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-ß) expression in lungs, together with an increased expression of TGF-ß 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 ß3. 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.

Application of Proteomics in Sarcoidosis.

Sarcoidosis is a multisystem disease with heterogeneity in manifestations and outcomes. System-level studies leveraging "omics" technologies are expected to define mechanisms contributing to sarcoidosis heterogeneous manifestations and course. With improvements in mass spectrometry (MS) and bioinformatics, it is possible to study protein abundance for a large number of proteins simultaneously. Contemporary fast-scanning MS enables the acquisition of spectral data for deep coverage of the proteins with data-dependent or data-independent acquisition MS modes. Studies leveraging MS-based proteomics in sarcoidosis have characterized BAL fluid (BALF), alveolar macrophages, plasma, and exosomes. These studies identified several differentially expressed proteins, including protocadherin-2 precursor, annexin A2, pulmonary surfactant A2, complement factors C3, vitamin-D-binding protein, cystatin B, and amyloid P, comparing subjects with sarcoidosis with control subjects. Other studies identified ceruloplasmin, complement factors B, C3, and 1, and others with differential abundance in sarcoidosis compared with other interstitial lung diseases. Using quantitative proteomics, most recent studies found differences in PI3K/Akt/mTOR, MAP kinase, pluripotency-associated transcriptional factor, and hypoxia response pathways. Other studies identified increased clathrin-mediated endocytosis and Fcγ receptor-mediated phagocytosis pathways in sarcoidosis alveolar macrophages. Although studies in mixed BAL and blood cells or plasma are limited, some of the changes in lung compartment are detected in the blood cells and plasma. We review proteomics for sarcoidosis with a focus on the existing MS data acquisition strategies, bioinformatics for spectral data analysis to infer protein identity and quantity, unique aspects about biospecimen collection and processing for lung-related proteomics, and proteomics studies conducted to date in sarcoidosis.

Lung CD4+ Vα2.3+ T-cells in sarcoidosis cohorts with Löfgren's syndrome.

BACKGROUND: Sarcoidosis is diagnosed by a combination of typical clinical and radiological findings together with biopsy proof of non-caseating epithelioid cell granulomas in affected tissues and/or the cell distribution in bronchoalveolar lavage fluid (BALF). We aimed at investigating the usefulness of measuring the proportion of T-cell receptor (TCR) CD4+ Vα2.3+ T-cells in BALF as an additive marker to CD4/CD8-ratio to confirm the diagnosis. METHODS: From a register consisting of 749 sarcoidosis patients [Löfgren's syndrome (LS) n = 274, non-LS n = 475] with information on Vα2.3+ T-cells, an expansion of CD4+ Vα2.3+ T-cells (CD4+ Vα2.3+ T cells > 10.5% in BALF) was seen in 268 (36%). Controls were healthy volunteers (n = 69) and patients with other pulmonary conditions (n = 39), investigated because of suspicion of sarcoidosis. RESULTS: A proportion of CD4+ Vα2.3+ T-cells in BALF > 10.5% was highly specific for sarcoidosis, with a specificity of 97% and with a sensitivity of 36% (p < 0.0001). Receiver operating characteristic (ROC) curves show that testing for CD4+ Vα2.3+ T-cells in BALF was a more useable test in individuals with LS [area under the curve (AUC) 0.82, p < 0.0001] compared to the whole patient group (AUC 0.64, p < 0.0001). CONCLUSION: In this study, we show that an increased proportion of CD4+ Vα2.3+ T-cells in BALF is highly specific for sarcoidosis. This suggests that this T-cell subset could be used as an additional tool to the CD4/CD8-ratio to support the sarcoidosis diagnosis, particularly in patients with LS but also in patients with non-LS.

The Role of Urinary Calcium and Chitotriosidase in a Cohort of Chronic Sarcoidosis Patients.

