Annegret Reinhold

Effects of volatile and intravenous anesthesia on the alveolar and systemic inflammatory response in thoracic surgical patients.

Background:One-lung ventilation (OLV) results in alveolar proinflammatory effects, whereas their extent may depend on administration of anesthetic drugs. The current study evaluates the effects of different volatile anesthetics compared with an intravenous anesthetic and the relationship between pulmonary and systemic inflammation in patients undergoing open thoracic surgery. Methods:Sixty-three patients scheduled for elective open thoracic surgery were randomized to receive anesthesia with 4 mg · kg−1 · h−1 propofol (n = 21), 1 minimum alveolar concentration desflurane (n = 21), or 1 minimum alveolar concentration sevoflurane (n = 21). Analgesia was provided by remifentanil (0.25 &mgr;g · kg−1 · min−1). After intubation, all patients received pressure-controlled mechanical ventilation with a tidal volume of approximately 7 ml · kg−1 ideal body weight, a peak airway pressure lower than 30 cm H2O, a respiratory rate adjusted to a Paco2 of 40 mmHg, and a fraction of inspired oxygen lower than 0.8 during OLV. Fiberoptic bronchoalveolar lavage of the ventilated lung was performed immediately after intubation and after surgery. The expression of inflammatory cytokines was determined in the lavage fluids and serum samples by multiplexed bead-based immunoassays. Results:Proinflammatory cytokines increased in the ventilated lung after OLV. Mediator release was more enhanced during propofol anesthesia compared with desflurane or sevoflurane administration. For tumor necrosis factor-&agr;, the values were as follows: propofol, 5.7 (8.6); desflurane, 1.6 (0.6); and sevoflurane, 1.6 (0.7). For interleukin-8, the values were as follows: propofol, 924 (1680); desflurane, 390 (813); and sevoflurane, 412 (410). (Values are given as median [interquartile range] pg · ml−1). Interleukin-1&bgr; was similarly reduced during volatile anesthesia. The postoperative serum interleukin-6 concentration was increased in all patients, whereas the systemic proinflammatory response was negligible. Conclusions:OLV increases the alveolar concentrations of proinflammatory mediators in the ventilated lung. Both desflurane and sevoflurane suppress the local alveolar, but not the systemic, inflammatory responses to OLV and thoracic surgery.

Role of dipeptidyl peptidase IV (DP IV)-like enzymes in T lymphocyte activation: investigations in DP IV/CD26-knockout mice.

Abstract Background: Dipeptidyl peptidase IV (DP IV, CD26) and DP IV-like enzymes, such as dipeptidyl peptidase II (DP II), dipeptidyl peptidase 8 (DP8), and dipeptidyl peptidase 9 (DP9), have been recognized to regulate T lymphocyte activation. Lys[Z(NO2)]-thiazolidide (LZNT) and Lys[Z(NO2)]-pyrrolidide (LZNP), non-selective inhibitors of DP IV-like activity known to target DP IV as well as DP II, DP8, and DP9, suppress T lymphocyte proliferation in vitro. Moreover, these inhibitors are capable of attenuating the severity of autoimmune diseases, such as experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis, and experimental arthritis, a model of human rheumatoid arthritis, in vivo, particularly in combination with inhibitors of aminopeptidase N (APN, CD13) enzymatic activity. Methods: Here, we studied the influence of non-selective and selective inhibitors of DP IV-like enzymes on DNA synthesis in mitogen-stimulated splenocytes from wild-type C57BL/6 mice and DP IV/CD26-knockout (DP IV/CD26-KO) mice. Results: LZNT and LZNP, the non-selective inhibitors of DP IV-like activity, suppressed the DNA synthesis in stimulated splenocytes from wild-type and DP IV/CD26-KO mice to a comparable extent. Further, a selective inhibitor of DP8/DP9 activity was capable of suppressing DNA synthesis in mitogen-stimulated splenocytes of both wild-type and knockout mice to the same extent. In contrast, selective inhibitors of DP IV and DP II lacked this suppressive activity. Conclusions: Our data support the hypothesis that DP8 and/or DP9 represent additional pharmacological targets for the suppression of T cell proliferation and for anti-inflammatory therapy. Clin Chem Lab Med 2009;47:268–74.

