Apoptotic Effects of Antilymphocyte Globulins on Human Pro-inflammatory CD4+CD28− T-cells

BACKGROUND: Pro-inflammatory, cytotoxic CD4(+)CD28(-) T-cells with known defects in apoptosis have been investigated as markers of premature immuno-senescence in various immune-mediated diseases. In this study we evaluated the influence of polyclonal antilymphocyte globulins (ATG-Fresenius, ATG-F) on CD4(+)CD28(-) T-cells in vivo and in vitro. PRINCIPAL FINDINGS: Surface and intracellular three colour fluorescence activated cell sorting analyses of peripheral blood mononuclear cells from 16 consecutive transplant recipients and short-term cell lines were performed. In vivo, peripheral levels of CD3(+)CD4(+)CD28(-) T-cells decreased from 3.7 ± 7.1% before to 0 ± 0% six hours after ATG-F application (P = 0.043) in 5 ATG-F treated but not in 11 control patients (2.9 ± 2.9% vs. 3.9 ± 3.0%). In vitro, ATG-F induced apoptosis even in CD4(+)CD28(-) T-cells, which was 4.3-times higher than in CD4(+)CD28(+) T-cells. ATG-F evoked apoptosis was partially reversed by the broad-spectrum caspase inhibitor benzyloxycarbonyl (Cbz)-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk) and prednisolon-21-hydrogensuccinate. ATG-F triggered CD25 expression and production of pro-inflammatory cytokines, and induced down-regulation of the type 1 chemokine receptors CXCR-3, CCR-5, CX3CR-1 and the central memory adhesion molecule CD62L predominately in CD4(+)CD28(-) T-cells. CONCLUSION: In summary, in vivo depletion of peripheral CD3(+)CD4(+)CD28(-) T-cells by ATG-F in transplant recipients was paralleled in vitro by ATG-F induced apoptosis. CD25 expression and chemokine receptor down-regulation in CD4(+)CD28(-) T-cells only partly explain the underlying mechanism

The cell-type specificity of mitochondrial dynamics.

International audienceRecent advances in mitochondrial imaging have revealed that in many cells mitochondria can be highly dynamic. They can undergo fission/fusion processes modulated by various mitochondria-associated proteins and also by conformational transitions in the inner mitochondrial membrane. Moreover, precise mitochondrial distribution can be achieved by their movement along the cytoskeleton, recruiting various connector and motor proteins. Such movement is evident in various cell types ranging from yeast to mammalian cells and serves to direct mitochondria to cellular regions of high ATP demand or to transport mitochondria destined for elimination. Existing data also demonstrate that many aspects of mitochondrial dynamics, morphology, regulation and intracellular organization can be cell type-/tissue-specific. In many cells like neurons, pancreatic cells, HL-1 cells, etc., complex dynamics of mitochondria include fission, fusion, small oscillatory movements of mitochondria, larger movements like filament extension, retraction, fast branching in the mitochondrial network and rapid long-distance intracellular translocation of single mitochondria. Alternatively, mitochondria can be rather fixed in other cells and tissues like adult cardiomyocytes or skeletal muscles with a very regular organelle organization between myofibrils, providing the bioenergetic basis for contraction. Adult cardiac cells show no displacement of mitochondria with only very small-amplitude rapid vibrations, demonstrating remarkable, cell type-dependent differences in the dynamics and spatial arrangement of mitochondria. These variations and the cell-type specificity of mitochondrial dynamics could be related to specific cellular functions and demands, also indicating a significant role of integrations of mitochondria with other intracellular systems like the cytoskeleton, nucleus and endoplasmic reticulum (ER)

Real-Time Live Confocal Fluorescence Microscopy as a New Tool for Assessing Platelet Vitality