BACKGROUND: Calcium metabolism alterations are quite common in sarcoidosis and have been correlated with disease activity. OBJECTIVES: The aim of the study was to investigate the clinical significance of calcium metabolism alterations in patients with chronic sarcoidosis. We paid particular attention to associations with specific disease phenotypes and chitotriosidase (CTO) expression. METHODS: 212 chronic sarcoidosis patients (mean age 56.07 ± 12 years; 97 males) were retrospectively recruited. Demographic, clinical, functional, and radiological data, and serum-urinary calcium metabolism were entered into an electronical database for analysis. Levels of CTO and angiotensin-converting enzyme (ACE) were measured and bone mineral density and lung function tests were conducted. RESULTS: Hypercalciuria and hypercalcemia were observed in 18.8 and 1.8% of patients, respectively. Urinary calcium levels correlated with CTO activity (r = 0.33, p = 0.0042). Patients with worsening persistent disease showed the highest levels of urinary calcium. Diffusing capacity of the lung for carbon monoxide (DLCO) percentage correlated inversely with urinary calcium (r = 0.1482; p = 0.0397). CONCLUSIONS: Calcium metabolism alteration, particularly hypercalciuria, was observed in a significant percentage of patients of sarcoidosis. Urinary calcium was correlated with clinical status, DLCO, and serum CTO activity, suggesting its potential role as a biomarker of the activity and severity of sarcoidosis.

TREM-1 and TREM-2 Expression on CD14+ Cells in Bronchoalveolar Lavage Fluid in Pulmonary Sarcoidosis and Hypersensitivity Pneumonitis in the Context of T Cell Immune Response.

BACKGROUND: Sarcoidosis and hypersensitivity pneumonitis (HP) are immunologically mediated processes caused by hypersensitivity reaction accompanied by similar features including lymphocytic alveolitis and granuloma formation. Recent studies describe the role of TREM receptors in T cell activation, differentiation, and granuloma formation. Alveolar macrophages activation via TREM receptors may be the key factor mediating subsequent immune response. The aim of the study was to analyse TREM-1 and TREM-2 expression to identify further molecular mechanisms participating in the immunopathogenesis of sarcoidosis and HP. METHODS: Flow cytometry was performed to analyse TREM-1 and TREM-2 expression on CD14+ cells in bronchoalveolar lavage fluid from patients having sarcoidosis or HP and a control group. RESULTS: The study proved increased TREM-1 expression on alveolar macrophages in pulmonary sarcoidosis and diminished TREM-1 expression in HP-Sarcoidosis: median: 76.7; HP: median: 29.9; control: median: 53.3, (sarcoidosis versus HP: p < 0.001; sarcoidosis versus control: p < 0.05). TREM-2 expression was increased in both, sarcoidosis and HP-sarcoidosis: median: 34.79; HP: median: 36.00; control: median: 12.98, (sarcoidosis versus control: p < 0.05; HP versus control: p < 0.05). Correlation analysis showed negative correlation between TREM-1 and total number of CD8+ cytotoxic T cells. In sarcoidosis TREM-1 expression decreased with changes of HRCT image, decrease in CD4/CD8 ratio and decrease in DLCO. CONCLUSIONS: Differences in TREM receptor expression in sarcoidosis (increase in TREM-1 and TREM-2) and HP (increase in TREM-2) and correlation analysis suggests that activation via TREM may participate in typical immunological characteristics of sarcoidosis and HP.

Alveolar Macrophage ABCG1 Deficiency Promotes Pulmonary Granulomatous Inflammation.

Pulmonary granuloma formation is a complex and poorly understood response to inhaled pathogens and particulate matter. To explore the mechanisms of pulmonary granuloma formation and maintenance, our laboratory has developed a multiwall carbon nanotube (MWCNT)-induced murine model of chronic granulomatous inflammation. We have demonstrated that the MWCNT model closely mimics pulmonary sarcoidosis pathophysiology, including the deficiency of alveolar macrophage ATP-binding cassette (ABC) lipid transporters ABCA1 and ABCG1. We hypothesized that deficiency of alveolar macrophage ABCA1 and ABCG1 would promote pulmonary granuloma formation and inflammation. To test this hypothesis, the effects of MWCNT instillation were evaluated in ABCA1, ABCG1, and ABCA1/ABCG1 myeloid-specific knockout (KO) mice. Histological examination revealed significantly larger pulmonary granulomas in ABCG1-KO and ABCA1/ABCG1 double-KO animals when compared with wild-type animals. Evaluation of BAL cells indicated increased expression of CCL2 and osteopontin, genes shown to be involved in the formation and maintenance of pulmonary granulomas. Single deficiency of alveolar macrophage ABCA1 did not affect MWCNT-induced granuloma formation or proinflammatory gene expression. These observations indicate that the deficiency of alveolar macrophage ABCG1 promotes pulmonary granulomatous inflammation and that this is augmented by additional deletion of ABCA1.