CCR7-mediated LFA-1 functions in T cells are regulated by 2 independent ADAP/SKAP55 modules

The β2-integrin lymphocyte function-associated antigen-1 (LFA-1) plays a crucial role within the immune system. It regulates the interaction between T cells and antigen-presenting cells and facilitates T-cell adhesion to the endothelium, a process that is important for lymphocyte extravasation and homing. Signals mediated via the T-cell receptor and the chemokine receptor CCR7 activate LFA-1 through processes known as inside-out signaling. The molecular mechanisms underlying inside-out signaling are not completely understood. Here, we have assessed the role of the ADAP/SKAP55 module for CCR7-mediated signaling. We show that loss of the module delays homing and reduces intranodal T-cell motility in vivo. This is probably because of a defect in CCR7-mediated adhesion that affects both affinity and avidity regulation of LFA-1. Further analysis of how the ADAP/SKAP55 module regulates CCR7-induced integrin activation revealed that 2 independent pools of the module are expressed in T cells. One pool interacts with a RAPL/Mst1 complex, whereas the other pool is linked to a RIAM/Mst1/Kindlin-3 complex. Importantly, both the RAPL/Mst1 and the RIAM/Mst1/Kindlin-3 complexes require ADAP/SKAP55 for binding to LFA-1 upon CCR7 stimulation. Hence, 2 independent ADAP/SKAP55 modules are essential components of the signaling machinery that regulates affinity and avidity of LFA-1 in response to CCR7.

Expression of SKAP‐HOM in DCs is required for an optimal immune response in vivo

The cytosolic adaptor molecule SKAP‐HOM, similar to the T cell‐specific homologue SKAP55, interacts directly with ADAP, and both molecules are involved in inside‐out signaling. Previous studies have shown that in the absence of SKAP‐HOM, antigen receptor‐triggered integrin‐mediated adhesion is impaired severely in B cells but not in T cells. In addition, loss of SKAP‐HOM results in a less severe clinical course of EAE. DCs are the most potent APCs and express SKAP‐HOM. However, the role of SKAP‐HOM in DCs remains unknown. Here, we assessed whether the reduced severity of EAE observed in SKAP‐HOM‐deficient mice is at least partially a result of an impaired cooperation between APCs and T cells. We demonstrate that migration of LC in vivo and the spontaneous motility of BMDCs in vitro are increased in the absence of SKAP‐HOM. In contrast, triggering of the integrin results in a drastic decrease of DC motility and in enhanced actin polymerization in SKAP‐HOM‐deficient DCs. Furthermore, the antigen‐dependent conjugate formed between wild‐type T cells and SKAP‐HOM−/− DCs is delayed in comparison with wild‐type DCs. Strikingly, fewer antigen‐specific T cells are induced by immunization with SKAP‐HOM−/− BMDCs as compared with wild‐type BMDCs in vivo. Thus, these findings suggest that SKAP‐HOM expression in DCs is required for the induction of an optimal immune response.

Plasma concentrations of zinc, copper, interleukin-6 and interferon-γ, and plasma dipeptidyl peptidase IV activity in chronic hepatitis C

