Pathogenesis Of Pulmonary Sarcoidosis Research Articles

Myeloid ABCG1 Deficiency Enhances Apoptosis and Initiates Efferocytosis in Bronchoalveolar Lavage Cells of Murine Multi-Walled Carbon Nanotube-Induced Granuloma Model.

The use of carbon nanotubes has increased in the past few decades. Carbon nanotubes are implicated in the pathogenesis of pulmonary sarcoidosis, a chronic granulomatous inflammatory condition. We developed a murine model of chronic granulomatous inflammation using multiwall carbon nanotubes (MWCNT) to investigate mechanisms of granuloma formation. Using this model, we demonstrated that myeloid deficiency of ATP-binding cassette (ABC) cholesterol transporter (ABCG1) promotes granuloma formation and fibrosis with MWCNT instillation; however, the mechanism remains unclear. Our previous studies showed that MWCNT induced apoptosis in bronchoalveolar lavage (BAL) cells of wild-type (C57BL/6) mice. Given that continual apoptosis causes persistent severe lung inflammation, we hypothesized that ABCG1 deficiency would increase MWCNT-induced apoptosis thereby promoting granulomatous inflammation and fibrosis. To test our hypothesis, we utilized myeloid-specific ABCG1 knockout (ABCG1 KO) mice. Our results demonstrate that MWCNT instillation enhances pulmonary fibrosis in ABCG1 KO mice compared to wild-type controls. Enhanced fibrosis is indicated by increased trichrome staining and transforming growth factor-beta (TGF-β) expression in lungs, together with an increased expression of TGF-β related signaling molecules, interleukin-13 (IL-13) and Smad-3. MWCNT induced more apoptosis in BAL cells of ABCG1 KO mice. Initiation of apoptosis is most likely mediated by the extrinsic pathway since caspase 8 activity and Fas expression are significantly higher in MWCNT instilled ABCG1 KO mice compared to the wild type. In addition, TUNEL staining shows that ABCG1 KO mice instilled with MWCNT have a higher percentage of TUNEL positive BAL cells and more efferocytosis than the WT control. Furthermore, BAL cells of ABCG1 KO mice instilled with MWCNT exhibit an increase in efferocytosis markers, milk fat globule-EGF factor 8 (MFG-E8) and integrin β3. Therefore, our observations suggest that ABCG1 deficiency promotes pulmonary fibrosis by MWCNT, and this effect may be due to an increase in apoptosis and efferocytosis in BAL cells.

TGF-β and SMADs mRNA Expression in Pulmonary Sarcoidosis.

Lung fibrosis is a complication of sarcoidosis, in which TGF-β/Smad pathway may play an important role. We evaluated gene expression of TGF-β1, SMAD2, 3 and 7 in bronchoalveolar lavage (BAL) cells and peripheral blood (PB) lymphocytes of sarcoidosis patients (n=94) to better understand the mechanisms of sarcoid inflammation. The relative gene expression was analyzed by qPCR method. Selected clinical/radiological features and biochemical markers were taken into account in the analysis. We found that TGF-β1 and SMAD3 expressions in PB lymphocytes were significantly higher in sarcoidosis patients. Up-regulation of SMAD7 (inhibitory Smad) and down-regulation of SMAD3 in BAL cells in all subgroups were found. The expression of TGF-β1 in PB lymphocytes was the highest in patients with lung parenchymal involvement and in the insidious onset phenotype. The expression of TGF-β1 in BAL cells was higher in patients with abnormal spirometry (p=0.012), and TGF-β1 and SMAD3 in patients with restrictive pattern (p=0.034 and 0.031, respectively). Several statistically significant negative correlations were found between the expression levels of SMAD2 and 3 in BAL cells and various LFT parameters. We conclude that TGF-β/Smad pathway is involved in the pathogenesis of pulmonary sarcoidosis. These biomarkers (especially TGF-β1, SMAD2 and 3) are of a negative prognostic value.

