Joachim Müller-Quernheim

Sarcoidosis is associated with a truncating splice site mutation in BTNL2.

Sarcoidosis is a polygenic immune disorder with predominant manifestation in the lung. Genome-wide linkage analysis previously indicated that the extended major histocompatibility locus on chromosome 6p was linked to susceptibility to sarcoidosis. Here, we carried out a systematic three-stage SNP scan of 16.4 Mb on chromosome 6p21 in as many as 947 independent cases of familial and sporadic sarcoidosis and found that a 15-kb segment of the gene butyrophilin-like 2 (BTNL2) was associated with the disease. The primary disease-associated variant (rs2076530; PTDT = 3 × 10−6, Pcase-control = 1.1 × 10−8; replication PTDT = 0.0018, Pcase-control = 1.8 × 10−6) represents a risk factor that is independent of variation in HLA-DRB1. BTNL2 is a member of the immunoglobulin superfamily and has been implicated as a costimulatory molecule involved in T-cell activation on the basis of its homology to B7-1. The G → A transition constituting rs2076530 leads to the use of a cryptic splice site located 4 bp upstream of the affected wild-type donor site. Transcripts of the risk-associated allele have a premature stop in the spliced mRNA. The resulting protein lacks the C-terminal IgC domain and transmembrane helix, thereby disrupting the membrane localization of the protein, as shown in experiments using green fluorescent protein and V5 fusion proteins.

Common patterns and disease-related signatures in tuberculosis and sarcoidosis

In light of the marked global health impact of tuberculosis (TB), strong focus has been on identifying biosignatures. Gene expression profiles in blood cells identified so far are indicative of a persistent activation of the immune system and chronic inflammatory pathology in active TB. Definition of a biosignature with unique specificity for TB demands that identified profiles can differentiate diseases with similar pathology, like sarcoidosis (SARC). Here, we present a detailed comparison between pulmonary TB and SARC, including whole-blood gene expression profiling, microRNA expression, and multiplex serum analytes. Our analysis reveals that previously disclosed gene expression signatures in TB show highly similar patterns in SARC, with a common up-regulation of proinflammatory pathways and IFN signaling and close similarity to TB-related signatures. microRNA expression also presented a highly similar pattern in both diseases, whereas cytokines in the serum of TB patients revealed a slightly elevated proinflammatory pattern compared with SARC and controls. Our results indicate several differences in expression between the two diseases, with increased metabolic activity and significantly higher antimicrobial defense responses in TB. However, matrix metallopeptidase 14 was identified as the most distinctive marker of SARC. Described communalities as well as unique signatures in blood profiles of two distinct inflammatory pulmonary diseases not only have considerable implications for the design of TB biosignatures and future diagnosis, but they also provide insights into biological processes underlying chronic inflammatory disease entities of different etiology.

Serum CC-Chemokine Ligand 18 Concentration Predicts Outcome in Idiopathic Pulmonary Fibrosis

RATIONALE Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with a poor prognosis. There is great effort to find predictors of outcome. Conclusive data for any serum biomarker are lacking. We have recently documented that serum CCL18 concentrations correlate with the course of pulmonary function data in patients with pulmonary fibrosis of various causes. OBJECTIVES To test the value of serum CCL18 concentrations in IPF, we included 72 patients in a prospective study. METHODS IPF was defined according to the ATS/ERS criteria. Serum CCL18 concentrations were measured by a commercially available ELISA. Patients were followed for 24 months. Pulmonary function tests were performed at least every 6 months. MEASUREMENTS AND MAIN RESULTS Baseline serum CCL18 concentrations predicted the change in TLC and FVC at the 6-month follow-up. Receiver operating characteristics (ROC) revealed a significant relation between survival and baseline CCL18 concentrations. By ROC analysis, the cutoff value with the highest diagnostic accuracy was defined as 150 ng/ml (sensitivity, 0.83; specificity, 0.77). There was a significantly higher mortality in patients with serum CCL18 concentrations above 150 ng/ml (P < 0.0001). The hazard proportional ratio adjusted for age, sex, and baseline pulmonary function data was 8.0. There was a higher incidence of disease progression in the group with high serum CCL18 concentrations. CONCLUSIONS Our data demonstrate that serum CCL18 concentrations have a predictive value in IPF and may be a useful tool in the clinical management of patients with IPF and in clinical trials.