Copper and zinc are essential trace elements which play an important role in various biological processes. Along with various cytokines, glucocorticoids, glucagon and insulin, the acute-phase protein metallothionein is involved in the regulation of immune cell functions. Metallothionein is the central protein regulating zinc concentration. Zinc deficiency is often found in patients with chronic liver disease, chronic kidney disease and diabetes mellitus, and in those with acute infectious diseases. In contrast, copper deficiency is rarely reported. In order to determine whether there is a correlation between zinc and/or copper and selected immunological parameters in patients with chronic liver disease, we investigated plasma levels of zinc and copper, concentrations of interleukin-6 (IL-6) and interferon-γ (IFN-γ), and the enzymatic activity of dipeptidyl peptidase IV (DP IV, CD26) in patients with chronic hepatitis C and in healthy control subjects. Whereas zinc plasma levels did not differ between patients and control subjects, copper concentrations revealed gender-specific differences. The mean copper concentration was higher in female patients with chronic hepatitis C and in female controls compared with the respective male groups. The immunological parameters of IFN-γ concentration and DP IV activity showed similar levels in the patient and control groups. Of note, the mean IL-6 plasma concentrations were significantly elevated in patients with chronic hepatitis C compared with healthy control subjects. In summary, there was no correlation between either zinc or copper concentrations and the immunological parameters measured (IL-6 and IFN-γ levels and DP IV activity) in patients with chronic hepatitis C and in healthy control subjects.

Increased carcinogenic potential of myeloid tumor cells induced by aberrant TGF-β1-signaling and upregulation of cathepsin B

Abstract The TGF-β signaling pathways are implicated in cancer. Cysteine cathepsins can contribute to the carcinogenic potential of tumor cells. The aim of this study was to investigate the regulation of cysteine cathepsin expression by TGF-β1 and the functional implications in tumor cells. We found an upregulation of cathepsin B (CathB, 2- to 5-fold) in different myeloid tumor cells (THP-1, MonoMac-1, MonoMac-6) after incubation with TGF-β1. No upregulation was found in monocytes, and there was suppression of CathB expression in epithelial tumor cells (A549). Increased cathepsin B activity led to enhanced carcinogenic potential, which was reflected by increased migration and invasion of the cells and resistance to inhibitor-induced apoptosis. Analysis of the TGF-β signaling pathways showed no alterations in TGF-β/BMP receptor expression or SMAD2/3 phosphorylation, and no influence of MAP kinase pathways. However, a reduction in SMAD1 expression was detected. The lack of BMP action on cysteine cathepsin expression in myeloid tumor cells, but not in epithelial tumor cells, suggests a defect in the Smad1/Smad5 pathway. We located a related TGF-β1-responsive element within the first intron of the CathB gene. In conclusion, alterations in the TGF-β1 signaling pathway lead to upregulation of CathB, which contributes to the carcinogenic potential of tumor cells.

miR-20a Inhibits TCR-Mediated Signaling and Cytokine Production in Human Naïve CD4 + T Cells

Upon TCR stimulation by peptide-MHC complexes, CD4+ T cells undergo activation and proliferation. This process will ultimately culminate in T-cell differentiation and the acquisition of effector functions. The production of specific cytokines by differentiated CD4+ T cells is crucial for the generation of the appropriate immune response. Altered CD4+ T-cell activation and cytokine production result in chronic inflammatory conditions and autoimmune disorders. miRNAs have been shown to be important regulators of T-cell biology. In this study, we have focused our investigation on miR-20a, a member of the miR-17-92 cluster, whose expression is decreased in patients suffering from multiple sclerosis. We have found that miR-20a is rapidly induced upon TCR-triggering in primary human naïve CD4+ T cells and that its transcription is regulated in a Erk-, NF-κB-, and Ca++-dependent manner. We have further shown that overexpression of miR-20a inhibits TCR-mediated signaling but not the proliferation of primary human naïve CD4+ T cells. However, miR-20a overexpression strongly suppresses IL-10 secretion and moderately decreases IL-2, IL-6 and IL8 production, which are crucial regulators of inflammatory responses. Our study suggests that miR-20a is a new player in the regulation of TCR signaling strength and cytokine production.