Disordered Toll-like receptor 2 responses in the pathogenesis of pulmonary sarcoidosis

In this study, we hypothesized that the granulomatous disorder sarcoidosis is not caused by a single pathogen, but rather results from abnormal responses of Toll-like receptors (TLRs) to conserved bacterial elements. Unsorted bronchoalveolar lavage (BAL) cells from patients with suspected pulmonary sarcoidosis and healthy non-smoking control subjects were stimulated with representative ligands of TLR-2 (in both TLR-2/1 and TLR-2/6 heterodimers) and TLR-4. Responses were determined by assessing resulting production of tumour necrosis factor (TNF)-α and interleukin (IL)-6. BAL cells from patients in whom sarcoidosis was confirmed displayed increased cytokine responses to the TLR-2/1 ligand 19-kDa lipoprotein of Mycobacterium tuberculosis (LpqH) and decreased responses to the TLR-2/6 agonist fibroblast stimulating ligand-1 (FSL)-1. Subsequently, we evaluated the impact of TLR-2 gene deletion in a recently described murine model of T helper type 1 (Th1)-associated lung disease induced by heat-killed Propionibacterium acnes. As quantified by blinded scoring of lung pathology, P. acnes-induced granulomatous pulmonary inflammation was markedly attenuated in TLR-2(-/-) mice compared to wild-type C57BL/6 animals. The findings support a potential role for disordered TLR-2 responses in the pathogenesis of pulmonary sarcoidosis.

Therapeutic efficacy of Sunitinib and other broad spectrum receptor tyrosine kinase inhibitors (RTKI) in bleomycin-induced pulmonary fibrosis

Platelet-derived growth factor (PDGF) has ample evidence to support its importance in pulmonary fibrosis. Expression of PDGF in the lungs of IPF patients as well as in BAL samples from animal models of lung fibrosis support its involvement during disease development. Vascular endothelial growth factor (VEGF) being a key regulator of angiogenesis and its receptors have been shown to be involved in the pathogenesis of pulmonary sarcoidosis and fibrosis. The hypothesis under investigation is that receptor tyrosine kinase inhibitors (e.g. Sutent) will slow both the fibrotic processes associated with PDGF signaling and the angiogenic processes regulated by VEGF. We tested two RTKi compounds from Pfizer’s internal portfolio (SU-11248 (Sutent), SU-14813) in the bleomycin-induced pulmonary fibrosis mouse model. Prophylactic as well as therapeutic dosing protocols were conducted. Endpoints included histology, body weights, wet lung weights, lung HO-Pro levels and tissue and serum samples for biomarker and lung exposure analyses. Statistically significant reductions in histological changes were demonstrated in lungs treated with both RTK inhibitors in both percentage of total collagen and inflammatory collagen present. RTK inhibitors demonstrated a statistically significant reduction in both wet lung weight and lung HO-Pro content when dosed prophylactically and therapeutically compared to controls. We have demonstrated that broad-spectrum RTK inhibition is efficacious in inhibiting established pulmonary fibrosis in the bleomycin-induced mouse model. Sutent and other broad spectrum RTKis may provide therapeutic benefit to IPF patients by slowing both the fibrotic and angiogenic processes regulated by tyrosine kinases.

2-color analysis of lymphocyte subpopulation of bronchoalveolar lavage fluid and peripheral blood in patients with sarcoidosis

By means of 2-color analysis, we investigated the lymphocyte subpopulation of bronchoalveolar lavage fluid (BALF) and peripheral blood (PB) of 13 patients with pulmonary sarcoidosis. The BALF-CD4+ T cells of normal subjects and patients with sarcoidosis consisted almost completely of CD4+4B4+ cells (helper inducer T cells) and BALF-CD8+ T cells were almost completely composed of CD8+CD11- cells (cytotoxic T cells). The BALF findings of sarcoidosis were characterized by increased percentages of CD4+4B4+ cells (54.9 +/- 15.7%), decreased percentage of CD8+CD11- cells (12.0 +/- 8.4%) and increased percentage of CD4+HLA-DR+ cells (27.8 +/- 13.1%). These findings suggest that activated CD4+4B4+ cells (helper inducer T cells) may primarily contribute to the pathogenesis of pulmonary sarcoidosis. The in vivo or in vitro functions of CD4+4B4+ cells in sarcoidosis need further investigation.

