Jak3 negatively regulates dendritic-cell cytokine production and survival

Cytokines are critical in regulating the development and function of diverse cells. Janus kinase 3 (Jak3) is a tyrosine kinase expressed in hematopoietic cells that associates with the common gamma chain (gammac) and is required for signaling for a family of cytokines including interleukin-2 (IL-2), IL-4, IL-7, IL-9, IL-15, and IL-21; deficiency of either Jak3 or gammac results in severe combined immunodeficiency (SCID). While Jak3 is essential for lymphoid-cell development, the potential roles for Jak3 in regulating dendritic cells (DCs) were unclear. Herein, we show that although CD8+CD11c+ splenic DCs are absent in Jak3-/- mice, bone marrow-derived DCs developed normally in vitro from Jak3-/- precursor cells. In fact, the survival of Jak3-/- DCs was enhanced, and they expressed lower levels of proapoptotic proteins. Jak3-/- DCs exhibited normal antigen uptake and up-regulation of costimulatory molecules. However, Jak3-/- DCs produced more IL-12 and IL-10 in response to Toll-like receptor ligands, which correlated with enhanced T helper 1 (Th1) differentiation in vivo. In summary, Jak3 is not essential for DC development but unexpectedly appears to be an important negative regulator. These results may be relevant clinically for patients with SCID who have undergone hematopoietic stem cell transplantation and for patients who might be treated with a Jak3 inhibitor.     

Expression of KIR and C-type lectin receptors in Behcet's disease

OBJECTIVE: Beh?et's disease (BD) is a multisystemic disorder with a possible underlying pathology of immune-mediated vasculitis. Genetic susceptibility associated with HLA-B*51 and B*2702 has been implicated in its pathogenesis. Considering the recently defined regulatory mechanisms of NK cells through HLA class I binding receptors, we hypothesized that interactions of NK and T cells through the NK receptors may be important in the pathogenesis of BD. METHODS: The impact of different expression patterns of HLA-recognizing receptors on NK or T cells was analysed in 51 patients with BD and 32 healthy controls. We used flow cytometry to investigate the expression of KIR3DL1 from the polymorphic killer immunoglobulin-like receptor (KIR) family, which binds a shared HLA-Bw4 motif on HLA-B51 and *2702 alleles, and CD94 from the conserved C-type lectin receptor family, which binds HLA-E. Thirty-three of the BD patients and 19 of the controls carried the same HLA-Bw4 motif. RESULTS: CD3(+) T cells were increased in patients with BD compared with controls (81 vs 75%, P = 0.001), whereas the NK cells did not show any difference between the two groups. Increased expression of CD94 in BD was observed on CD16(+)CD56(+) cells (66 vs 57, P = 0.04) and on CD3(+) (7.7 vs 4.0, P < 0.001) and CD3(+)CD56(+) (44 vs 35, P = 0.02) T cells. KIR3DL1 expression on the NK and T cells was not statistically different between the two groups. No effect of HLA-Bw4 motif was observed on the expression of CD94 and KIR3DL1 in both the patients and the controls. CONCLUSION: The absence of a correlation between KIR3DL1 expression and HLA-Bw4 motif confirms previous work reporting that the expression of these molecules is regulated separately. Increased expression of CD94 may suggest that NK receptors play a pathogenic or regulatory role in BD.     

A novel Syk-dependent mechanism of platelet activation by the C-type lectin receptor CLEC-2

The snake venom rhodocytin has been reported to bind to integrin alpha2beta1 and glycoprotein (GP) Ibalpha on platelets, but it is also able to induce activation independent of the 2 receptors and of GPVI. Using rhodocytin affinity chromatography, we have identified a novel C-type lectin receptor, CLEC-2, in platelets that confers signaling responses to rhodocytin when expressed in a cell line. CLEC-2 has a single tyrosine residue in a YXXL motif in its cytosolic tail, which undergoes tyrosine phosphorylation upon platelet activation by rhodocytin or an antibody to CLEC-2, but not to collagen, thrombin receptor agonist peptide (TRAP), or convulxin. Tyrosine phosphorylation of CLEC-2 and other signaling proteins by rhodocytin is inhibited by the Src family kinase inhibitor PP2. Further, activation of murine platelets by rhodocytin is abolished in the absence of Syk and PLCgamma2, and partially reduced in the absence of LAT, SLP-76, and Vav1/Vav3. These findings define a novel signaling pathway in platelets whereby activation of CLEC-2 by rhodocytin leads to tyrosine phosphorylation of its cytosolic tail, binding of Syk and initiation of downstream tyrosine phosphorylation events, and activation of PLCgamma2. CLEC-2 is the first C-type lectin receptor to be found on platelets which signals through this novel pathway.     