IL-13-regulated Macrophage Polarization during Granuloma Formation in an In Vitro Human Sarcoidosis Model.

The mechanisms underlying abnormal granuloma formation in patients with sarcoidosis are complex and remain poorly understood. A novel in vitro human granuloma model was used to determine the molecular mechanisms of granuloma genesis in patients with sarcoidosis in response to putative disease-causing mycobacterial antigens. Peripheral blood mononuclear cells (PBMCs) from patients with active sarcoidosis and from normal, disease-free control subjects were incubated for 7 days with purified protein derivative-coated polystyrene beads. Molecular responses, as reflected by differential expression of genes, extracellular cytokine patterns, and cell surface receptor expression, were analyzed. Unbiased systems biology approaches were used to identify signaling pathways engaged during granuloma formation. Model findings were compared with human lung and mediastinal lymph node gene expression profiles. Compared with identically treated PBMCs of control subjects (n = 5), purified protein derivative-treated sarcoidosis PBMCs (n = 6) were distinguished by the formation of cellular aggregates resembling granulomas. Ingenuity Pathway Analysis of differential expression gene patterns identified molecular pathways that are primarily regulated by IL-13, which promotes alternatively activated (M2) macrophage polarization. M2 polarization was further demonstrated by immunohistochemistry performed on the in vitro sarcoidosis granuloma-like structures. IL-13-regulated gene pathways were confirmed in human sarcoidosis lung and mediastinal lymph node tissues. The in vitro human sarcoidosis granuloma model provides novel insights into early granuloma formation, particularly IL-13 regulation of molecular networks that regulate M2 macrophage polarization. M2 macrophages are predisposed to aggregation and multinucleated giant cell formation, which are characteristic features of sarcoidosis granulomas. Clinical trial registered with www.clinicaltrials.gov (NCT01857401).

Serum amyloid A - a review.

Serum amyloid A (SAA) proteins were isolated and named over 50 years ago. They are small (104 amino acids) and have a striking relationship to the acute phase response with serum levels rising as much as 1000-fold in 24 hours. SAA proteins are encoded in a family of closely-related genes and have been remarkably conserved throughout vertebrate evolution. Amino-terminal fragments of SAA can form highly organized, insoluble fibrils that accumulate in "secondary" amyloid disease. Despite their evolutionary preservation and dynamic synthesis pattern SAA proteins have lacked well-defined physiologic roles. However, considering an array of many, often unrelated, reports now permits a more coordinated perspective. Protein studies have elucidated basic SAA structure and fibril formation. Appreciating SAA's lipophilicity helps relate it to lipid transport and metabolism as well as atherosclerosis. SAA's function as a cytokine-like protein has become recognized in cell-cell communication as well as feedback in inflammatory, immunologic, neoplastic and protective pathways. SAA likely has a critical role in control and possibly propagation of the primordial acute phase response. Appreciating the many cellular and molecular interactions for SAA suggests possibilities for improved understanding of pathophysiology as well as treatment and disease prevention.

Increased T-helper 17.1 cells in sarcoidosis mediastinal lymph nodes.