CXCR3 ligands are augmented during the pathogenesis of pulmonary sarcoidosis

We and other investigators have hypothesised that the CXC chemokine receptor (CXCR)3/CXCR3 ligand biological axis is involved in the formation of sarcoid lung granulomas; however, significant discrepancies in the current literature remain. In an effort to clarify previous conflicting findings, we performed the largest observational study to date of interferon-inducible ELR(-) (lacking the sequence glutamic acid-leucine-arginine) CXC chemokines in sarcoid bronchoalveolar fluid (BALF). BALF chemokine levels from sarcoid patients (n = 72) and healthy controls (n = 8) were measured with the ELISA method. Immunohistochemical staining was performed for CXCR3 and its ligands. BALF CXC chemokine ligand (CXCL)10 levels from sarcoid patients were not significantly increased compared with controls. BALF CXCL11 levels from sarcoid patients demonstrated a trend towards elevation; subgroup analysis by stage showed significant BALF CXCL11 elevation in stage I sarcoid patients compared with controls. BALF CXCL9 levels were elevated from sarcoid patients compared with controls. CXC11, CXCL9 and CXCR3 were expressed from epithelioid histiocytes, multinucleated giant cells and other inflammatory cells forming sarcoid lung granulomas. Our data suggest that CXCL9 and CXCL11 are important mediators in recruiting CXCR3-expressing cells. Importantly, we have made the novel observation that both lymphocytes and cells of monocyte linage express CXCR3 and are involved in the formation of sarcoid lung granulomas.

Gene Expression Profiling Identifies MMP-12 and ADAMDEC1 as Potential Pathogenic Mediators of Pulmonary Sarcoidosis

Little is known about the genetic regulation of granulomatous inflammation in sarcoidosis. To determine if tissue gene array analysis would identify novel genes engaged in inflammation and lung remodeling in patients with sarcoidosis. Gene expression analysis was performed on tissues obtained from patients with sarcoidosis at the time of diagnosis (untreated) (n = 6) compared with normal lung tissue (n = 6). Expression of select genes was further confirmed in lung tissue from a second series of patients with sarcoidosis and disease-free control subjects (n = 11 per group) by semi-quantitative RT-PCR. Interactive gene networks were identified in patients with sarcoidosis using Ingenuity Pathway Analysis (Ingenuity Systems, Inc., Redwood, CA) software. The expression of proteins corresponding to selected overexpressed genes was determined using fluorokine multiplex analysis, and immunohistochemistry. Selected genes and proteins were then analyzed in bronchoalveolar lavage fluid in an independent series of patients with sarcoidosis (n = 36) and control subjects (n = 12). A gene network engaged in Th1-type responses was most significantly overexpressed in the sarcoidosis lung tissues, including genes not previously reported in the context of sarcoidosis (e.g., IL-7). MMP-12 and ADAMDEC1 transcripts were most highly expressed (> 25-fold) in sarcoidosis lung tissues, corresponding with increased protein expression by immunohistochemistry. MMP-12 and ADAMDEC1 gene and protein expression were increased in bronchoalveolar lavage samples from patients with sarcoidosis, correlating with disease severity. Tissue gene expression analyses provide novel insights into the pathogenesis of pulmonary sarcoidosis. MMP-12 and ADAMDEC1 emerge as likely mediators of lung damage and/or remodeling and may serve as markers of disease activity.

Th1 cytokine pattern in sarcoidosis is expressed by bronchoalveolar CD4+ and CD8+ T cells.