Potentially probiotic bacteria induce efficient maturation but differential cytokine production in human monocyte-derived dendritic cells

AIM： To analyze the ability of nine different potentially probiotic bacteria to induce maturation and cytokine production in human monocyte-derived dendritic cells （moDCs）.
METHODS： Cytokine production and maturation of moDCs in response to bacterial stimulation was analyzed with enzyme-linked immunosorbent assay （ELISA） and flow cytometric analysis （FACS）, respectively. The kinetics of mRNA expression of cytokine genes was determined by Northern blotting. The involvement of different signaling pathways in cytokine gene expression was studied using specific pharmacological signaling inhibitors.
RESULTS： All studied bacteria induced the maturation of moDCs in a dose-dependent manner. More detailed analysis with S. thermophilus THS, B. breve Bb99, and L. lactis subsp, cremoris ARH74 indicated that these bacteria induced the expression of moDC maturation markers HLA class Ⅱ and CD86 as efficiently as pathogenic bacteria. However, these bacteria differed in their ability to induce moDC cytokine gene expression. S. thermophilus induced the expression of pro-inflammatory （TNF-α, IL-12, IL-6, and CCL20） and Thl type （IL-12 and IFN-γ） cytokines, while B. breve and L. lactis were also potent inducers of antiinflammatory IL-10. Mitogen-activated protein kinase （MAPK） p38, phosphatidylinositol 3 （PI3） kinase, and nuclear factor-kappa B （NF-κB） signaling pathways were shown to be involved in bacteria-induced cytokine production. CONCLUSION： Our results indicate that potentially probiotic bacteria are able to induce moDC maturation, but their ability to induce cytokine gene expression varies significantly from one bacterial strain to another,     

Continuous and intermittent walking alters HDL(2)-C and LCATa

Purpose

The purpose of this study was to determine if exercise, whether continuous (CE: completed all in one session) or intermittent (completed in either two (IE 2) or three (IE 3) exercise sessions) expending the same number of calories alters reverse cholesterol transport or low density lipoprotein (LDL) particle size.

Methods

Sixteen healthy (22 ± 2.1 year old) men (VO₂ max = 37.0 ± 3.3 mL/kg/min) randomly completed three exercise trials, CE, IE 2 and IE 3, expending 450 calories. Blood samples were drawn immediately post-exercise (IPE) and 24 and 48 h following exercise and analyzed for total cholesterol (TC), triglycerides (TG), low density lipoprotein (LDL-C), high density lipoprotein (HDL-C) and subfractions (HDL₂-C, HDL₃-C). Samples were also analyzed to determine LDL-C particle size, lecithin cholesterol acyl transferase activity (LCATa) and cholesterol ester transfer protein activity (CETPa).

Results

HDL₂-C increased significantly 48 h post-exercise in the CE and IE 2 groups. Additionally, the IE 3 group had significant increases in HDL₂-C at 24 (39%) and 48 h post-exercise by 66%. This change in HDL₂-C was significantly and positively correlated (r = 0.62; p < 0.05) to the changes in LCATa which increased compared to baseline at 48 h post-exercise in the CE and IE 3 groups. No significant changes in LDL particle size or alterations in CETPa were seen.

Conclusions

The results of this study indicate that whether the exercise is continuous or intermittent, keeping calorie expenditure the same, causes significant changes in the HDL₂-C subfraction, which was augmented by an increase in LCATa.     

Increased renal production of C-type natriuretic peptide (CNP) in patients with cirrhosis and functional renal failure

BACKGROUND/AIMS: C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, is considered to be involved in the regulation of vascular tone. Furthermore, the recent demonstration of CNP in human kidney and urine may indicate a role for CNP in fluid and electrolyte homeostasis. Therefore, the aim of the present study was to investigate the possible role of CNP in renal function disturbances in patients with cirrhosis of the liver. METHODS: Peripheral venous and urinary concentrations of CNP were determined in samples from 11 healthy controls, 20 cirrhotic patients with normal renal function (creatinine clearance 117 (8) ml/min), and 20 cirrhotic patients with impaired renal function (creatinine clearance 35 (4) ml/min). In a second protocol, arterial and renal venous plasma concentrations of CNP were determined in 37 patients with cirrhosis of the liver to estimate renal extraction ratios of CNP. A sensitive and specific radioimmunoassay was applied after solid phase extraction of samples. RESULTS: Plasma CNP was lower in cirrhotic patients with normal and impaired renal function than in controls (3.0 (0.4) and 2.7 (0.2) v. 4.2 (0.4) pg/ml, respectively; p<0.05; mean (SEM)). In contrast, urinary CNP was higher in patients with impaired renal function compared with those with normal renal function and healthy controls (47.2 (7.4) v. 20.8 (1.9) and 17.0 (3.0) ng CNP/g creatinine, respectively; p<0.05). Urinary CNP was found to be inversely related to urinary sodium excretion in cirrhotic patients (r=-0.56; p<0.01). No differences were observed between arterial and renal venous concentrations of CNP in cirrhosis (2.4 (0.2) v. 2.4 (0.2) pg/ml). In cirrhotic patients with hepatorenal syndrome or refractory ascites (n=5), urinary CNP decreased from 132 (59) to 38 (7) ng/g creatinine (p<0.05) one week after either ornipressin infusion or insertion of a transjugular intrahepatic portosystemic shunt together with an increase in urinary sodium excretion from 27 (17) to 90 (34) mmol/24 hours. CONCLUSIONS: Increased urinary CNP in cirrhotic patients in the absence of renal arteriovenous concentration gradients suggests enhanced renal CNP production in cirrhosis. Furthermore, an inverse relation between urinary CNP and urinary sodium excretion suggests a role for this peptide in renal sodium handling in patients with cirrhosis.     

