Upregulation Of CD36 Research Articles

C-Reactive Protein Isoforms Differ in Their Effects on Thrombus Growth

We studied the impact of native (natCRP) and modified CRP (mCRP) isoforms on platelet adhesion and thrombus growth under arterial flow. Blood was perfused over type I collagen at a wall shear rate of 1500 s(-1), and platelet deposition and thrombus growth were evaluated by confocal microscopy. natCRP and mCRP were either incubated with blood before perfusion experiments or immobilized in the collagen surface and exposed to flowing blood. mCRP significantly increased platelet adhesion and thrombus growth when directly incubated with blood and when immobilized on a collagen surface (P<0.05). In contrast, natCRP did not exert any effect. Confocal immunohistochemistry revealed the presence of CRP on the surface of adhered platelets and within the thrombus and showed an upregulation of P-selectin and CD36 in effluent platelets preincubated with mCRP (P<0.05). Flow cytometry analysis of agonist-induced platelet activation demonstrated that mCRP, but not natCRP, significantly increased platelet surface P-selectin (P<0.05) without modifying CD63 and PAC-1. Our data indicate that whereas serum natCRP may not affect thrombus growth, mCRP displays a prothrombotic phenotype enhancing not only platelet deposition, but also thrombus growth under arterial flow conditions.

Pioglitazone promotes peripheral nerve remyelination after crush injury through CD36 upregulation

In our previous study, we found that CD36-deficient mice showed significant delays in peripheral nerve remyelination after sciatic nerve crush injury and suggested that CD36 played an important role in the restoration of injured peripheral nerves. The aim of this study was to investigate whether CD36 upregulation can promote peripheral nerve remyelination. We made crush injury that caused demyelination and mild axonal degeneration to sciatic nerves and investigated the effect of pioglitazone (PIO) on the remyelination post-injury in C57Bl/6 wild-type and CD36-deficient mice. The immunohistochemistry with anti-CD36 antibody showed that CD36 was upregulated in macrophages infiltrating peripheral nerves from the wild-type mice by PIO administration at 1 week post-injury. The lectin histochemistry represented that infiltrating macrophages lessened in the wild-type mice at 3 weeks post-injury by PIO administration. General histopathology and morphometry indicated that thinly myelinated fibers and naked axons diminished in PIO-treated wild-type mice compared with non-treated wild-type mice at 3 weeks post-injury. No significant differences were observed in remyelination and number of infiltrating macrophages between PIO-treated and non-treated CD36-deficient mice. These results indicate that PIO promotes peripheral nerve remyelination possibly through CD36. It may be possible to apply PIO to the remedy against demyelinating neuropathies.

Macrophage Secretory Phospholipase A2 Group X Enhances Anti-inflammatory Responses, Promotes Lipid Accumulation, and Contributes to Aberrant Lung Pathology

Secreted phospholipase A2 group X (sPLA(2)-X) is one of the most potent enzymes of the phospholipase A(2) lipolytic enzyme superfamily. Its high catalytic activity toward phosphatidylcholine (PC), the major phospholipid of cell membranes and low-density lipoproteins (LDL), has implicated sPLA(2)-X in chronic inflammatory conditions such as atherogenesis. We studied the role of sPLA(2)-X enzyme activity in vitro and in vivo, by generating sPLA(2)-X-overexpressing macrophages and transgenic macrophage-specific sPLA(2)-X mice. Our results show that sPLA(2)-X expression inhibits macrophage activation and inflammatory responses upon stimulation, characterized by reduced cell adhesion and nitric oxide production, a decrease in tumor necrosis factor (TNF), and an increase in interleukin (IL)-10. These effects were mediated by an increase in IL-6, and enhanced production of prostaglandin E(2) (PGE(2)) and 15-deoxy-Delta12,14-prostaglandin J(2) (PGJ(2)). Moreover, we found that overexpression of active sPLA(2)-X in macrophages strongly increases foam cell formation upon incubation with native LDL but also oxidized LDL (oxLDL), which is mediated by enhanced expression of scavenger receptor CD36. Transgenic sPLA(2)-X mice died neonatally because of severe lung pathology characterized by interstitial pneumonia with massive granulocyte and surfactant-laden macrophage infiltration. We conclude that overexpression of the active sPLA(2)-X enzyme results in enhanced foam cell formation but reduced activation and inflammatory responses in macrophages in vitro. Interestingly, enhanced sPLA(2)-X activity in macrophages in vivo leads to fatal pulmonary defects, suggesting a crucial role for sPLA(2)-X in inflammatory lung disease.