The pathogenesis of pulmonary sarcoidosis has been related to an increased production of Th1-like cytokines. However, cytokine expression in sarcoidosis has not been systematically studied at a single-cell level. We therefore investigated the expression of IL-2, IL-4, IL-13, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) intracellularly in bronchoalveolar lavage (BAL) and peripheral blood CD3+ T lymphocytes from patients with pulmonary sarcoidosis (radiologic stage II-III, n = 8) and normal controls (n = 9) by flow cytometry. In contrast to IL-4 and IL-13, the percentage of T lymphocytes expressing intracellular IL-2 (49.3 +/- 21.3% versus 14.5 +/- 15.6%), IFN-gamma (75.5 +/- 14.9% versus 32.6 +/- 18.7%) and TNF-alpha (68.3 +/- 18.7% versus 36.8 +/- 20.8%) was significantly higher in patients with sarcoidosis than in normal controls (each P < 0.005). In contrast to BAL lymphocytes, expression of these cytokines in peripheral blood lymphocytes did not differ between patients with sarcoidosis and normal controls. Close correlations were observed between the percentages of BAL lymphocytes expressing intracellular IL-2, IFN-gamma and TNF-alpha, but not for IL-4 or IL-13. Analysis of the expression of these cytokines in T lymphocyte subsets revealed IL-2, IFN-gamma, and TNF-alpha in CD4+ as well as CD8+ T lymphocytes, suggesting a contribution of TC1 cells to the production of proinflammatory cytokines in sarcoidosis. We conclude that a Th1-like cytokine pattern can be observed in CD4+ as well as in CD8+ BAL T lymphocytes in patients with pulmonary sarcoidosis.

CXCL9 and 11 in patients with pulmonary sarcoidosis: a role of alveolar macrophages

Interferon-inducible protein-10 (IP-10)/CXCL10, which is a ligand for CXC chemokine receptor 3 (CXCR3), is known to be involved in the pathogenesis of pulmonary sarcoidosis. However, the roles of monokine induced by interferon gamma (Mig)/CXCL9 and interferon-inducible T cell alpha chemoattractant (I-TAC)/CXCL11, which are also CXCR3 ligands, remain unclear. Mig/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11 in both bronchoalveolar lavage fluid (BALF) and serum in patients with pulmonary sarcoidosis were measured by enzyme-linked immunosorbent assay (ELISA). The expression of these chemokines in alveolar macrophages was examined using ELISA, quantitative real-time polymerase chain reaction and immunostaining. In BALF, Mig/CXCL9 and IP-10/CXCL10 were significantly elevated in stage II sarcoidosis as compared with the levels in healthy volunteers. In serum, Mig/CXCL9 and I-TAC/CXCL11 were increased in stage II of the disease. The levels of all CXCR3 ligands in BALF were correlated with the numbers of both total and CD4(+) lymphocytes. Alveolar macrophages were stained positive for all CXCR3 ligands and produced increased amounts of these chemokines. Positive staining of the three chemokines was also observed in the epithelioid and giant cells in the sarcoid lungs. These findings suggest that Mig/CXCL9 and I-TAC/CXCL11 as well as IP-10/CXCL10 play important roles in the accumulation of Th1 lymphocytes in sarcoid lungs.

Increased IL-6 and IL-8 in bronchoalveolar lavage fluids (BALF) from patients with sarcoidosis: correlation with the clinical parameters.

A variety of cytokines have been implicated in the pathogenesis of pulmonary sarcoidosis, but the exact roles of IL-6 and IL-8 are not yet clear. We studied these cytokine levels in BALF from patients with pulmonary sarcoidosis, idiopathic pulmonary fibrosis (IPF), systemic screlosis (SSc) with interstitial lung disease and control subjects. IL-6 and IL-8 levels were significantly elevated in sarcoidosis, IPF and SSc with interstitial lung disease compared with control subjects. Subjects with sarcoidosis had significantly increased levels of both cytokines compared with controls when the cytokine values were corrected by the total albumin content and the two cytokine levels correlated with each other (r = 0.876). BALF IL-6 levels correlated with percent lymphocytes and percent CD3+ cells. Moreover, when sarcoidosis patients were divided into three groups, those who needed steroid therapy or had progressive disease showed increased cytokine levels in BALF over stable or improved patients. These observations suggest that locally derived IL-6 and IL-8 were increased in sarcoidosis and correlated with activity of this granulomatous lung disease.