Efficacy of infliximab in extrapulmonary sarcoidosis: results from a randomised trial

The aim of the present study was to investigate the efficacy of infliximab for the treatment of extrapulmonary sarcoidosis. A prospective, randomised, double-blind, placebo-controlled trial was conducted, with infliximab at 3 and 5 mg·kg−1 body weight administered over 24 weeks. Extrapulmonary organ severity was determined by a novel severity tool (extrapulmonary physician organ severity tool; ePOST) with an adjustment for the number of organs involved (ePOSTadj). In total, 138 patients enrolled in the trial of infliximab versus placebo for the treatment of chronic corticosteroid-dependent pulmonary sarcoidosis. The baseline severity of extrapulmonary organ involvement, as measured by ePOST, was similar across treatment groups. After 24 weeks of drug-therapy study, the change from baseline to week 24 in ePOST was greater for the combined infliximab group compared with the placebo group. After adjustment for the number of extrapulmonary organs involved, the improvement in ePOSTadj observed in the combined infliximab group was also greater than that observed in placebo-treated patients, after 24 weeks of therapy. The improvements in ePOST and ePOSTadj were not maintained during a subsequent 24-week washout period. Infliximab may be beneficial compared with placebo in the treatment of extrapulmonary sarcoidosis in patients already receiving corticosteroids, as assessed by the severity tool described in the present study.

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.

Treatment of chronic sarcoidosis with an azathioprine/ prednisolone regimen

In a few patients with chronic sarcoidosis, prolonged, unacceptably high doses of corticosteroids are required to achieve symptomatic relief. In these cases, a corticosteroid-sparing drug might be administered to allow long-term treatment without the adverse effects of corticosteroids. This study examines azathioprine as a prednisolone-sparing treatment. In an open study, the course of 11 patients with chronic sarcoidosis was analysed. In an induction phase, 2 mg azathioprine x kg body weight (BW)(-1) x day(-1) in combination with 0.6-0.8 mg prednisolone x kg BW(-1) x day(-1) were administered with prednisolone being reduced to 0.1 mg x kg BW(-1) x day(-1) within 2-3 months. This was followed by a 21-22-month maintenance phase with 2 mg azathioprine x kg BW(-1) x day(-1) and 0.1 mg prednisolone x kg BW(-1) day(-1). Clinical parameters and immunological findings of bronchoalveolar lavage (BAL) were analysed. All patients had significant symptomatic relief and improvements or resolutions of physiological, serological and radiographic findings without suffering from serious adverse effects. Nine of 11 patients completed therapy after 19-26 months, and 2/11 patients terminated therapy after 8 and 12 months, respectively. Eight patients had remissions lasting 4-73 months. Three relapses occurred after 8, 18, and 22 months. During the induction phase, BAL cell composition changed and their activity in terms of cytokine release was suppressed. This preliminary study suggests that azathioprine may be effective as a corticosteroid-sparing agent in long-term therapy of sarcoidosis, but a much larger study is necessary to give the definitive answer.

Bronchoalveolar and serological parameters reflecting the severity of sarcoidosis

The aim of the present study was to determine which bronchoalveolar lavage fluid (BALF) and serological parameters reflect the severity of newly diagnosed pulmonary sarcoidosis. Seventy-four previously untreated sarcoid patients were categorised into three groups: 10 patients with Löfgrens syndrome, 51 patients with stable disease and 13 patients with progressing disease requiring systemic steroid treatment. Total BALF cell count, percentage of alveolar lymphocytes and lymphocyte CD4/CD8 ratio were not associated with severity of disease. Interestingly, a significant increase in percentages of BALF neutrophils (5.2±1.1%) and eosinophils (1.7±0.6%) was observed in sarcoid patients with progressing disease. Elevated percentages of these two cell types were the only BALF parameters associated with a more frequent necessity for systemic steroid therapy. This association between an elevated percentage of BALF neutrophils and the necessity for steroid treatment was observed in advanced as well as early sarcoidosis (radiological types I and II). Serum levels of soluble interleukin‐2 receptor and neopterin were significantly elevated in progressing disease compared to stable disease or Löfgrens syndrome. The present results demonstrate that increased percentages of neutrophils (>3.0%) and eosinophils (>1%) in bronchoalveolar lavage fluid from newly diagnosed pulmonary sarcoidosis is associated with a significantly higher risk of necessity for steroid therapy and may be helpful markers of progressive disease. Furthermore, of the serological parameters investigated, only serum levels of soluble interleukin‐2 receptor and neopterin were associated with disease severity.