Background: Assessment of platelet vitality is important for patients presenting with inherited or acquired disorders of platelet function and for quality assessment of platelet concentrates. Methods: Herein we combined live stains with intra-vital confocal fluorescence microscopy in order to obtain an imaging method that allows fast and accurate assessment of platelet vitality. Three fluorescent dyes, FITC-coupled wheat germ agglutinin (WGA), tetramethylrhodamine methyl ester perchlorate (TMRM) and acetoxymethylester (Rhod-2), were used to assess platelet morphology, mitochondrial activity and intra-platelet calcium levels. Microscopy was performed with a microlens-enhanced Nipkow spinning disk-based system allowing live confocal imaging. Results: Comparison of ten samples of donor platelets collected before apheresis and platelets collected on days 5 and 7 of storage showed an increase in the percentage of Rhod-2positive platelets from 3.6 to 47 and finally to 71%. Mitochondrial potential was demonstrated in 95.4% of donor platelets and in 92.5% of platelets stored for 7 days. Conclusion: Such fast and accurate visualization of known key parameters of platelet function could be of relevance for studies addressing the quality of platelets after storage and additional manipulation, such as pathogen inactivation, as well as for the analysis of inherited platelet function disorders.Hintergrund: Die Vitalitätsbestimmung von Blutplättchen ist sowohl für die Analyse angeborener Plättchendefekte als auch für die Qualitätsbestimmung von Plättchenkonzentraten von zentraler Bedeutung. Methoden: In der vorliegenden Arbeit stellen wir eine Methode vor, die mittels einer Kombination von Vitalfarbstoffen und konfokaler «Real time»-Mikroskopie neue Einblicke in die Vitalitätsbestimmung lebender Plättchen ermöglicht. Mittels der Zugabe von FITC-gekoppeltem Weizenkeimlektin (WGA), Tetramethylrhodamin-Methylesterperchlorat (TMRM) und Acetoxymethylester (Rhod-2) wurde bei lebenden Blutplättchen deren Morphologie, mitochondriale Aktivität und Veränderungen im Calcium-Haushalt im Rahmen der Lagerung analysiert. Für die Mikroskopie wurde ein Nipkow-System gewählt, das eine konfokale Mikroskopie lebender Zellen ermöglicht. Ergebnisse: Der Vergleich von 10 humanen Blutplättchenproben zu Beginn bzw. nach 5 und 7 Tagen Lagerung zeigte einen Anstieg der Rhod-2-positiven Plättchen von 3,6 über 47 auf 71%. Die Anzahl der Blutplättchen mit TMRM-positiven Mitochondrien hingegen lag vor der Lagerung bei 95,4% und nach den 7 Tagen Lagerung bei 92,5%. Schlussfolgerung: Die hier vorgestellte Methodik der Bildgebung zur Bestimmung vitaler Parameter von Blutplättchen eignet sich als ergänzende Analysemodalität für eine bessere Bestimmung der Blutplättchenqualität.Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

Peroxisome proliferator-activated receptor-alpha activation inhibits Langerhans cell function

Epidermal Langerhans cells (LC) play a pivotal role in initiating and maintaining primary immune responses in the skin. In the present study, we asked whether peroxisome proliferator-activated receptor-alpha (PPARalpha) activation modulates LC function. Our results show that PPARalpha is expressed in immature LC and is down-regulated in mature LC suggesting that an early decrease of PPARalpha expression in LC may allow them to mature after contact with an Ag. We further show that pharmacologic PPARalpha activation inhibits LC maturation, migratory capacity, cytokine expression, and the ability to drive T cell proliferation. Moreover, PPARalpha activation inhibits NF-kappaB but not stress-activated protein kinase/JNK, p38MAPK, and ERK1/2. In conclusion, PPARalpha activation by endogenous ligands may provide a molecular signal that allows LC to remain in an immature state within the epidermis for extended periods of time despite minor environmental stimuli

Treatment with polyclonal antilymphocyte globulins induces depletion of circulating CD3⁺CD4⁺CD28⁻ T-cells in transplant recipients.

<p>Prevalences of peripheral circulating CD3⁺CD4⁺CD28⁻ T-cells in 16 age- and sex-matched healthy controls, 5 allograft recipients before and 6 hours after the application of ATG-F and 11 control patients before and 6 hours after organ transplantation. Data are given as mean±standard error of the mean (SEM).</p

Production of pro-inflammatory cytokines by polyclonal antilymphocyte globulins in CD4⁺CD28⁻ T-cells.