Inter-relationship between tumour necrosis factor-alpha (TNF-α) and TNF soluble receptors in pulmonary sarcoidosis

BACKGROUNDThe importance of tumour necrosis factor-alpha (TNF-α) in the pathogenesis of pulmonary sarcoidosis has remained uncertain because of the paucity of clinical features associated with excessive levels of this cytokine....

Soluble CD14 is increased in bronchoalveolar lavage of active sarcoidosis and correlates with alveolar macrophage membrane-bound CD14.

CD14 is a myeloid differentiation antigen which exists in a membrane-bound (55 kD) and a soluble (48 kD) form. This antigen is a receptor for lipopolysaccharide (LPS) structures and triggers the production of various cytokines. The aim of this study was to evaluate whether in active sarcoidosis, a disease with increased proportions of alveolar macrophages (AM) with CD14 expression in BAL fluid, the soluble form of CD14 (sCD14) is also increased. The sCD14 levels were measured in BAL fluid with an ELISA, and membrane-bound CD14 was determined by an immunoperoxidase assay, in active sarcoidosis (n = 13), inactive sarcoidosis (n = 9), idiopathic pulmonary fibrosis (IPF) (n = 6), and control subjects (n = 8). Higher concentrations of sCD14 were present in BAL fluid of patients with active sarcoidosis (58 +/- 34 ng/ml) than in those with inactive disease (13 +/- 10 ng/ml), patients with IPF (5 +/- 5 ng/ml), or control subjects (10 +/- 8% ng/ml) (p < 0.01). Similarly, the proportions of AM expressing membrane-bound CD14 were increased in active sarcoidosis (91 +/- 6%) compared with inactive sarcoidosis (82 +/- 6%), patients with IPF (76 +/- 13%), and control subjects (79 +/- 9%) (p < .05). In sarcoidosis, a significant correlation was found between the sCD14 concentration in BAL fluid and AM membrane expression of CD14 (r = 0.57, p < 0.01). We conclude that sCD14 is increased in BAL of active sarcoidosis suggesting a potential role for this substance as marker of activity and in the pathogenesis of pulmonary sarcoidosis.

The role of angiotensin II in the pathogenesis of pulmonary sarcoidosis

The role of angiotensin II in the pathogenesis of pulmonary sarcoidosis

Down-regulation of Arachidonic Acid Metabolism By Sarcoid Alveolar Macrophages

Macrophages (MØs) are known to produce various arachidonic acid (AA) metabolic products, which may either enhance or suppress inflammatory processes. 1, 2 The amount and type of AA metabolites differ according to the state of activation of the MØ. 1-3 In animal models, immunologically activated MØs produce fewer cyclooxygenase (CO) and lipoxygenase (LO) products than resident MØs. 3-4 Limited data exist, however, concerning AA metabolism by human alveolar MØs (AMs). Since activated AMs appear to be important in the pathogenesis of pulmonary sarcoidosis and idiopathic pulmonary fibrosis (IPF), we investigated AA metabolism by AMs obtained from patients with these interstitial lung diseases.

Bronchoalveolar cells from sarcoid patients demonstrate enhanced antigen presentation.

