Gernot Zissel

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.

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.

The Serotoninergic Receptors of Human Dendritic Cells: Identification and Coupling to Cytokine Release

The neurotransmitter 5-hydroxytryptamine (5-HT), commonly known as serotonin, is stored at peripheral sites in mast cells and released from this peripheral source upon IgE cross-linking. In this study, we investigated the expression of serotoninergic receptors (5-HTR), the signaling pathway, and biological activity of 5-HT on human dendritic cells (DC), showing that immature and mature DC expressed mRNA for different serotoninergic receptors. Thereby, the mRNA of 5-HTR1B, 5-HTR1E, 5-HTR2A, 5-HTR2B, one splicing variant of the 5-HTR3, 5-HTR4, and 5-HTR7 receptors were detected. Immature DC preferentially expressed mRNA for the heptahelical 5-HTR1B, 5-HTR1E, and 5-HTR2B receptors, while mature DC mostly expressed 5-HTR4 and 5-HTR7. The mRNA expression level of the ligand-gated cation channel 5-HTR3 and the heptahelical 5-HTR2A did not significantly change during maturation. Isotype-selective receptor agonists allowed us to show that 5-HT stimulated 5-HTR3-dependent Ca2+ influx in immature and mature DC. Moreover, we revealed that 5-HTR1 and 5-HTR2 receptor stimulation induced intracellular Ca2+ mobilization via Gi/o proteins in immature, but not mature, DC. Activation of 5-HTR4 and 5-HTR7 induced cAMP elevation in mature DC. Functional studies indicated that activation of 5-HTR4 and 5-HTR7 enhanced the release of the cytokines IL-1β and IL-8, while reducing the secretion of IL-12 and TNF-α in mature DC. In summary, our study shows that 5-HT stimulated, in a maturation-dependent manner, different signaling pathways in DC. These data point to a role for 5-HT in regulating the immune response at peripheral sites.

Extracellular Adenosine Triphosphate and Chronic Obstructive Pulmonary Disease

RATIONALE Extracellular ATP promotes inflammation, but its role in chronic obstructive pulmonary disease (COPD) is unknown. OBJECTIVES To analyze the expression of ATP and its functional consequences in never-smokers, asymptomatic smokers, and patients with COPD. METHODS ATP was quantified in bronchoalveolar lavage fluid (BALF) of never-smokers, asymptomatic smokers, and patients with COPD of different severity. The expression of specific ATP (purinergic) receptors was measured in airway macrophages and blood neutrophils from control subjects and patients with COPD. The release of mediators by macrophages and neutrophils and neutrophil chemotaxis was assessed after ATP stimulation. MEASUREMENTS AND MAIN RESULTS Chronic smokers had elevated ATP concentrations in BALF compared with never-smokers. Acute smoke exposure led to a further increase in endobronchial ATP concentrations. Highest ATP concentrations in BALF were present in smokers and ex-smokers with COPD. In patients with COPD, BALF ATP concentrations correlated negatively with lung function and positively with BALF neutrophil counts. ATP induced a stronger chemotaxis and a stronger elastase release in blood neutrophils from patients with COPD, as compared with control subjects. In addition, airway macrophages from patients with COPD responded with an increased secretion of proinflammatory and tissue-degrading mediators after ATP stimulation. These findings were accompanied by an up-regulation of specific purinergic receptors in blood neutrophils and airway macrophages of patients with COPD. CONCLUSIONS COPD is characterized by a strong and persistent up-regulation of extracellular ATP in the airways. Extracellular ATP appears to contribute to the pathogenesis of COPD by promoting inflammation and tissue degradation.

P2X7 receptor signaling in the pathogenesis of smoke-induced lung inflammation and emphysema.

Extracellular ATP is up-regulated in the airways of patients with chronic obstructive pulmonary disease, and may contribute to the pathogenesis of the disease. However, the precise mechanisms are poorly understood. Our objective was to investigate the functional role of the ATP receptor P2X(7) in the pathogenesis of cigarette smoke (CS)-induced lung inflammation and emphysema in vivo. Expression of the P2X(7) receptor (P2X(7)R) was measured in lung tissue und immune cells of mice with CS-induced lung inflammation. In a series of experiments using P2X(7) antagonists and genetically engineered mice, the functional role of the P2X(7)R in CS-induced lung inflammation was explored. CS-induced inflammation was associated with an up-regulation of the P2X(7)R on blood and airway neutrophils, alveolar macrophages, and in whole lung tissue. Selective intrapulmonary inhibition of the P2X(7)R reduced CS-induced lung inflammation and prevented the development of emphysema. Accordingly, P2X(7)R knockout mice showed a reduced pulmonary inflammation after acute CS exposure. Experiments with P2X(7)R chimera animals revealed that immune cell P2X(7)R expression plays an important role in CS-induced lung inflammation and emphysema. Extracellular ATP contributes to the development of CS-induced lung inflammation and emphysema via activation of the P2X(7)R. Inhibition of this receptor may be a new therapeutic target for the treatment of chronic obstructive pulmonary disease.