Candida albicans-Induced DC Activation Partially Restricts HIV Amplification in DCs and Increases DC to T-Cell Spread of HIV

Dendritic cells (DCs) are central to the innate and adaptive responses needed to control pathogens, yet HIV exploits DCs to promote infection. The influence of other pathogens on DC-HIV interplay has not been extensively studied. We used Candida albicans (Candida) as a model pathogen which elicits innate DC responses that are likely important in controlling Candida by healthy immune systems. HIV did not impede Candida-specific DC activation. Candida-induced CD80 and CD83 upregulation was greater in DCs that had captured HIV, coinciding with increased amplification in presence of T cells and reduced but persistent low-level DC infection. In contrast, HIV-infected DCs matured normally in response to Candida, but this did not shut down HIV replication in DCs, and again Candida augmented HIV amplification in DC-T-cell mixtures. HIV-infected DCs secreted more IL-10 and IL-1beta earlier than uninfected DCs and initially induced a higher frequency of CD4CD25FoxP3 T-regulatory cells in response to Candida. Elevated early IL-10 production in cocultures was evident only when azidothymidine (AZT) was included to limit T-regulatory cell infection and destruction. Therefore, HIV manipulates the DC's innate and adaptive responses to Candida to further augment HIV spread, ultimately destroying the cells needed to limit candidiasis.

Low-Intensity Exercise Exerts Beneficial Effects on Plasma Lipids via PPARγ

An important mechanism by which physical activity reduces the risk of cardiovascular disease is through regulating plasma lipids. We investigated whether low-intensity exercise modulates lipid metabolism and the transcription factors peroxisome proliferator-activated receptor gamma (PPARgamma) and liver X receptor alpha (LXRalpha) responsible for controlling reverse cholesterol transport (RCT). Thirty-four sedentary adults, mean age 45.6 +/- 11.1 yr, participated in an 8-wk low-intensity exercise program consisting of walking 10,000 steps, three times a week. Subjects were randomly allocated to either an exercise group or a sedentary control group, and serum lipid or lipoprotein concentrations were determined. Compared with controls, there was a significant decrease in total cholesterol (preexercise, 5.73 +/- 1.39 mmol x L; postexercise, 5.32 +/- 1.28 mmol x L) and a significant increase in HDL (preexercise, 1.46 +/- 0.47 mmol x L; postexercise, 1.56 +/- 0.50 mmol x L) after the exercise program. There was a significant increase in serum oxidized LDL (oxLDL) concentrations in the exercise group before and after exercise (0 wk, 554 +/- 107 ng x mL; 4 wk, 698 +/- 134 ng x mL; 8 wk, 588 +/- 145 ng x mL). A significant increase in leukocyte mRNA expression for PPARgamma (4 wk, 1.8 +/- 0.9-fold; 8 wk, 4.3 +/- 1.9-fold) was observed, which was reinforced by increased PPARgamma DNA-binding activity postexercise (preexercise, 0.22 +/- 0.09 OD units; postexercise, 1.13 +/- 0.29 OD units). A significant increase in gene expression was observed for the oxLDL scavenger receptor CD36 (4 wk, 3.8 +/- 0.6-fold; 8 wk, 2.7 +/- 0.5-fold) and LXRalpha (8 wk, 3.5 +/- 0.8-fold). Two LXRalpha-regulated genes involved in RCT, namely, ATP-binding cassette transporters A1 and GI (ABCA1 and ABCG1, respectively), were significantly up-regulated postexercise (8 wk: ABCA1, 3.46 +/- 0.56-fold; ABCG1, 3.06 +/- 0.47-fold). We propose that the net effect of these changes may be to increase oxLDL uptake, to stimulate RCT, and thus to promote clearance of proatherogenic lipids from the vasculature, ultimately contributing to the cardiovascular benefits of low-intensity aerobic exercise.

Nrf2 regulates the alternative first exons of CD36 in macrophages through specific antioxidant response elements

We previously demonstrated that Nrf2 regulates oxidized LDL-mediated CD36 expression in macrophages. The current study aimed to determine the mechanism of Nrf2-mediated macrophage CD36 induction. Treatment with the Nrf2 activator diethylmaleate, but not PPARγ specific ligands, caused marked upregulation of CD36 in mouse macrophage RAW264.7 cells. Similarly, Nrf2 activators induced CD36 expression in bone marrow-derived macrophages in a Nrf2-dependent manner. Induced expression of the three alternative first exons of mouse CD36, deemed 1A, 1B, and 1C, occurred upon Nrf2 activation with exon1A mainly contributing to the CD36 expression. Four antioxidant response elements (AREs) lie within close proximity to these three exons, and chromatin immunoprecipitation assays demonstrated that two AREs upstream of exon1A, the distal 1A-ARE1, and the proximal 1A-ARE2, were Nrf2-responsive. Luciferase reporter assays conclusively demonstrated that 1A-ARE2 is the critical regulatory element for the Nrf2-mediated gene expression. Thus Nrf2 directly regulates CD36 gene expression by binding to 1A-ARE2.