The recognition of foreign antigens by T lymphocytes in association with lung antigen-presenting cells may be critical in the initiation of the mononuclear alveolitis and granuloma formation of pulmonary sarcoidosis. However, it has been shown that bronchoalveolar cells (BAC) from normal volunteers function poorly as antigen-presenting cells. Therefore, the ability of sarcoid BAC to serve as accessory cells for antigen-dependent autologous T cell proliferation, as measured by tritiated thymidine uptake, was compared with that of normal BAC. Although irradiated sarcoid BAC supported antigen-induced T cell proliferation, normal BAC did so poorly (p less than 0.005). Because it has been shown that sarcoid BAC produce more interleukin 1 (IL 1) than normal BAC, it was considered that the enhancement of antigen-induced proliferative responses could result from an increased amount of IL 1, and that contaminating monocytes in the peripheral blood T cell preparations displayed the antigen for T cell recognition. Therefore, it was necessary to establish that antigen-induced T cell responses required HLA-D region compatibility between the sarcoid BAC and T lymphocytes. BAC from sarcoid patients stimulated antigen-specific proliferation in T cells lines matched for at least one HLA-D-region antigen, but failed to stimulate T cell lines that were unmatched for both antigens. This finding indicates that cells in bronchoalveolar lavage fluids from sarcoid patients were fully capable of acting as antigen-presenting cells. The identification of antigen-presenting cells in the lungs of patients with sarcoidosis together with the previous findings of activated T cells, enhanced IL 1 production, and spontaneous interleukin 2 release in sarcoid patients is compatible with the hypothesis that local cell-mediated immunity is involved in the pathogenesis of pulmonary sarcoidosis.

Pulmonary sarcoidosis: flow cytometry measurement of lung T cell activation.

Lung T cell activation is considered a major factor in the pathogenesis of pulmonary sarcoidosis. Our study was designed to investigate several parameters of T cell activation among blood and alveolar cell populations, including expression of HLA-DR or MLR antigens, increased cell size, and presence of dividing cells. Blood sampling and bronchoalveolar lavages were performed in 20 patients with pulmonary sarcoidosis. Cell populations were analyzed by flow cytometry using immunofluorescence labeling with monoclonal antibodies to lymphocyte differentiation or activation antigens. Cell types were identified by their light-scattering properties. Cell cycle analysis was done after staining with acridine orange. Bronchoalveolar lavage contained a higher proportion of small T4-positive lymphocytes, and large cells of the same phenotype were detected in three patients. T cells bearing HLA-DR antigens were detected in six of 14 bronchoalveolar lavage samples. A marked increased of MLR-positive cells was found in the peripheral blood of eight of eight patients and in the bronchoalveolar lavage of five of seven patients. Increased percentages of cells in the S + G2 + M phases were found in blood lymphocytes from three patients and in half the bronchoalveolar lavage samples. Therefore, a variety of activation markers may be expressed by alveolar T cells. Their qualitative and quantitative assessment may provide additional criteria for staging the intensity of the alveolitis, and the possible relationship between these markers and disease progression or activity deserves long-term clinical investigation.

Spontaneous release of interleukin-2 by lung T lymphocytes in active pulmonary sarcoidosis.

We investigated the possible role of interleukin-2, a T-cell product that stimulates the clonal increase of responsive T lymphocytes, in the pathogenesis of pulmonary sarcoidosis. We obtained mononuclear effector cells from the lungs of 10 patients with sarcoidosis and high-intensity alveolitis, 17 patients with sarcoidosis and low-intensity alveolitis, 3 patients with idiopathic pulmonary fibrosis, and 10 normal controls. Lung cells from the group with sarcoidosis and low-intensity alveolitis, from the group with idiopathic pulmonary fibrosis, and from the controls produced insignificant amounts of interleukin-2. However, lung cells from 9 of 10 patients with sarcoidosis and high-intensity alveolitis spontaneously released interleukin-2, and in a proportion that correlated with the proportion of T cells in the lung washings (P less than 0.01). Blood T cells from the same patients did not release interleukin-2. To determine whether release of interleukin-2 by the lung T cells had a biologic effect in vivo, we measured T-lymphocyte replication in the lungs of patients and controls. The lung T lymphocytes replicated at a rate that was several times higher in the patients with sarcoidosis and high-intensity alveolitis than in the other patient groups or the controls (P less than 0.01). These observations suggest that the release of interleukin-2 by lung T cells has a central role in increasing the numbers of lung T cells in active pulmonary sarcoidosis.