The lung-draining mediastinal lymph nodes (MLNs) are currently widely used to diagnose sarcoidosis. We previously reported that T-helper (Th) 17.1 cells are responsible for the exaggerated interferon-γ production in sarcoidosis lungs. In this study, we aimed to investigate 1) whether Th17.1 cells are also increased in the MLNs of sarcoidosis patients and 2) whether frequencies of the Th17.1 cells at diagnosis may correlate with disease progression.MLN cells from treatment-naive pulmonary sarcoidosis patients (n=17) and healthy controls (n=22) and peripheral blood mononuclear cells (n=34) and bronchoalveolar lavage fluid (BALF) (n=36) from sarcoidosis patients were examined for CD4+ T-cell subset proportions using flow cytometry.Higher proportions of Th17.1 cells were detected in sarcoidosis MLNs than in control MLNs. Higher Th17.1 cell proportions were found in sarcoidosis BALF compared with MLNs and peripheral blood. Furthermore, BALF Th17.1 cell proportions were significantly higher in patients developing chronic disease than in patients undergoing resolution within 2 years of clinical follow-up.These data suggest that Th17.1 cell proportions in pulmonary sarcoidosis can be evaluated as a diagnostic and/or prognostic marker in clinical practice and could serve as a new therapeutic target.

Soluble epoxide hydrolase derived lipid mediators are elevated in bronchoalveolar lavage fluid from patients with sarcoidosis: a cross-sectional study.

BACKGROUND: Sarcoidosis is a systemic inflammatory multi-organ disease almost always affecting the lungs. The etiology remains unknown, but the hallmark of sarcoidosis is formation of non-caseating epithelioid cells granulomas in involved organs. In Scandinavia, > 30% of sarcoidosis patients have Löfgren's syndrome (LS), an acute disease onset mostly indicating a favorable prognosis. The impact of dysregulation of lipid mediators, which has been investigated in other inflammatory disorders, is still unknown. METHODS: Using three different liquid chromatography coupled to tandem mass spectrometry targeted platforms (LC-MS/MS), we quantified a broad suite of lipid mediators including eicosanoids, sphingolipids and endocannabinoids in bronchoalveolar lavage (BAL) fluid from pulmonary sarcoidosis patients (n = 41) and healthy controls (n = 16). RESULTS: A total of 47 lipid mediators were consistently detected in BAL fluid of patients and controls. After false discovery rate adjustment, two products of the soluble epoxide hydrolase (sEH) enzyme, 11,12-dihydroxyeicosa-5,8,14-trienoic acid (11,12-DiHETrE, p = 4.4E-5, q = 1.2E-3, median fold change = 6.0) and its regioisomer 14,15-dihydroxyeicosa-5,8,11-trienoic acid (14,15-DiHETrE, p = 3.6E-3, q = 3.2E-2, median fold change = 1.8) increased in patients with sarcoidosis. Additional shifts were observed in sphingolipid metabolism, with a significant increase in palmitic acid-derived sphingomyelin (SM16:0, p = 1.3E-3, q = 1.7E-2, median fold change = 1.3). No associations were found between these 3 lipid mediators and LS, whereas levels of SM 16:0 and 11,12-DiHETrE associated with radiological stage (p < 0.05), and levels of 14,15-DiHETrE were associated with the BAL fluid CD4/CD8 ratio. CONCLUSIONS: These observed shifts in lipid mediators provide new insights into the pathobiology of sarcoidosis and in particular highlight the sEH pathway to be dysregulated in disease.

Dual Inhibition of Rip2 and IRAK1/4 Regulates IL-1ß and IL-6 in Sarcoidosis Alveolar Macrophages and Peripheral Blood Mononuclear Cells.

Sarcoidosis is a multisystem granulomatous disease of unknown etiology that primarily affects the lungs. Our previous work indicates that activation of p38 plays a pivotal role in sarcoidosis inflammatory response. Therefore, we investigated the upstream kinase responsible for activation of p38 in sarcoidosis alveolar macrophages (AMs) and PBMCs. We identified that sustained p38 phosphorylation in sarcoidosis AMs and PBMCs is associated with active MAPK kinase 4 but not with MAPK kinase 3/6. Additionally, we found that sarcoidosis AMs exhibit a higher expression of IRAK1, IRAK-M, and receptor interacting protein 2 (Rip2). Surprisingly, ex vivo treatment of sarcoidosis AMs or PBMCs with IRAK1/4 inhibitor led to a significant increase in IL-1ß mRNA expression both spontaneously and in response to TLR2 ligand. However, a combination of Rip2 and IRAK-1/4 inhibitors significantly decreased both IL-1ß and IL-6 production in sarcoidosis PBMCs and moderately in AMs. Importantly, a combination of Rip2 and IRAK-1/4 inhibitors led to decreased IFN-γ and IL-6 and decreased percentage of activated CD4(+)CD25(+) cells in PBMCs. These data suggest that in sarcoidosis, both pathways, namely IRAK and Rip2, are deregulated. Targeted modulation of Rip2 and IRAK pathways may prove to be a novel treatment for sarcoidosis.