Oxidised LDL up-regulate CD36 expression by the Nrf2 pathway in 3T3-L1 preadipocytes

The effect of oxLDL on CD36 expression has been assessed in preadipocytes induced to differentiate. Novel evidence is provided that oxLDL induce a peroxisome proliferator-activated receptor γ-independent CD36 overexpression, by up-regulating nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2). The nuclear translocation of Nrf2 appeared to depend on PKC pathway activation. In adipocytes, the CD36 up-regulation may indicate a compensation mechanism to meet the demand of excess oxLDL and oxidised lipids in blood, reducing the risk of atherogenesis. Besides strengthening the hypothesis that oxLDL can contribute to the onset of insulin-resistance, data herein presented highlight the significance of oxLDL-induced CD36 overexpression within the cellular defence response.

Increased expression and internalization of the endotoxin coreceptor CD14 in enterocytes occur as an early event in the development of experimental necrotizing enterocolitis

Background The early signaling events in the development of necrotizing enterocolitis (NEC) remain undefined. We have recently shown that the endotoxin (lipopolysaccharide [LPS]) receptor toll-like receptor 4 (TLR4) on enterocytes is critical in the pathogenesis of experimental NEC. Given that the membrane receptor CD14 is known to facilitate the activation of TLR4, we now hypothesize that endotoxemia induces an early upregulation of CD14 in enterocytes and that this participates in the early intestinal inflammatory response in the development of NEC. Methods IEC-6 enterocytes were treated with LPS (50 μg/mL), and the subcellular localization of CD14 and TLR4 was assessed by confocal microscopy. C57/Bl6 or CD14−/− mice were treated with LPS (5 mg/kg), whereas experimental NEC was induced using a combination of gavage formula feeding and intermittent hypoxia. CD14 expression was determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and reverse transcriptase–polymerase chain reaction, and interleukin 6 was quantified by enzyme-linked immunosorbent assay and reverse transcriptase–polymerase chain reaction. Results Exposure of IEC-6 enterocytes to LPS led to an initial, transient increase in CD14 expression. The early increase in CD14 expression was associated with internalization of CD14 to a perinuclear compartment where increased colocalization with TLR4 was noted. The in vivo significance of these findings is suggested as treatment of mice with LPS led to an early increase in CD14 expression in the intestinal mucosa, whereas the persistent endotoxemia of experimental NEC was associated with decreased CD14 expression within enterocytes. Conclusions LPS signaling in the enterocyte is marked by an early, transient increase in expression of CD14 and redistribution of the receptor. This process may contribute to the early activation of the intestinal inflammatory response that is observed in the development of NEC.

Oxidation as a crucial reaction for cholesterol to induce tissue degeneration: CD36 overexpression in human promonocytic cells treated with a biologically relevant oxysterol mixture

Oxidative stress, inflammation and altered cholesterol metabolism and levels are among the pathogenetic mechanisms of cognitive impairment that may accompany aging. Within the research area of hypercholesterolemia and age-related disease processes, the molecular mechanisms of cholesterol interaction with the inflammatory cells of the macrophage lineage are yet to be elucidated. We thus investigated the effect of both non-oxidized and oxidized cholesterol on monocytic cell differentiation and foam cell formation, as it occurs within vascular lesions during progression of atherosclerosis. In vitro experiments performed on human U937 promonocytic cells showed that a biologically representative mixture of oxysterols markedly stimulated CD36 expression and synthesis. In contrast, non-oxidized cholesterol did not exert any effect on CD36 mRNA and protein levels. Furthermore, the oxysterol-induced up-regulation of CD36 appeared to be based on the subsequent activation of protein kinase Cdelta (PKCdelta), extracellular signal-regulated kinase 1/2 (ERK1/2) and peroxisome proliferator-activated receptor gamma (PPARgamma). Cells overexpressing CD36 were indeed able to actively take up oxidized low-density lipoproteins, and become foam cells. The essential role of ERK pathway and CD36 receptor in oxysterol-induced foam cell formation was proved by the prevention of the latter event when monocytic cells were incubated in the presence of MEK1/2 selective inhibitor or anti-CD36 specific antibody. These experimental findings point to cholesterol oxidation as an essential reaction for this sterol to exert cellular stress and tissue damage in age-related diseases in which inflammation represents a main driving force.