<p><i>(</i><b><i>A</i></b><i>)</i> To test whether ATG-F treatment leads to cytokine production of CD4⁺ T-cell subsets <i>in vitro</i>, stimulation with rabbit IgG (rIgG), phorbol 12-myristate 13-acetate (PMA)/ionomycin, and ATG-F at a dose of 300 µg/ml or 1000 µg/ml and intracellular cytokine staining for IFN-γ, TNF-α and IL-4 was performed (n = 6). Whiskers box plots show 50% of cases within the boxes and all data excluding mavericks between the end-points of the whiskers (lines). An asterisk indicates significant differences (P<0.05) between CD28⁺ and CD28⁻CD4⁺ T-cell subsets. Depicting significances between rabbit IgG, PMA/ionomycin and ATG-F induced cytokine expression broken and continuous lines were used for CD28⁺ and CD28⁻CD4⁺ T-cells, respectively. <i>(</i><b><i>B</i></b><i>)</i> Representative dot plot and histograms depicting ATG-F induced production of TNF-α after 4 hours of stimulation in supramitogenic dosages of 300 µg/ml and 1000 µg/ml in comparison to stimulation with unspecific rIgG and PMA/ionomycin in the presence of brefeldin A. Gates were set on lymphocytes (forward and sideward scatter) as well as on CD4⁺CD28⁺ and CD4⁺CD28⁻ cells, and markers according to the negative control.</p

Down-regulation of Th1 type chemokine and leukocyte homing receptors by antilymphocyte globulins in CD4⁺CD28⁻ T-cells.

<p>Dose dependent <i>in vitro</i> effects of ATG-F on surface expression of the type 1 chemokine receptors <i>(</i><b><i>A</i></b><i>)</i> CCR-5, <i>(</i><b><i>B</i></b><i>)</i> CXCR-3, <i>(</i><b><i>C</i></b><i>)</i> CX3CR-1, the type 2 chemokine receptor <i>(</i><b><i>D</i></b><i>)</i> CCR-4, and the central memory receptors <i>(</i><b><i>E</i></b><i>)</i> CD62L and <i>(</i><b><i>F</i></b><i>)</i> CCR-7 on CD4⁺ T-cell subsets in comparison to rabbit IgG (rIgG) (n = 6). Data are given as mean (bars) and standard deviation (lines) for CD4⁺CD28⁺ (white) and CD4⁺CD28⁻ T-cells (grey). An asterisk indicates significant differences (P<0.05) between CD28⁺ and CD28⁻CD4⁺ T-cell subsets. Depicting significances between rabbit IgG and ATG-F induced modulation of chemokine receptor expression broken and continuous lines were used for CD28⁺ and CD28⁻CD4⁺ T-cells, respectively.</p

Antilymphocyte globulin-triggered apoptosis of CD4⁺ T-cells partially depends on activation of caspases.

<p>To investigate the underlying mechanisms of apoptosis induction in CD4⁺ T-cells by ATG-F <i>(</i><b><i>A</i></b><i>)</i> a caspase-mediated mechanism (n = 5) was analysed by AnnexinV staining in flow cytometry. <i>(</i><b><i>B</i></b><i>)</i> As the immunosuppressive agents prednisolon-21-hydrogensuccinate (glucocorticoid), FK506 and cyclosporine A are known to interfere with the interleukin (IL)-2 pathway, their influence on ATG-F mediated apoptosis was examined in CD28⁺ and CD28⁻CD4⁺ T-cell subsets (n = 6). Data are given as mean (bars) and standard deviation (lines) for CD4⁺CD28⁺ (white) and CD4⁺CD28⁻ T-cells (grey). An asterisk indicates significant differences (P<0.05) between CD28⁺ and CD28⁻CD4⁺ T-cell subsets. Depicting significances broken and continuous lines were used for CD28⁺ and CD28⁻CD4⁺ T-cells, respectively.</p