Michael Lichtenauer

Primary sources and immunological prerequisites for sST2 secretion in humans

AIMS Serum levels of the soluble growth stimulation gene-2 (sST2) are elevated in heart and pulmonary diseases. However, the relationship of the sST2/interleukin (IL)-33 axis and its triggers as well as its organ distribution is still not known. This study was thus designed to investigate the cellular origin and regulation of sST2 and IL-33 in vitro and in vivo. METHODS AND RESULTS sST2 and IL-33 gene expression and protein secretion were analysed in pooled organ-specific cDNAs and in primary cell cultures, respectively, by RT-PCR and ELISA technology. The strongest sST2 mRNA expression was detected in heart and lung tissues, which correlated with spontaneous secretion of sST2 protein in vitro. The inflammatory cytokines IL-1alpha, IL-1beta, and tumour necrosis factor alpha as well as supernatants of lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells led to an enhanced secretion of sST2 in cultured cardiac myocytes and lung alveolar epithelial cells. These cytokines enhanced sST2 secretion via an NFkappaB-dependent mechanism. In addition, LPS stimulation in humans in vivo induced a short-term inflammatory response that was followed by a massive enhancement of sST2 secretion. CONCLUSION These results identify the primary sources and inflammatory triggers for the enhancement of sST2 secretion and demonstrate a relationship between inflammation and the secretion of a bioactive member of the IL-1R family, both in vitro and in vivo.

Intravenous and intramyocardial injection of apoptotic white blood cell suspensions prevents ventricular remodelling by increasing elastin expression in cardiac scar tissue after myocardial infarction

Congestive heart failure developing after acute myocardial infarction (AMI) is a major cause of morbidity and mortality. Clinical trials of cell-based therapy after AMI evidenced only a moderate benefit. We could show previously that suspensions of apoptotic peripheral blood mononuclear cells (PBMC) are able to reduce myocardial damage in a rat model of AMI. Here we experimentally examined the biochemical mechanisms involved in preventing ventricular remodelling and preserving cardiac function after AMI. Cell suspensions of apoptotic cells were injected intravenously or intramyocardially after experimental AMI induced by coronary artery ligation in rats. Administration of cell culture medium or viable PBMC served as controls. Immunohistological analysis was performed to analyse the cellular infiltrate in the ischaemic myocardium. Cardiac function was quantified by echocardiography. Planimetry of the infarcted hearts showed a significant reduction of infarction size and an improvement of post AMI remodelling in rats treated with suspensions of apoptotic PBMC (injected either intravenously or intramoycardially). Moreover, these hearts evidenced enhanced homing of macrophages and cells staining positive for c-kit, FLK-1, IGF-I and FGF-2 as compared to controls. A major finding in this study further was that the ratio of elastic and collagenous fibres within the scar tissue was altered in a favourable fashion in rats injected with apoptotic cells. Intravenous or intramyocardial injection of apoptotic cell suspensions results in attenuation of myocardial remodelling after experimental AMI, preserves left ventricular function, increases homing of regenerative cells and alters the composition of cardiac scar tissue. The higher expression of elastic fibres provides passive energy to the cardiac scar tissue and results in prevention of ventricular remodelling.

T cell senescence and contraction of T cell repertoire diversity in patients with chronic obstructive pulmonary disease

Pathogenetic mechanisms leading to chronic obstructive pulmonary disease (COPD) remain poorly understood. Because clonogenic T cells (CD4+CD28null) were shown to be increased in autoimmune diseases we hypothesized that CD4+CD28null T cells play a role in COPD. Here we describe that enhanced presence of CD4+CD28null cells is associated with impaired lung function. Sixty‐four patients and controls were included. T cell phenotype was analysed using flow cytometry. Enzyme‐linked immunosorbent assays were utilized to determine cytokines. Statistical evaluations were performed using non‐parametric group comparisons and correlations. A logistic regression model was used to determine predictive values of CD4+CD28null in the diagnosis of COPD. Populations of CD4+ T cells lacking surface co‐stimulatory CD28 were enlarged significantly in evaluated patients when compared with controls. Natural killer (NK)‐like T cell receptors (CD94, 158) and intracellular perforin, granzyme B were increased in CD4+CD28null cells. Cytokine production after triggering of peripheral blood mononuclear cells (PBMCs) was elevated in patients at early disease stages. Receiver operating characteristic curve plotting revealed that presence of CD4+CD28null T cells has a diagnostic value. These CD4+CD28null T cells show increased expression of NK‐like T cell receptors (CD94, 158) and intracellular perforin and granzyme B. Furthermore, triggering of PBMCs obtained from patients with mild COPD led to increased interferon‐γ and tumour necrosis factor‐α production in vitro compared with controls. Our finding of increased CD4+CD28null T cells in COPD indicates that chronic antigen exposure, e.g. through contents of smoke, leads to loss of CD28 and up‐regulation of NK cell receptors expression on T cells in susceptible patients.