Protective effect of bicyclol on acute alcohol-induced liver injury in mice

Oxidative stress, cytokine over expression and Kupffer cell activation are well-documented pathological factors in the development of alcoholic liver disease. Bicyclol is a novel synthetic anti-hepatitis drug with anti-oxidative and anti-inflammatory property. The present study was to investigate the effect of bicyclol on acute alcohol-induced liver injury and related mechanisms in mice. Bicyclol (200, 300 mg/kg) was given to mice by gavage for three times. Alcohol (6 g/kg) was administered orally 1 h after the last dose of bicyclol. All animals were sacrificed at different time points after alcohol administration. Liver injury was evaluated by biochemical and histopathological examination. Lipid peroxidation and the activity of antioxidants were measured by spectrophotometric method. Expression of cytokines and CD14 were determined by enzyme-linked immunosorbent assay, reverse transcriptional-polymerase chain reaction and immunohistochemical staining. As a result, bicyclol pretreatment significantly protected against acute alcohol-induced liver injury as evidenced by the decrease of elevated serum alanine aminotransferase and hepatic triglyceride, and the attenuation of histopathological changes in mice. In addition, bicyclol remarkably alleviated the formation of thiobarbituric acid-reactive substance and restored impaired antioxidants, including glutathione, superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase. Over-expression of cytokines, such as tumor necrosis factor α and interleukin 1β, elevated plasma endotoxin level, and up-regulation of CD14 were also suppressed by bicyclol in alcohol-intoxicated mice. The protective effect of bicyclol on alcohol-induced hepatotoxicity was mainly due to its ability to attenuate oxidative stress, suppress the cytokine expression at both protein and gene level, and inhibit the activation of Kupffer cells by decreasing plasma endotoxin level and CD14 expression.

Rosiglitazone but not losartan prevents Nrf-2 dependent CD36 gene expression up-regulation in an in vivo atherosclerosis model

BackgroundThiazolidinediones exert anti-inflammatory and anti-oxidative roles and attenuate atherosclerosis by mechanisms partially independent of their metabolizing actions. High doses of angiotensin type 1 receptor (AT1R) blocker losartan (LST) seem to promote fat cell formation by preserving PPARγ activity.MethodsC57BL/6J diet-induced atherosclerotic susceptible mice randomly received a normal or a high-fat high-cholesterol (HFHC) diet and were treated with rosiglitazone (RG), LST or a vehicle for 12 weeks.ResultsHFHC was associated with increased PPARγ gene expression without an over regulation of PPARγ responsive genes, whereas RG and LST treatments were found to maintain PPARγ activity without resulting in increased PPARγ gene expression. A better anti-inflammatory and antioxidant profile in mice treated with RG regarding LST was observed in spite of a similar PPARγ preserved activity. Chromatin immunoprecipitation (ChIP) assays revealed that animals under HFHC diet treated with RG showed a significant nuclear factor erythroid 2-like 2 (Nrf2)-dependent down-regulation of the expression of the CD36 gene.ConclusionThe PPARγ agonist RG exerts antioxidant properties that significantly reduced Nrf-2-dependent CD-36 up-regulation in mice under HFHC diet. Because LST treatment was also associated with a preserved PPARγ activity, our data suggests that these RG antioxidant effects are partially independent of its PPARγ metabolizing properties.

Differential costimulation through CD137 (4–1BB) restores proliferation of human virus-specific “effector memory” (CD28− CD45RAHI) CD8+ T cells

In healthy carriers of human cytomegalovirus (HCMV), the virus-specific memory CD8(+) T-cell population is often dominated by CD28(-) CD45RA(hi) cells that exhibit direct ex vivo cytotoxicity but whose capacity for proliferation and generation of further memory cells has been questioned. We show that when highly purified CD28(-) CD45RA(hi) CD8(+) T cells are stimulated with viral peptide presented by autologous monocytes, the virus-specific T cells show early up-regulation of CD137 (4-1BB) and CD278 (ICOS), re-express CD28, and proliferate with similarly high cloning efficiency in limiting dilution analysis as CD28(+) CD45RO(hi) cells or CD28(-) CD45RO(hi) cells. Using peptide-pulsed autologous fibroblasts transfected with individual costimulatory ligands as antigen presenting cells, we showed CD137L to be a key costimulatory ligand for proliferation of CD28(-) CD45RA(hi) CD8(+) T cells and not CD80, CD86, or CD275 (ICOSL). Therefore, CD28(-) CD45RA(hi) CD8(+) T cells were not terminally differentiated but required a specific costimulatory signal for proliferation.