Purinergic Receptor Inhibition Prevents the Development of Smoke-Induced Lung Injury and Emphysema

Extracellular ATP acts as a “danger signal” and can induce inflammation by binding to purinergic receptors. Chronic obstructive pulmonary disease is one of the most common inflammatory diseases associated with cigarette smoke inhalation, but the underlying mechanisms are incompletely understood. In this study, we show that endogenous pulmonary ATP levels are increased in a mouse model of smoke-induced acute lung inflammation and emphysema. ATP neutralization or nonspecific P2R-blockade markedly reduced smoke-induced lung inflammation and emphysema. We detected an upregulation the purinergic receptors subtypes on neutrophils (e.g., P2Y2R), macrophages, and lung tissue from animals with smoke-induced lung inflammation. By using P2Y2R deficient (−/−) animals, we show that ATP induces the recruitment of blood neutrophils to the lungs via P2Y2R. Moreover, P2Y2R deficient animals had a reduced pulmonary inflammation following acute smoke-exposure. A series of experiments with P2Y2R−/− and wild type chimera animals revealed that P2Y2R expression on hematopoietic cell plays the pivotal role in the observed effect. We demonstrate, for the first time, that endogenous ATP contributes to smoke-induced lung inflammation and then development of emphysema via activation of the purinergic receptor subtypes, such as P2Y2R.

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.

Immunologic Response of Sarcoidosis

Sarcoidosis is a chronic granulomatous disorder characterized by an accumulation of T lymphocytes, macrophages, and immune-effector cells within affected organs. The pathogenesis of sarcoidosis involves a complex interplay between immune and nonimmune cells, with myriad cytokines and chemokines involved in the orchestration and evolution of the granulomatous/fibrotic process. The etiology of sarcoidosis is not known, but a granulomatous response to antigen(s) is likely. Genetic polymorphisms influence the susceptibility to sarcoidosis as well as the evolution/prognosis of the disease among patients with sarcoidosis. This article discusses the complex immunological events and immune effector cells that are critical to the induction, evolution, and resolution of the sarcoid granulomatous response.

Genetics of Sarcoidosis

Sarcoidosis is a multigenic and multifactorial disease. Predisposing genes have been identified and fast progress in molecular technologies including systematic genome-wide association studies and large-scale resequencing will aid the discovery of further risk loci and variants. The exploration of the molecular epidemiology of genetic variants in the pathogenesis of sarcoidosis will allow an assessment of their prognostic usefulness. To this end, different granulomatous disorders of known and unknown etiology should be investigated jointly by genetic, immunobiological, and proteomic approaches. The definition of individual genetic risk profiles in sarcoidosis and other chronic inflammatory disorders seems achievable and a useful route for clinical translation.

Diagnoses of chronic beryllium disease within cohorts of sarcoidosis patients

An increase in chronic beryllium disease (CBD) has been suggested due to higher industrial use of beryllium alloys. Since occupational CBD is a perfect phenocopy of sarcoidosis, it might be misdiagnosed as sarcoidosis. In the current it was hypothesised that CBD exists in cohorts of sarcoidosis patients. In a prospective case study, sarcoidosis patients were evaluated for potential beryllium exposure. In those patients in whom beryllium exposure was confirmed and beryllium hypersensitivity demonstrated, the diagnosis of sarcoidosis was rejected and corrected to CBD. In 84 patients seen for re-evaluation or making a diagnosis of sarcoidosis, beryllium exposure was recognised and a diagnosis of CBD was made in 34 out of 84 patients. The time lag between clinical diagnosis of sarcoidosis and the final diagnosis of CBD ranged 0–18 yrs (median 3 yrs) and the mean (range) age at time of diagnosis of CBD was 43.9(25–80) yrs. Beryllium-contaminated workplaces causing disease encompassed a wide spectrum of industries and technical trades in which beryllium-exposure is generally not perceived as a health hazard. In conclusion, chronic beryllium disease still belongs to the spectrum of differential diagnoses of granulomatous disorders.