T Cell–Independent Modulation of Experimental Autoimmune Encephalomyelitis in ADAP-Deficient Mice

The adhesion- and degranulation-promoting adaptor protein (ADAP), expressed in T cells, myeloid cells, and platelets, is known to regulate receptor-mediated inside-out signaling leading to integrin activation and adhesion. In this study, we demonstrate that, upon induction of active experimental autoimmune encephalomyelitis (EAE) by immunization with the myelin oligodendrocyte glycoprotein35–55 peptide, ADAP-deficient mice developed a significantly milder clinical course of EAE and showed markedly less inflammatory infiltrates in the CNS than wild-type mice. Moreover, ADAP-deficient recipients failed to induce EAE after adoptive transfer of myelin oligodendrocyte glycoprotein–specific TCR-transgenic T cells (2D2 T cells). In addition, ex vivo fully activated 2D2 T cells induced significantly less severe EAE in ADAP-deficient recipients. The ameliorated disease in the absence of ADAP was not due to expansion or deletion of a particular T cell subset but rather because of a strong reduction of all inflammatory leukocyte populations invading the CNS. Monitoring the adoptively transferred 2D2 T cells over time demonstrated that they accumulated within the lymph nodes of ADAP-deficient hosts. Importantly, transfer of complete wild-type bone marrow or even bone marrow of 2D2 TCR–transgenic mice was unable to reconstitute EAE in the ADAP-deficient animals, indicating that the milder EAE was dependent on (a) radio-resistant nonhematopoietic cell population(s). Two-photon microscopy of lymph node explants revealed that adoptively transferred lymphocytes accumulated at lymphatic vessels in the lymph nodes of ADAP-deficient mice. Thus, our data identify a T cell–independent mechanism of EAE modulation in ADAP-deficient mice.

Skap2 is required for β 2 integrin–mediated neutrophil recruitment and functions

Integrin activation is required for neutrophil functions. Impaired integrin activation on neutrophils is the hallmark of leukocyte adhesion deficiency (LAD) syndrome in humans, characterized by impaired leukocyte recruitment and recurrent infections. The Src kinase–associated phosphoprotein 2 (Skap2) is involved in integrin functions in different leukocyte subtypes. However, the role of Skap2 in &bgr;2 integrin activation and neutrophil recruitment is unknown. In this study, we demonstrate the crucial role of Skap2 in regulating actin polymerization and binding of talin-1 and kindlin-3 to the &bgr;2 integrin cytoplasmic domain, thereby being indispensable for &bgr;2 integrin activation and neutrophil recruitment. The direct interaction of Skap2 with the Wiskott–Aldrich syndrome protein via its SH3 domain is critical for integrin activation and neutrophil recruitment in vivo. Furthermore, Skap2 regulates integrin-mediated outside-in signaling events and neutrophil functions. Thus, Skap2 is essential to activate the &bgr;2 integrins, and loss of Skap2 function is sufficient to cause a LAD-like phenotype in mice.

Methodologic issues in the measurement of interleukin-16 in clinical blood samples using immunoassays.

Quantitation of interleukin-16 (IL-16) in clinical blood samples has strongly increased, since IL-16 appears to be involved in the pathogenesis of several inflammatory diseases. IL-16 is synthesized in the cell cytoplasm as precursor protein (pro-IL-16), which can be processed by caspase-3 into N-terminal (N-IL-16) and C-terminal (C-IL-16) fragments. C-IL-16 is described to be subsequently secreted. Using commercially available IL-16 ELISA, a pro-IL-16 ELISA and immunoprecipitation analysis, we investigated, whether type and handling of blood samples influence IL-16 quantitation and whether existing IL-16 ELISA are specific for C-IL-16. We observed that cell-rich plasma samples reflect falsely-elevated IL-16 concentrations due to cell contaminations. Interestingly, not C-IL-16, but pro-IL-16 represents the major IL-16 form in cell-rich plasma samples. Notably, commercially IL-16 ELISA could not distinguish between C-IL-16 and pro-IL-16. Thus, cell-rich plasma samples should not be used for IL-16 measurements and new methods are necessary for quantitation of C-IL-16 and pro-IL-16 uniquely.