Necrobiosis lipoidica associated with sarcoidosis.

We report the case of a 40-year-old African-American female with biopsy-proven pulmonary sarcoidosis who developed atrophic plaques on her shins, trunk, and scalp that were clinically and histologically consistent with necrobiosis lipoidica (NL). The lesions appeared 3 years after her diagnosis of sarcoidosis, and progressed despite chronic prednisone. Sarcoidosis and NL are granulomatous skin disorders reported to coexist in the same patient only 10 times in the literature. Including the current case, patients have been exclusively females around middle age, and have greater tendencies to develop typical cutaneous sarcoidosis. The incidence of diabetes is rare in this group. Like typical NL, NL associated with sarcoidosis tends to ulcerate, and is difficult to treat. Interestingly, there are six similar cases reported in the literature of patients with sarcoidosis who developed lesions clinically and behaviorally consistent with NL, but received a final histological diagnosis of sarcoidosis. These cases share very similar demographics and clinical features with cases of true NL associated with sarcoidosis, and often have more ambiguous histology containing features of both cutaneous sarcoidosis and NL. Comparing the two sets of cases raises the possibility of a final common disease pathway shared by these two granulomatous skin disorders.

Pulmonary sarcoidosis is associated with exosomal vitamin D-binding protein and inflammatory molecules.

BACKGROUND: Sarcoidosis is an inflammatory granulomatous disorder characterized by accumulation of TH1-type CD4+ T cells and immune effector cells within affected organs, most frequently the lungs. Exosomes are extracellular vesicles conveying intercellular communication with possible diagnostic and therapeutic applications. OBJECTIVES: We aimed to provide an understanding of the proinflammatory role of bronchoalveolar lavage fluid (BALF) exosomes in patients with sarcoidosis and to find candidates for disease biomarkers. METHODS: We performed a mass spectrometric proteomics characterization of BALF exosomes from 15 patients with sarcoidosis and 5 healthy control subjects and verified the most interesting results with flow cytometry, ELISA, and Western blot analyses in an additional 39 patients and 22 control subjects. RESULTS: More than 690 proteins were identified in the BALF exosomes, several of which displayed significant upregulation in patients, including inflammation-associated proteins, such as leukotriene A4 hydrolase. Most of the complement-activating factors were upregulated, whereas the complement regulator CD55 was seen less in patients compared with healthy control subjects. In addition, for the first time, we detected vitamin D-binding protein in BALF exosomes, which was more abundant in patients. To evaluate exosome-associated vitamin D-binding protein as a biomarker for sarcoidosis, we investigated plasma exosomes from 23 patients and 11 healthy control subjects and found significantly higher expression in patients. CONCLUSION: Together, these data contribute to understanding the role of exosomes in lung disease and provide suggestions for highly warranted sarcoidosis biomarkers. Furthermore, the validation of an exosome-associated biomarker in the blood of patients provides novel, and less invasive, opportunities for disease diagnosis.

[The diagnostic algorithm of practice in pulmonary and extrapulmonary sarcoidosis]. / Diagnostyczny algorytm postepowania w sarkoidozie plucnej i pozaplucnej.