Adhesion molecules and differentiation syndrome: phenotypic and functional analysis of the effect of ATRA, As2O3, phenylbutyrate, and G-CSF in acute promyelocytic leukemia

Differentiation Syndrome (DS) is a treatment complication which can occur in patients treated with acute promyelocytic leukemia (APL) with all transretinoic acid (ATRA) or As(2)O(3), and is characterized by enhanced leukocyte transmigration. As(2)O(3), Phenylbutyrate (PB) and G-CSF are known to potentiate ATRA effects. Our aim was to analyze the changes in expression and function of adhesion molecules induced by ATRA, As(2)O(3), G-CSF and PB, and their association. APL blasts and NB4 cells were treated with ATRA, As(2)O(3), PB, G-CSF or their association and the expression of adhesion molecules was determined by flow cytometry. Cell adhesion was evaluated in vitro using Matrigel and for the in vivo analysis, Balb-c mice were injected with NB4 cells pre-treated with ATRA, As(2)O(3), ATRA+G-CSF or ATRA+As(2)O(3). In addition, CD54 and CD18 knock-out mice were injected with NB4 cells and concomitantly treated with ATRA. In both models, the MPO activity in the lungs was determined 6 hours after the injection of the cells. In NB4 and APL blasts, ATRA and As(2)O(3) increased CD54 expression, but no synergism was detected. CD11b and CD18 were also up-regulated by ATRA in primary cells. PB and G-CSF had no effect, but the latter potentiated ATRA-induced CD18 up-regulation. These changes were accompanied by increased adhesion to Matrigel and to lung microvasculature, and reversed by anti-CD54, anti-CD18 antibodies. In CD54 and CD18 knock-out mice the ATRA effect was canceled. The use of As(2)O(3), PB and G-CSF in association with ATRA should not aggravate DS in APL.

Modulation of CD20 Expression in Rituximab-Sensitive (RSCL) and Rituximab Resistant Cell Lines (RRCL) Using IL-4 and Bryostatin-1.

While the use of rituximab in combination with chemotherapy resulted in an improved survival among various subtypes of B-cell lymphomas, a significant number of patients fail to respond or relapse as a consequence of intrinsic or acquired resistance. A change in CD20 antigen density expression is a potential mechanism to explain rituximab resistance. Moreover, several groups of investigators are focused in understanding the mechanisms that regulate CD20 expression and to develop therapeutic strategies to up-regulate CD20 expression (i.e. IL-4, GM-CSF or Bryostatin-1). Up-regulation of CD20 in DB and Ramos cells by Bryostatin-1 was found to be PKC and Erk dependent. In an attempt to characterize the mechanisms responsible for rituximab resistance we developed several RRCL derived from rituximab-sensitive RL and Raji cells. We have demonstrated a significant down-regulation of CD20mRNA and CD20 surface antigen in RRCL when compared to RSCL. In our present work we evaluated the mechanisms involved in the mRNA down-regulation of CD20 and the potential of IL-4 and Bryostatin-1 in modulating CD20 antigen expression among a panel of RRCL. To this end, RSCL and RRCL were treated with either 5ng/ml of IL-4 or 3 different doses of Bryostatin-1(1, 3 or 5ng/ml). Nuclear and cytosolic extract were also obtained from RSCL RL and RRCL RL-4RH after 24, 48 and 74 hrs exposure to IL-4 (5ng/ml) or control. Differences in the expression of key regulatory transcription factors for B-cell lymphocyte development (PU.1, Oct-2, Pax5, E2A and EBF) were studied by Western Blotting. Previously, we found a significant down-regulation of CD20 antigen in the RRCL. An up-regulation of cytosolic and surface CD20 was detected in RRCL exposed to either IL-4 or Bryostatin by western blotting and flow cytometric analysis. Besides, a higher expression of Pax5, PU.1, EBF was found in nuclear fractions of RRCL when compared to RSCL. In vitro exposure of RRCL to IL-4 decreases the expression of PU.1, Oct-2, Pax5 and EBF in nuclear extracts from RSCL or RRCL when compared with controls treated cells. Our data suggest that rituximab resistance is associated with the up-regulation of transcription factors PU.1, Oct-2, Pax5 and EBF and concomitant suppression of CD20 antigen. Future study of how these agents induce CD20 expression in RRCL will provide a potential means to reversing resistance towards rituximab treatment.

Clonal Analysis of Progressive Maturation of Erythroid Cells from Human Embryonic Stem Cell-Derived Definitive Hematopoiesis.