Oxidative stress in the pathogenesis of diffuse lung diseases: A review

Oxidative stress is an imbalance between oxidants (reactive oxygen and nitrogen species) and antioxidants that may affect lipids, DNA, carbohydrates and proteins. The lung is continuously exposed to endogenous and exogenous oxidants (cigarette smoke, mineral dust, ozone, radiation). Reactive oxygen and nitrogen species are mainly produced by phagocytes as well as by polymorphonuclear, alveolar, bronchial and endothelial cells. A potential role of oxidative stress in the pathogenesis of diffuse lung diseases (particularly idiopathic pulmonary fibrosis) has been demonstrated. Increased oxidant levels and decreased antioxidant defences can contribute to the progression of idiopathic pulmonary fibrosis and other diffuse lung diseases. The growing number of papers on the different aspects of oxidant/antioxidant imbalance in diffuse lung diseases in the last decade reflects increasing interest in this topic and suggests that specific DLDs may be characterized by specific patterns of oxidation and antioxidant responses. The study of oxidative stress can provide insights into etiopathogenesis and favour the discovery of new treatments. In this review of the literature on oxidants and antioxidants in diffuse lung diseases, the focus is on idiopathic pulmonary fibrosis, sarcoidosis, pneumoconiosis and pulmonary fibrosis associated with systemic sclerosis.

Inhaled Vasoactive Intestinal Peptide Exerts Immunoregulatory Effects in Sarcoidosis

RATIONALE Previous studies suggest an important immunoregulatory role of vasoactive intestinal peptide (VIP) in experimental models of chronic noninfectious inflammation. Sarcoidosis is characterized by noncaseating epitheloid cell granulomas, where excessive tumor necrosis factor-alpha production by pulmonary macrophages plays a critical role in granuloma formation and disease progression, which may lead to fatal organ dysfunction. OBJECTIVES To test whether inhaled VIP has an immunoregulatory role. Sarcoid alveolitis was used as a prototype of immune-mediated chronic lung inflammation. METHODS In an open clinical phase II study, we treated 20 patients with histologically proved sarcoidosis and active disease with nebulized VIP for 4 weeks. MEASUREMENTS AND MAIN RESULTS VIP inhalation was safe, well-tolerated, and significantly reduced the production of tumor necrosis factor-alpha by cells isolated from bronchoalveolar lavage fluids of these patients. VIP treatment significantly increased the numbers of bronchoalveolar lavage CD4(+)CD127(-)CD25(+) T cells, which showed regulatory activities on conventional effector T cells. In vitro experiments demonstrated the capacity of VIP to convert naive CD4(+)CD25(-) T cells into CD4(+)CD25(+)FoxP3(+) regulatory T cells, suggesting the generation of peripheral regulatory T cells by VIP treatment. CONCLUSIONS This study is the first to show the immunoregulatory effect of VIP in humans, and supports the notion of inhaled VIP as an attractive future therapy to dampen exaggerated immune responses in lung disorders. Thus, the inhalation of neuropeptides may be developed into a new therapeutic principle for chronic inflammatory lung disorders in humans.

CARD15 gene mutations in sarcoidosis

Sarcoidosis, Blau syndrome and Crohns disease are complex disorders, characterised by granulomatous inflammation affecting a variety of organs. Mutations of the CARD15 gene, on chromosome 16, have been shown to contribute significantly to Crohns disease and to cause Blau syndrome. These factors prompted the current authors to study CARD15 mutations in sarcoidosis. A total of 138 families were genotyped, including 302 patients with sarcoidosis and 127 patients without a family history of sarcoidosis (together with their parents), for four main coding CARD15 polymorphisms associated with increased risk of Crohns disease. Furthermore, the gene segment that harbours Blau syndrome mutations was sequenced in 39 selected patients from 39 families with affected siblings identical for one or two parental chromosomes 16s and in eight patients from multi-case families. None of the reported Blau syndrome mutations and no new sequence alterations were found. There was an increased frequency of transmission of the rare allele of the polymorphic sites 802C>T (SNP5) and 2722G>C (SNP12) in at least one of the two study groups. In conclusion, CARD15 mutations, which are important in Crohns disease and Blau syndrome, play no major role in sarcoidosis in this study population. However, these mutations could be of limited importance, especially in patients without a family history of sarcoidosis.