes preferentially differentiate into suppressor of cytokine signaling 3 expressing aaMPhi, which perpetuate T(H)2 immune response. CONCLUSION: Our study shows the existence of an IL-10-producing monocyte subset, which is increased in atopic disease and which might facilitate allergic inflammation and fibrotic remodeling by differentiation into aaMPhi. CLINICAL IMPLICATIONS: Controlling aaMPhi in T(H)2-driven inflammatory processes might be a novel target for intervention strategies.

Endothelin-1 induces hypertrophy and inhibits apoptosis in human airway smooth muscle cells.

Endothelin-1 (ET-1), a G protein-coupled receptor-activating peptide, is increased in airway epithelium, plasma, and bronchoalveolar lavage fluid of asthmatic patients. We hypothesized that ET-1 may contribute to the increased airway smooth muscle mass found in severe asthma by inducing hypertrophy and inhibiting apoptosis of smooth muscle cells. To investigate this hypothesis, we determined that treatment of primary human bronchial smooth muscle cells with ET-1 dose dependently [10(-11)-10(-7) M] inhibited the apoptosis induced by serum withdrawal. ET-1 treatment also resulted in a significant increase in total protein synthesis, mediated through both ET(A) and ET(B) receptors, cell size, as well as increased expression of myosin heavy chain, alpha-smooth muscle actin, and calponin. ET-1-induced hypertrophy was accompanied by activation of JAK1/STAT-3 and MAPK1/2 (ERK1/2) cell signaling pathways. Inhibition of JAK1/STAT-3 pathways by piceatannol or ERK1/2 by the MAPK/ERK kinase 1/2 inhibitor U0126 blunted the increase in total protein synthesis. The hypertrophic effect of ET-1 was equivalent to that of the gp130 cytokine oncostatin M and greater than that induced by cardiotrophin-1. ET-1 induced release of IL-6 but not IL-11, leukemia inhibitory factor, oncostatin M, or cardiotrophin-1, although treatment of cells with IL-6 alone did not induce hypertrophy. These results suggest that ET-1 is a candidate mediator for the induction of increased smooth muscle mass in asthma and identify signaling pathways activated by this mediator.

A vicious circle of alveolar macrophages and fibroblasts perpetuates pulmonary fibrosis via CCL18.

RATIONALE: Recently, models of macrophage activation have been revised. Macrophages stimulated with Th2 cytokines have been classified as alternatively activated. OBJECTIVES: This article examines the expression and regulation of CC chemokine ligand 18 (CCL18), a marker of alternative activation, by human alveolar macrophages (AMs). METHODS: AM were obtained from bronchoalveolar lavage (BAL) fluid of patients with idiopathic pulmonary fibrosis, sarcoidosis, or hypersensitivity pneumonitis (n = 69) and healthy volunteers (n = 22). Expression of CCL18 was determined by quantitative reverse transcriptase-polymerase chain reaction, in situ hybridization, flow cytometry, and immunohistochemistry, respectively. MEASUREMENTS AND MAIN RESULTS: Spontaneous CCL18 production by BAL-derived cells was markedly increased in patients with pulmonary fibrosis and correlated negatively with pulmonary function test parameters. CCL18 gene expression and protein production were up-regulated in normal AMs after Th2 cytokine stimulation and/or coculture with human lung fibroblasts. Native collagen significantly up-regulated CCL18 expression in normal AMs activated with Th2 cytokines via a mechanism mediated by beta2-integrin/ scavenger receptor(s). Culture supernatants of AMs from patients with idiopathic pulmonary fibrosis increased collagen production by normal lung fibroblasts partly mediated via CCL18. CONCLUSIONS: Our findings suggest that AMs from patients with pulmonary fibrosis disclose a phenotype of alternative activation and might be a part of a positive feedback loop with lung fibroblasts perpetuating fibrotic processes.

Interleukin-18 expression by alveolar epithelial cells type II in tuberculosis and sarcoidosis.

The aims of the present study were to characterize the localization of interleukin-18 (IL-18) expression in lung tissue specimens from patients with pulmonary tuberculosis, sarcoidosis and controls, and to determine whether human alveolar epithelial cells type II (AEC-II) are able to generate IL-18 in primary culture. IL-18 was determined using semiquantitative reverse-transcription-PCR and localized in lungs using in situ hybridization. IL-18 protein levels were determined using the enzyme-linked immunosorbent assay and western blot analysis. Alveolar epithelial cells type II (AEC-II) were stimulated in vitro by proinflammatory cytokines, lipopolysaccharides, and whole cell lysate from Mycobacterium tuberculosis. IL-18 mRNA expression was significantly increased in the lungs affected by tuberculosis and sarcoidosis. In situ hybridization revealed different sites of expression in tuberculosis and sarcoidosis lungs, with AEC-II as one major source of IL-18 in the alveolar compartment. Basal IL-18 expression could be detected in normal AEC-II. Whole cell lysate from M. tuberculosis, but not lipopolysaccharide, led to a strong increase of IL-18 mRNA accumulation in AEC-II. Resting AEC-II secreted only small amounts of IL-18, but intracellular IL-18 protein levels increased in a time-dependent manner during culture. Proinflammatory cytokines tumour necrosis factor-alpha, IL-1beta, and interferon-gamma altered IL-18 mRNA expression and mature protein secretion of human AEC-II. These findings indicate a possible role for AEC-II and AEC-II-derived IL-18 in pathomechanisms of granulomatous lung diseases.

Mycobacterium bovis BCG attenuates surface expression of mature class II molecules through IL-10-dependent inhibition of cathepsin S.

We have previously shown that macrophage infection with Mycobacterium tuberculosis and M. bovis bacillus Calmette-Guérin (BCG) partially inhibits MHC class II surface expression in response to IFN-gamma. The present study examined the nature of class II molecules that do in fact reach the surface of infected cells. Immunostaining with specific Abs that discriminate between mature and immature class II populations showed a predominance of invariant chain (Ii)-associated class II molecules at the surface of BCG-infected cells suggesting that mycobacteria specifically block the surface export of peptide-loaded class II molecules. This phenotype was due to inhibition of IFN-gamma-induced cathepsin S (Cat S) expression in infected cells and the subsequent intracellular accumulation of alphabeta class II dimers associated with the Cat S substrate Ii p10 fragment. In contrast, infection with BCG was shown to induce secretion of IL-10, and addition of blocking anti-IL-10 Abs to cell cultures restored both expression of active Cat S and export of mature class II molecules to the surface of infected cells. Consistent with these findings, expression of mature class II molecules was also restored in cells infected with BCG and transfected with active recombinant Cat S. Thus, M. bovis BCG exploits IL-10 induction to inhibit Cat S-dependent processing of Ii in human macrophages. This effect results in inhibition of peptide loading of class II molecules and in reduced presentation of mycobacterial peptides to CD4(+) T cells. This ability may represent an effective mycobacterial strategy for eluding immune surveillance and persisting in the host.