Sarcoidosis (SA) is a granulomatous multisystem disease of unknown ethiology. Pulmonary, lymphadenopathy, liver, spleen, skin, and bone sarcoidosis are more frequent but also SA of the heart, central nervous system, eye, and hypercalcemia with following kidney failure also occur. Sarcoidosis may co-exist with extrapulmonary forms, which may overtake or precede each other. SA may occur as acute or chronic with the possibility of complete remission in the early stages of disease. Due to frequent occurrence of asymptomatic SA in threatening vital organs a diagnostic algorithm of practice in pulmonary and extrapulmonary sarcoidosis has been proposed by the author of the article. Diagnosis of SA is based on a correlation of clinical, radiological and histopathological pictures with the presence of non-caseating granuloma in material from the biopsy from at least one organ and having excluded tuberculosis. In all forms of SA, USG abdomen, ECG, ECHO heart, blood tests (blood count, calcium, creatinine, transaminases), level of calcium in a 24-hour urine samples, ophtalmoscopic examinations and lung function tests in pulmonary sarcoidosis should be undertaken to avoid overlooking any form of SA, especially in threatened vital organs. For this purpose, the multidisciplinary team providing an adequate care to the patient with SA has been created by the author of the article has been created by the author of the article in the University Clinical Center in Gdansk providing comprehensive care to patients with sarcoidosis.

Extensively disturbance of regulatory T cells - Th17 cells balance in stage II pulmonary sarcoidosis.

BACKGROUND: Sarcoidosis is a systemic inflammatory disorder characterized by granulomas. Not enough evidences correlate the derangement of CD4+ T subsets, which have an impact on the therapeutic effects of corticosteroids, with the radiographical staging of sarcoidosis. Here we show the disturbance of CD4+ T subsets in newly diagnosed stage II pulmonary sarcoidosis, which is the most common stage in which corticosteroids treatment is used. MATERIALS AND METHODS: 39 newly diagnosed and treatment-naïve patients and 9 subjects after corticosteroids treatment were included. CD4+ CD45RA+/ CD45RO+ cells, CCR4+ CCR6+ cells, and T regulatory cells (Tregs) were tested by Flow Cytometry Analysis. Th1/Th2, Tregs/Th17 related cytokines and mRNAs, SAA and CCL20 were also measured. The activation of PI3K/PTEN/Akt signaling pathway was detected. RESULTS: Percentages of CD4+CD45RO+ memory T cells and Tregs, serum levels of IL-17A, TGF-ß1, IL-6, IFN-γ, IL-10, SAA and CCL20, copies of T-bet, FoxP3, IL-17 and RORc in the periphery were elevated in newly diagnosed stage II pulmonary sarcoidosis patients. Additionally, PI3K/Akt signaling pathway was activated in bronchoalveolar lavage fluid cells. CONCLUSIONS: Disturbance of T memory cells, Th1/Th2, and Tregs/Th17 cells, and activation of PI3K/Akt signaling were seen in newly diagnosed stage II pulmonary sarcoidosis, which can be partly ameliorated by corticosteroids treatment.

Possible Role of IL-25 in Eosinophilic Lung Inflammation in Patients with Chronic Eosinophilic Pneumonia.

PURPOSE: Interleukin (IL)-25 and IL-33 induce IL-5 production by various types of cells, such as type 2 helper T (Th2) cells and type 2 innate lymphoid cells. The number of Th2 cells and concentration of IL-5 in the bronchoalveolar lavage fluid (BALF) are increased in patients with eosinophilic pneumonia (EP). To examine the contribution of IL-25 and IL-33 to eosinophilic inflammation of the lung in humans, we evaluated IL-5, IL-25 and IL-33 levels in the BALF of patients with EP. METHODS: IL-5, IL-25, and IL-33 concentrations in the BALF were measured by enzyme-linked immunosorbent assay in patients with acute eosinophilic pneumonia (AEP), chronic eosinophilic pneumonia (CEP), idiopathic pulmonary fibrosis (IPF), and sarcoidosis. RESULTS: The absolute number of eosinophils, and IL-5 levels, but not IL-33 levels, in the BALF were significantly higher in patients with EP than in patients with IPF and sarcoidosis. IL-25 levels in the BALF were significantly higher in patients with CEP, but not in patients with AEP, than in patients with IPF and sarcoidosis. The absolute number of eosinophils was significantly correlated with the IL-5 concentration in the BALF of patients with EP. IL-5 concentrations were significantly correlated with IL-25 concentrations in the BALF of patients with CEP, but not in patients with AEP. IL-5 levels were not correlated with IL-33 levels in the BALF of patients with EP. CONCLUSIONS: Our findings suggest that IL-25 plays an important role via IL-5 in eosinophilic lung inflammation in patients with CEP.