A critical issue for utilization of human embryonic stem cells (hESCs) in regenerative medicine is whether they can derive terminally mature progenies with normal function. We recently developed an efficient co-culture system of hESCs with murine fetal liver-derived stromal cells (mFLSCs), which enabled us to produce a large quantity of hESC-derived erythroid progenitor cells. By combining the hESC/mFLSC co-culture and a clonal culture, we analyzed the development of hESC-derived erythroid cells at a clone level. From day 10 of the co-culture, hESCs generated both erythroid colony- and erythroid burst-forming cells (E-CFCs and E-BFCs, respectively), and their numbers rapidly increased and reached a peak at day 14. By a clone-culture, we analyzed the erythroid cells in individually colonies and bursts by immunostaining. All erythroid cells in the colonies and bursts expressed alpha and gama globins, while beta and epsilon globins were expressed in a portion of these erythroid cells, but all the colonies and bursts contained a substantial number of beta and epsilon globin-expressing cells. We then examined the globin expression in individual bursts at day 12, 14, 16 and 18 of the co-culture. All erythroid cells in the bursts contained alpha and gama globins while beta globin-expressing erythroid cells gradually increased up to 100% until day 18, along with a decrease of epsilon globin-expressing erythroid cells in individual bursts. We then examined the globin expression of erythroid cells through day 12 to 18 in clonal culture of E-BFC at day 14 of hESC/mFLSC co-culture. Resultantly, beta globin-positive cells gradually increased up to 100% at day 18 of clonal culture, while epsilon globin-positive cells decreased. We equally divided erythroid cells in single bursts at day 12 of clonal culture; half was processed to the analysis of globin expression, and the other half was re-cultured for additional 6 days, and then their globin expression was examined. We again observed the time-dependent increase of beta globin and decrease of epsilon globin in hESC-derived erythroid cells at a clone level. On the other hand, hESC-derived erythroid cells could undergo encleation and their differentiation, when defined by co-expression of Glycophorin A, could be confirmed by up-regulation of CD71 in association with down-regulation of CD81 by flow cytometry, showing a similar pattern to human cord blood-derived erythroid cells. Furthermore, these hESC-derived erythroid cells could function as oxygen carrier, and had a sufficient glucose-6-phosphate dehydrogenase activity. The present study provides an experimental model for investigating early development of human erythropoiesis at a clone level. It enables us to conduct subtle and detailed experiment on hemoglobin switching, and the pathogenesis and discovery of drugs for hereditary diseases in human erythrocyte development. More importantly, the time-dependent maturation of hESC-derived erythroid cells indicates that hESCs can be a novel source for therapeutic transfusion.

Toll-Like Receptor-Induced Expression of the NKG2D Ligand MICA on Monocytes Modulates Their Interaction with NK Cells.

Reciprocal interactions of NK cells with Dendritic Cells (DC) have been shown to influence activation of NK cells as well as maturation or lysis of DC and subsequent adaptive immune responses. However, little is known about the interaction of peripheral blood Monocytes with NK cells. Here we report that stimulation of Toll-like receptors (TLR) on Monocytes using LPS, Pam3Cys, R848 and PolyI:C induced mRNA and surface expression of MICA but no relevant changes of the further ligands of the activating immunoreceptor NKG2D, MICB or ULBP1-3. MICA upregulation upon stimulation with TLR ligands correlated with CD80 and HLA-DR upregulation and increased secretion of the pro-inflammatory cytokines IL-6, IL-8 and TNF by Monocytes. Interestingly, high MICA surface expression on activated Monocytes was not accompanied by a release of MICA in soluble form. The LPS-induced MICA upregulation by Monocytes was highly sensitive since it was observed with as little as 4 ng/ml LPS after 15 hours and was associated with IL-6 secretion and inhibition of apoptosis indicating that Monocytes were activated and functional. To determine whether upregulated MICA expression on Monocytes was detected by NK cells we took advantage of the fact that NKG2D is down-regulated after interacting with its ligands. While NKG2D levels on NK cells were not substantially changed in cultures with unstimulated Monocytes, a marked reduction of NKG2D was observed in the presence of activated MICA-expressing Monocytes. The modulation of NKG2D did not occur when NK cells and monocytes were separated by a transwell filter, which demonstrates that the numerous cytokines produced by TLR-activated Monocytes are not responsible for the NKG2D modulation and confirmes the role of MICA in Monocyte-NK cell interaction. Importantly, TLR-induced MICA expression on Monocytes stimulated IFN-γ production of NK cells, which could be reduced by addition of blocking anti-MICA/B F(ab')2 fragments while isotype control had no effect. This demonstrates that the observed stimulation of NK cells was in fact specifically due to NKG2D-NKG2DL interaction. Our data indicate that NKG2D-MICA interaction provides a mechanism by which NK cells and Monocytes may communicate directly during innate immune responses to infections in humans.

Rapid Detection and Isolation of Viable Virus-Reactive CD4+ and CD8+ T Cells Based on Activation-Induced CD137 Expression.