The C-type lectin receptor CLEC9A mediates antigen uptake and (cross-)presentation by human blood BDCA3+ myeloid dendritic cells

CLEC9A is a recently discovered C-type lectin receptor involved in sensing necrotic cells. In humans, this receptor is selectively expressed by BDCA3(+) myeloid dendritic cells (mDCs), which have been proposed to be the main human cross-presenting mDCs and may represent the human homologue of murine CD8(+) DCs. In mice, it was demonstrated that antigens delivered with antibodies to CLEC9A are presented by CD8(+) DCs to both CD4(+) and CD8(+) T cells and induce antitumor immunity in a melanoma model. Here we assessed the ability of CLEC9A to mediate antigen presentation by human BDCA3(+) mDCs, which represent < 0.05% of peripheral blood leukocytes. We demonstrate that CLEC9A is only expressed on immature BDCA3(+) mDCs and that cell surface expression is lost after TLR-mediated maturation. CLEC9A triggering via antibody binding rapidly induces receptor internalization but does not affect TLR-induced cytokine production or expression of costimulatory molecules. More importantly, antigens delivered via CLEC9A antibodies to BDCA3(+) mDCs are presented by both MHC class I (cross-presentation) and MHC class II to antigen-specific T cells. We conclude that CLEC9A is a promising target for in vivo antigen delivery in humans to increase the efficiency of vaccines against infectious or malignant diseases.     

Triptolide, a constituent of immunosuppressive Chinese herbal medicine, is a potent suppressor of dendritic-cell maturation and trafficking

Triptolide (TPT) is a chemically defined, potent immunosuppressive compound isolated from an anti-inflammatory Chinese herbal medicine. TPT has been reported to inhibit autoimmunity, allograft rejection, and graft-versus-host disease (GVHD), and its efficacy was previously attributed to the suppression of T cells. Since dendritic cells (DCs) play a major role in the initiation of T-cell-mediated immunity, we studied the effects of TPT on the phenotype, function, and migration of human monocyte-derived DCs. TPT treatment, over a pharmacologic concentration range, inhibited the lipopolysaccharide (LPS)-induced phenotypic changes, characteristic of mature DCs and the production of interleukin-12p70 (IL-12p70). Consequently, the allostimulatory functions of DCs were impaired by TPT treatment. Furthermore, the calcium mobilization and chemotactic responses of LPS-stimulated DCs to secondary lymphoid tissue chemokine (SLC)/CC chemokine ligand 21 (CCL21) were significantly lower in TPT-treated than untreated DCs, in association with lower chemokine receptor 7 (CCR7) and higher CCR5 expression. Egress of Langerhans cells (LCs) from explanted mouse skin in response to macrophage inflammatory protein-3beta (MIP-3beta)/CCL19 was arrested by TPT. In vivo administration of TPT markedly inhibited hapten (fluorescein isothiocyanate [FITC])-stimulated migration of mouse skin LCs to the draining lymph nodes. These data provide new insight into the mechanism of action of TPT and indicate that the inhibition of maturation and trafficking of DCs by TPT contributes to its immunosuppressive effects.     

Evolution of the type-1 (Th1)-type-2 (Th2) cytokine paradigm

During the past 12 years, since the discovery of murine Th1 and Th2 clones, increasing numbers of cells that make type-1 and type-2 cytokines have been recognized, and a growing number of these cytokines have been described. Emphasis has shifted from the CD4+ T cell, as the source of Th1-Th2 cytokines, to the functional effect of the type-1 and type-2 cytokines, independent of their cell of origin. Appreciation of the complex interactions of CMI and humoral immune responses continues to evolve. If this new paradigm provides insight into infectious disease pathogenesis and prevention, then it should allow development of new vaccines and vaccine adjuvants against these diseases.     