Impaired Vascular Function in Sarcoidosis Patients.

A common feature of sarcoidosis and atherosclerosis is a chronic systemic inflammatory reaction. Our hypothesis was that sarcoidosis may negatively influence the vessel status. We addressed the issue by examining preatherosclerotic vascular alternations using an ultrasound-based speckle-tracking method in 72 sarcoidosis patients and 15 matched controls. To find potential factors which may have a deleterious influence on arterial performance, different subgroups of sarcoidosis, such as sarcoidosis with or without cortisone therapy, pulmonary sarcoidosis in early and advanced stages, pulmonary sarcoidosis alone or combined with extrapulmonary sarcoidosis, and sarcoidosis with or without elevated blood levels of angiotensin converting enzyme (ACE)/soluble interleukin 2 receptor (sIL-2R) were investigated. We found in the general collective of sarcoidosis patients that circumferential strain (2.68 ± 0.19%), circumferential strain rate (0.21 ± 0.01 1/s), and radial displacement (0.10 ± 0.01 mm) were significantly decreased compared to controls (3.77 ± 0.35%, 0.28 ± 0.02 1/s, and 0.14 ± 0.02 mm, respectively). Vascular strains were more impaired in patients with cortisone therapy, pulmonary sarcoidosis in stages III-IV, and in pulmonary sarcoidosis accompanied by extrapulmonary involvement. The level of ACE/sIL-2R had no relevant influence on the angiological parameters. In conclusion, sarcoidosis is associated with increased vascular stiffness. Cortisone therapy and advanced stages of pulmonary sarcoidosis with extrapulmonary manifestations may account for the impaired vascular function in this patient collective.

Osteoprotegerin/sRANKL Signaling System in Pulmonary Sarcoidosis: A Bronchoalveolar Lavage Study.

Osteoprotegerin (OPG), a soluble tumor necrosis factor receptor family molecule, protects endothelial cells from apoptosis in vitro and promotes neovascularization in vivo. Angiogenesis may be crucial for the course and outcome of sarcoidosis. In this study, we evaluated the clinical usefulness of OPG and its ligand, a soluble receptor activator of nuclear factor-kappaB (sRANKL), in bronchoalveolar lavage fluid (BALF) in patients with sarcoidosis (BBS, Besniera-Boeck-Schaumann disease). We studied 22 BBS patients and 15 healthy volunteers as a control group. The levels of OPG, sRANKL, and interleukin-18 (IL-18) were measured by the Elisa method. The BALF levels of sRANKL and IL-18 were higher in the BBS patients compared with controls [sRANKL: 2.12 (0.82-10.23) vs. 1.12 (0.79-4.39) pmol/l, p = 0.03; IL-18: 34.29 (12.50-133.70) vs. 13.05 (12.43-25.88) pg/ml, p = 0.001]. There were no significant differences between the concentration of OPG in the BBS patients and healthy controls [0.22 (0.14-0.81) vs. 0.23 (0.14-0.75) pmol/l]. In the BBS patients we found correlations between sRANKL and IL-18 in BALF (r = 0.742, p = 0.0001) and between OPG and lung diffusing capacity for carbon monoxide (DLCO) (r = -0.528, p = 0.029). Receiver-operating characteristic (ROC) curve was applied to find the cut-off for the BALF level of sRANKL (BBS vs. healthy: 1.32 pmol/l). We conclude that OPG and sRANKL may have usefulness in clinical evaluation of BBS patients.

Mathematical model of sarcoidosis.

Sarcoidosis is a disease involving abnormal collection of inflammatory cells forming nodules, called granulomas. Such granulomas occur in the lung and the mediastinal lymph nodes, in the heart, and in other vital and nonvital organs. The origin of the disease is unknown, and there are only limited clinical data on lung tissue of patients. No current model of sarcoidosis exists. In this paper we develop a mathematical model on the dynamics of the disease in the lung and use patients' lung tissue data to validate the model. The model is used to explore potential treatments.

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.