Current methods for the detection and isolation of antigen-specific CD4+ and CD8+ T cells require the availability of peptide/MHC multimers or are restricted to cells that produce cytokines after antigen contact. We have recently reported that de novo cell surface expression of the TNF receptor family member CD137 (4-1BB) identifies currently activated, but not resting, human alloreactive CD8+ T cells. This observation allowed us to develop a CD137-based technology for the depletion of alloreactive CD8+ T cells in vitro (Wehler et al. Blood 2007; 109:365–373). More recently, a similar approach has been described that uses activation-induced CD137 expression for the detection and enrichment of antigen-specific CD8+ T cells (Wolfl et al. Blood 2007; 110:201–210). In the current study we complement this work and demonstrate the transient up-regulation of CD137 directly on activated cytomegalovirus (CMV) or Epstein-Barr virus specific CD8+ T cells using peptide/HLA tetramer staining of PBMC from seropositive healthy individuals. Antigen-triggered CD137 expression was first detectable upon 6h of stimulation, and reached peak intensity at 24h, allowing the determination of a clear-cut population of CD137+ T cells at this time point. Most importantly, we also observed a similar CD137 expression kinetics (i.e. low baseline, maximum at 24h) on virus-specific CD4+ T cells upon activation with CMVpp65 peptides. The median frequencies of CMVpp65-reactive CD137+ cells measured ex vivo in 4 different CMV+ healthy donors after 24h of stimulation were 3.6% (range, 1.1–6.7) in CD8+ T cells and 2.7% (range, 0.8–6.4) in CD4+ T cells, respectively. We also analyzed PBMC derived from the same donors and left unstimulated, as well as PBMC from CMV-seronegative donors (n=3) stimulated with CMV peptides. None of these samples contained more than 0.3% CD137+ cells per total CD4+ and CD8+ T cells, thereby confirming the specificity of antigen-induced CD137 expression. We next established a two-step in vitro approach allowing the activation and subsequent CD137-based immunomagnetic cell sorting of virus-reactive CD4+ and CD8+ T cells at the same time. We demonstrated the suitability of this assay to isolate CMVpp65-reactive CD4+ and CD8+ T cells from PBMC of 6 CMV+ healthy individuals. Enriched fractions had a median purity of CD137+ cells of 69.4% (range 12.7–94.1) among CD4+ T cells and 70.6% (range, 28.5–93.4) among CD8+ T cells, respectively. The CD137+ populations could be expanded in vitro and showed CMVpp65-specific cytokine production by CD4+ and CD8+ T cells as well as a strong enrichment of CMVpp65/HLA tetramer-binding CD8+ T cells. We finally compared the efficiency of the CD137 assay with the IFN-γ secretion assay to isolate CMVpp65-specific CD4+ and CD8+ T cells from PBMC. Although both methods were performed at optimal conditions, the numbers of CD137+ T cells measured before and after enrichment were approximately 2-fold higher than those of IFN-γ+ cells (n=5), suggesting that CD137 might detect a broader repertoire of virus-reactive T cells. In conclusion, activation-induced CD137 expression provides a means for the rapid detection and isolation of viable virus-reactive CD4+ and CD8+ T cells. The CD137 assay is most attractive for the simultaneous targeting of both T-cell subsets in monitoring studies and adoptive immunotherapy trials.

A Phase I Study of Immune Gene Therapy for Patients with CLL Using a Membrane-Stable, Humanized CD154.