Irradiated cultured apoptotic peripheral blood mononuclear cells regenerate infarcted myocardium

Background  Acute myocardial infarction (AMI) is followed by post AMI cardiac remodelling, often leading to congestive heart failure. Homing of c‐kit+ endothelial progenitor cells (EPC) has been thought to be the optimal source for regenerating infarcted myocardium.

Alpha-Gal specific IgG immune response after implantation of bioprostheses.

BACKGROUND We have previously shown that the alpha-Gal (Galalpha1.3-Galbeta1-4GlcNAc-R) epitope is a relevant xenoantigen present on bioprostheses utilized in cardiac surgery and elicits an alpha-Gal specific IgM immune response. We sought to investigate whether that immune response continues after valve implantation. MATERIALS AND METHODS We collected plasma samples from patients who underwent bioprosthesis implantation (n = 19) or mechanical valve replacement (n = 8), respectively, prior to, at 10 days and at 3 months after cardiac surgery. ELISA was utilized to quantify alpha-Gal specific IgG and IgG subclasses. 3 bioprosthetic tissue samples were obtained from patients who had to undergo re-operation within 1 week (n = 1) or at 12-15 months (n = 2) after the initial operation. We utilized confocal laser scanning microscopy (CLSM) to detect the presence of alpha-Gal epitopes (IB4) and cell nuclei (DAPI). RESULTS alpha-Gal specific IgG was significantly increased 3 months after implantation of bioprostheses compared to preoperative values (p < 0.001) and was significantly higher than alpha-Gal specific IgG levels of the control group (p < 0.05). IgG3 was the major subclass directed against alpha-Gal (p < 0.05, pre- vs. postoperative values). In CLSM analysis we demonstrated that bioprostheses explanted 1 week after implantation contained IB4/DAPI positive cells within the collagen matrix. In contrast, in patients who underwent reoperation after 12 months, porcine tissue showed a complete lack of IB4/DAPI. CONCLUSION Our results indicate that the implantation of bioprostheses elicits a specific humoral immune response against alpha-Gal bearing cells compared to controls within 3 months after cardiac surgery. The complete absence of IB4/DAPI positive structures 12 months after implantation indicates a specific degradation of alpha-Gal bearing cells through previous exposure to the human blood circuit.

Increased soluble serum markers caspase-cleaved cytokeratin-18, histones, and ST2 indicate apoptotic turnover and chronic immune response in COPD.

Introduction: Chronic obstructive pulmonary disease (COPD) is a worldwide burden and a major cause of death. The disease is accompanied by chronic inflammation and increased cellular turnover that is partly due to an overwhelming induction of apoptosis. In this study, we hypothesized that systemic markers of apoptosis are altered in patients with mild‐to‐severe COPD.

Simulated temporary hypoxia triggers the release of CD31+/Annexin+ endothelial microparticles: A prospective pilot study in humans

INTRODUCTION Endothelial microparticles (EMP) are small membrane vesicles that originate from activated or apoptotic endothelial cells. Although the exact mechanism of EMP function is still relatively unknown, it has been shown that they modulate inflammatory processes, coagulation and vascular function. In this study we hypothesized that transient hypoxia may act as a trigger for the release of EMP into circulation. MATERIALS AND METHODS Fourteen healthy volunteers were subjected to transient normobaric hypoxia in an air-conditioned chamber simulating an oxygen concentration of a height of up to 5500 meters. Blood samples were evaluated for EMP using flow cytometry. RESULTS During the experiment oxygen concentration was adjusted to a value equivalent to a height of 5500 meters to achieve hypoxic conditions. Oxygen saturation decreased to 78% . At the final height a significant increase of CD31+/Annexin+ EMP levels was evident (increase from 0.03% ± 0.01% SEM to 0.12% ± 0.04% SEM, p = 0.0188). CONCLUSIONS These experimental results show that temporary hypoxic conditions can trigger the release of CD31+/ Annexin+ EMP also in healthy volunteers. In our previous studies we have shown that apoptotic bodies can confer pro-survival signals to cardiomyocytes during myocardial ischemia. Based on the experimental results of this current study we believe that the release of CD31+/Annexin+ EMP during hypoxia might act as an endogenous survival signal.