C-type natriuretic peptide (CNP) in rats and humans

We have established a specific radioimmunoassay (RIA) for C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, and have elucidated its tissue distribution and molecular form. In rats, high concentrations of CNP-like immunoreactivity (-LI) were detected in the anterior lobe (19.8 +/- 8.6 pmol/g) and neurointermediate lobe (4.64 +/- 0.74 pmol/g) of the pituitary gland. CNP-LI was present throughout the brain with its high concentrations in the hypothalamus and cerebellum. Small amounts of CNP-LI were also detected in the lower part of gastrointestinal tract and the kidney. However, no significant amount of CNP-LI was present in other organs including the heart. Considerable amounts of CNP-LI were detected throughout the human brain. High performance-gel permeation chromatography coupled with the RIA detected two peaks of CNP-LI in the rat brain; CNP and presumably its N-terminally elongated form with 53 amino-acid residues, CNP-53. These findings indicate that the tissue distribution and processing pattern of CNP are clearly different from those of atrial natriuretic peptide and brain natriuretic peptide and suggest possible roles of CNP as a neurotransmitter or neuromodulator rather than as a cardiac hormone.     

A cyclooxygenase-2 inhibitor alters Th1/Th2 cytokine balance and suppresses autoimmune myocarditis in rats

Acute myocarditis is a clinically serious disease; however, no effective treatment has been elucidated. Cyclooxygenase (COX)-2 is a key factor for progression of inflammation. Although inflammation is an essential pathological feature of acute myocarditis, the role of COX-2 in this process remains unclear. Thus, the purpose of this study was to clarify the role of COX-2 in acute myocarditis. We used a rat experimental autoimmune myocarditis (EAM) model and a specific COX-2 inhibitor in this study. Lewis rats were immunized on day 0 with porcine cardiac myosin to establish EAM. We administered the COX-2 inhibitor (meloxicam, 0.1 mg/kg per day) daily; the rats were killed on day 21. Echocardiograms, body and heart weight, heart rate, blood pressure, and histological and molecular examinations were performed. Cytokine expression in the hearts and cell proliferation against cardiac myosin were also analyzed. The COX-2 inhibition during the immune response (late) phase attenuated EAM development; however, the inhibition during the antigen priming (early) phase did not attenuate EAM. The COX-2 inhibitor altered Th1/Th2 cytokine balance and inhibited cell proliferation in vitro. The COX-2 inhibitor suppresses the development of EAM. COX-2 regulation is promising for treating acute myocarditis.     

C型凝集素CLEC4M对HCVcc易感性的影响

目的利用HCV体外培养细胞模型,观察C型凝集素CLEC4M对于HCVcc易感性的影响。方法构建GV166-CLEC4M慢病毒表达载体,脂质体转染293T细胞,获取的慢病毒转染Huh7.5细胞,获得稳定过表达CLEC4M的Huh7.5细胞系(即Huh7.5-CLEC4M),Western印迹和RT-PCR检测CLEC4M的过表达情况。利用HCVcc病毒液感染过表达CLEC4M的Huh7.5细胞系,并设置单克隆抗体干扰组及甘露聚糖干扰组,检测CLEC4M对HCVcc感染性的变化。结果 Huh7.5细胞系获得稳定过表达的CLEC4M。Huh7.5-CLEC4M获得对HCVcc的高度易感性。经鼠抗人CLEC4M单克隆抗体和甘露聚糖干扰后,Huh7.5细胞的感染效率较前降低。结论 C型凝集素CLEC4M影响细胞对HCVcc的易感性,可能与HCV的嗜肝性有关。     

NKG2D receptor regulates human effector T-cell cytokine production

Although innate immune signals shape the activation of naive T cells, it is unclear how innate signals influence effector T-cell function. This study determined the effects of stimulating the NKG2D receptor in conjunction with the TCR on human effector CD8(+) T cells. Stimulation of CD8(+) T cells through CD3 and NKG2D simultaneously or through a chimeric NKG2D receptor, which consists of NKG2D fused to the intracellular region of CD3ζ, activated β-catenin and increased expression of β-catenin-induced genes, whereas T cells stimulated through the TCR or a combination of the TCR and CD28 did not. Activation by TCR and NKG2D prevented expression and production of anti-inflammatory cytokines IL-10, IL-9, IL-13, and VEGF-α in a β-catenin- and PPARγ- dependent manner. NKG2D stimulation also modulated the cytokine secretion of T cells activated simultaneously through CD3 and CD28. These data indicate that activating CD8(+) T cells through the NKG2D receptor along with the TCR modulates signal transduction and the production of anti-inflammatory cytokines. Thus, human effector T cells alter their function depending on which innate receptors are engaged in conjunction with the TCR complex.