Chronic lymphocytic leukemia (CLL) is an ideal disease for therapeutic vaccine strategies. While the leukemia cells are usually stealth-like, avoiding T cell recognition, they can be manipulated through ligation of CD40 on their surface to become very effective antigen presenting cells (APCs). Ligation of CD40 leads to expression of CD80, CD86 and upregulation of CD54. Other biochemical changes occur upon ligation of CD40, including upregulation of CD95, DR5, and expression of Bid, predisposing the leukemia cells to death-receptor-induced apoptosis. CLL cells can be made to express CD154 (CD40-ligand) using a replication-defective adenovirus. A phase I clinical trial with autologous CLL cells transduced to express murine CD154 previously demonstrated tolerability and clinical activity with this strategy (Blood 96:2917, 2000). More recently, a recombinant CD154 (ISF35) was produced, based on the human CD154 backbone, incorporating murine sequences needed for expression on CLL cells and with the proteinase cleavage site removed. We evaluated this new transgene in a phase I clinical trial, expecting to have similar tolerability to the murine CD154. Transduction of CLL cells results in expression of ISF35, ligation of CD40 on transduced and bystander cells, and the resultant downstream changes needed for antigen presentation and sensitivity to death receptor-induced apoptosis. We conducted a phase I study of a single infusion of autologous CLL cells transduced to express ISF35. Three dose levels were evaluated with 3 patients (pts) each: 1×108, 3×108, & 1×109 transduced cells. Infusions were well tolerated, no acute infusion-related toxicities were observed. ISF35-related toxicities consisted of grade 1–2 flu-like symptoms that occurred several hrs after the infusion and consisted of fever, arthralgia, myalgia, nausea, vomiting, and fatigue lasting 2–4 days and resolving in all cases. There were no dose-limiting toxicities at any dose level. Biologic responses were seen at all doses, there was no dose-response relationship. There were consistent decreases in absolute lymphocyte counts at all dose levels, indicating a therapeutic effect. This was not dose-related, and ALC returned to pre-treatment level after 1–2 months post-infusion. There was consistent induction of CD95 and DR5 expression on bystander cells in vivo by 3 days following infusion, which lasted 2–3 weeks. Furthermore, consistent induction of Bid expression in bystander cells was seen by wk 1, also lasting 2–3 wks. Finally, consistent increases in absolute T cell counts (both CD4+ & CD8+) were seen, peaking 1–4 wks post infusion. These results demonstrate that ISF34-transduced autologous leukemia cells can be given safely at up to 1×109 transduced cells, without dose-limiting toxicities, and resulting in phenotypic and biochemical changes in bystander leukemia cells in vivo that render the cells able to present antigen and priming them for death-receptor-induced apoptosis. Furthermore, clinical responses were seen with reduction in leukemia counts and increases in absolute T cell counts. We expect that multiple, sequential doses will be needed for maximal therapeutic effect with this strategy. Given these results, we have developed a phase II trial of repeated doses of autologous ISF35-transduced leukemia cells for patients with CLL.

A Novel Mechanism for TNFR-Associated Factor 6-Dependent CD40 Signaling

Members of the TNFR family play critical roles in the regulation of the immune system. One member of the family critical for efficient activation of T-dependent humoral immune responses is CD40, a cell surface protein expressed by B cells and other APC. The cytoplasmic domain of CD40 interacts with several members of the TNFR-associated factor (TRAF) family, which link CD40 to intracellular signaling pathways. TRAF2 and 6 appear to play particularly important roles in CD40 signaling. Previous studies suggest that the two molecules have certain overlapping roles in signaling, but that unique roles for each molecule also exist. To better define the roles of TRAF2 and TRAF6 in CD40 signaling, we used somatic cell gene targeting to generate TRAF-deficient mouse B cell lines. A20.2J cells deficient in TRAF6 exhibit marked defects in CD40-mediated JNK activation and the up-regulation of CD80. Our previous experiments with TRAF2-deficient B cell lines suggest that TRAF6 and TRAF2 may have redundant roles in CD40-mediated NF-kappaB activation. Consistent with this hypothesis, we found CD40-mediated activation of NF-kappaB intact in TRAF6-deficient cells and defective in cells lacking both TRAF2 and TRAF6. Interestingly, we found that TRAF6 mutants defective in CD40 binding were able to restore CD40-mediated JNK activation and CD80 up-regulation in TRAF6-deficient cells, indicating that TRAF6 may be able to contribute to certain CD40 signals without directly binding CD40.

Toll‐Like Receptors in Older Adults

Toll-like receptors (TLRs) recognize a limited number of conserved elements in pathogens and, by activating antigen-presenting cells such as dendritic cells and monocytes and macrophages, play a crucial role in the immune response to infection and vaccination. Most data on TLR function in the context of human aging focus on responses to lipopolysaccharide, an integral component of gram-negative bacteria, which signals through TLR4. However, such studies have not led to a consensus conclusion and are limited by differences in epidemiological and laboratory methods. A recent comprehensive evaluation of TLR function in monocytes from older adults was conducted using a multivariable mixed statistical model to account for covariates. It was found that cytokine production after TLR1/2 engagement, which is essential for the recognition of triacylated lipopeptides found in a variety of bacteria, is substantially lower in monocytes from older adults. The upregulation of costimulatory proteins such as CD80, essential for optimal activation of T cells, on monocytes from older adults was less for all TLR ligands tested than for cells from young individuals, and the extent of CD80 upregulation predicted subsequent antibody response to influenza immunization. These and other consequences of aging on human TLR function may impair activation of the immune response and contribute to poorer vaccine responses and greater morbidity and mortality from infectious diseases in older adults. Such age-associated alterations have particular relevance in view of the interest in TLR agonists as therapeutic agents not only for infections, but also for allergic, autoimmune, and malignant disease.