Secretion of soluble ST2 - possible explanation for systemic immunosuppression after heart surgery.

BACKGROUND Cardiopulmonary bypass is known to affect cytokine release leading to a generalized endogenous immune reaction similar to that described in sepsis, without having been explored in great detail. Therefore we evaluated the anti- and pro-inflammatory cytokine responses after heart surgery. METHODS 16 patients who underwent coronary artery bypass graft (CABG) surgery with extracorporeal circulation were included. ST2, IL-4 and IL-10 served as markers for TH2 cytokine response; IL-6, IL-8 and IFN-gamma as TH1 markers. Furthermore, total immunoglobulin subtype analysis (IgM, IgG, IgE) was performed. RESULTS Serum levels of soluble ST2 started to climb at 60 minutes (from 38 +/- 14 preoperatively to 1 480 +/- 890 pg/ml) and peaked 24 hours after surgery (13 360 +/- 2 840 pg/ml, P < 0.001). IL-10 reached a maximum at 60 minutes and returned to baseline levels 24 hours later. IL-6 and IL-8 levels peaked 60 minutes after surgery. IL-4 and IFN-gamma did not change. Only IgM showed a significant peak on day eight ( P < 0.001). CONCLUSION Our results demonstrate that CABG surgery induces a massive long-lasting secretion of ST2, a protein related to immune suppression.

Consequences of a Wait-and-See Strategy for Benign Metastasizing Leiomyomatosis of the Lung

Pulmonary benign metastasizing leiomyomatosis (BML) is a rare smooth-muscle cell disorder of the lung. Most BML lesions stay constant in size for a long time. The prevailing treatment option is primary excision of the nodules or if unresectable, long-time hormone therapy. Herein, we present a case of BML in which a wait-and-see strategy after diagnosis was decided. Fourteen years later a routine chest roentgenogram revealed multiple bi-lobar BML lesions with a giant cyst filling the whole left lung cavity. We conclude that a wait-and-see procedure for BML is feasible, but primary resection of the BML tumor masses is preferable to avoid complications as described in our case.

Secretome of apoptotic peripheral blood cells (APOSEC) attenuates microvascular obstruction in a porcine closed chest reperfused acute myocardial infarction model: role of platelet aggregation and vasodilation.

Although epicardial blood flow can be restored by an early intervention in most cases, a lack of adequate reperfusion at the microvascular level is often a limiting prognostic factor of acute myocardial infarction (AMI). Our group has recently found that paracrine factors secreted from apoptotic peripheral blood mononuclear cells (APOSEC) attenuate the extent of myocardial injury. The aim of this study was to determine the influence of APOSEC on microvascular obstruction (MVO) in a porcine AMI model. A single dose of APOSEC was intravenously injected in a closed chest reperfused infarction model. MVO was determined by magnetic resonance imaging and cardiac catheterization. Role of platelet function and vasodilation were monitored by means of ELISA, flow cytometry, aggregometry, western blot and myographic experiments in vitro and in vivo. Treatment of AMI with APOSEC resulted in a significant reduction of MVO. Platelet activation markers were reduced in plasma samples obtained during AMI, suggesting an anti-aggregatory capacity of APOSEC. This finding was confirmed by in vitro tests showing that activation and aggregation of both porcine and human platelets were significantly impaired by co-incubation with APOSEC, paralleled by vasodilator-stimulated phosphoprotein (VASP)-mediated inhibition of platelets. In addition, APOSEC evidenced a significant vasodilatory capacity on coronary arteries via p-eNOS and iNOS activation. Our data give first evidence that APOSEC reduces the extent of MVO during AMI, and suggest that modulation of platelet activation and vasodilation in the initial phase after myocardial infarction contributes to the improved long-term outcome in APOSEC treated animals.