Transendothelial migration of CD16+ monocytes in response to fractalkine under constitutive and inflammatory conditions

CD16+ monocytes represent 5-10% of circulating monocytes in healthy individuals and are dramatically expanded in several pathological conditions including AIDS and HIV-1-associated dementia (HAD). CD16+ monocytes constitutively produce high levels of pro-inflammatory cytokines and neurotoxic factors that may contribute to the pathogenesis of these disorders. Monocyte recruitment into the central nervous system (CNS) and other peripheral tissues in response to locally produced chemokines is a critical event in immune surveillance and inflammation and involves monocyte arrest onto vascular beds and subsequent diapedesis. Here we investigate the ability of CD16+ monocytes to undergo transendothelial migration (TEM) under constitutive and inflammatory conditions. CD16+ monocytes underwent TEM across unstimulated human umbilical vascular (HUVEC) and brain microvascular endothelial (BMVEC) cell monolayers in response to soluble fractalkine (FKN/CX3CL1). Stimulation with tumor necrosis factor (TNF) and interferon-gamma (IFN-gamma) induced high and low expression of membrane-bound FKN on HUVEC and BMVEC, respectively, together with expression of VCAM-1 and intercellular adhesion molecule-1 (ICAM)-1. By contrast, only HUVEC expressed CD62E while BMVEC remained negative. Both CD16- and CD16+ monocyte subsets adhered to TNF/IFN-gamma-stimulated HUVEC with higher frequency than to unstimulated HUVEC. Monocyte chemoattractant protein-1 (MCP-1) triggered efficient TEM of CD16- monocytes across TNF/IFN-gamma-stimulated HUVEC, whereas soluble FKN failed to induce TEM of CD16+ monocytes across stimulated HUVEC. These results demonstrate that stimulation with TNF and IFN-gamma triggers expression of membrane-bound FKN on both HUVEC and BMVEC, but prevents TEM of CD16+ monocytes in response to soluble FKN. Thus, pro-inflammatory CD16+ monocytes may contribute to the pathogenesis of HAD and other inflammatory CNS diseases by affecting the integrity of the blood-brain barrier as a consequence of their massive accumulation onto inflamed brain vascular endothelial cells expressing FKN and other adhesion molecules.     

Autoreactive natural killer T cells: promoting immune protection and immune tolerance through varied interactions with myeloid antigen-presenting cells

Natural killer T (NKT) cells are innate T lymphocytes that are restricted by CD1d antigen‐presenting molecules and recognize lipids and glycolipids as antigens. NKT cells have attracted attention for their potent immunoregulatory effects. Like other types of regulatory lymphocytes, a high proportion of NKT cells appear to be autoreactive to self antigens. Thus, as myeloid antigen‐presenting cells (APCs) such as monocytes, dendritic cells (DCs) and myeloid‐derived suppressor cells (MDSCs) constitutively express CD1d, NKT cells are able to interact with these APCs not only during times of immune activation but also in immunologically quiescent periods. The interactions of NKT cells with myeloid APCs can have either pro‐inflammatory or tolerizing outcomes, and a central question is how the ensuing response is determined. Here we bring together published results from a variety of model systems to highlight three critical factors that influence the outcome of the NKT–APC interaction: (i) the strength of the antigenic signal delivered to the NKT cell, as determined by antigen abundance and/or T‐cell receptor (TCR) affinity; (ii) the presence or absence of cytokines that costimulate NKT cells [e.g. interleukin (IL)‐12, IL‐18 and interferon (IFN)‐α]; (iii) APC intrinsic factors such as differentiation state (e.g. monocyte versus DC) and Toll‐like receptor (TLR) stimulation. Together with recent findings that demonstrate new links between NKT cell activation and endogenous lipid metabolism, these results outline a picture in which the functions of NKT cells are closely attuned to the existing biological context. Thus, NKT cells may actively promote tolerance until a critical level of danger signals arises, at which point they switch to activating pro‐inflammatory immune responses.     

Hantavirus and acute appendicitis—The diagnosis behind the diagnosis?

A 33-year-old man with a history of acute lower abdominal pain was admitted to the emergency room. After laparoscopic appendectomy and pathological confirmed acute appendicitis the patient developed thrombocytopenia and acute renal failure. Serological testing for hantaviruses revealed a positive result for PUUV IgG and IgM. Immunohistochemical work-up detected PUUV antigen in endothelial cells of capillaries and larger vessels. The high percentage of patients with hantavirus infection and severe abdominal pain is remarkable and, up to now, unexplained. To our knowledge this is the first report demonstrating PUUV antigen in the human intestine. Further studies are warranted whether hantaviruses are setting the stage for a secondary bacterial infection or cause an inflammation itself.     

真核表达载体pEGFP-GPC3的构建及表达

目的:构建磷脂酰肌醇蛋白聚糖3(GPC3)真核表达载体pEGFP-GPC3,并在小鼠树突状细胞(DC)表达.方法:将质粒pCMV-SPORT6-GPC3和载体pEGFP-N1分别进行双酶切获得目的基因GPC3片段和空载体,回收纯化后用T4连接酶将两者连接并转化E.coli DH5α菌株,用EcoR I、BamH I双酶切鉴定后通过脂质体法转染到DC中,然后进行荧光检测和Western blot分析.结果:构建的真核表达载体pEGFP-GPC3,转染DC后,荧光显微镜下可见转染的DC中有EGFP-GPC3融合蛋白的表达,Western blot分析发现有相对分子质量(M,)大小约为67 000的蛋白条带.结论:成功地构建了真核表达载体pEGFP-GPC3并在转染DC后能在其中表达,为进一步研究GPC3基因的功能提供实验基础.     

Innate immune signaling in defense against intestinal microbes

The gastrointestinal system is a common entry point for pathogenic microbes to access the inner environment of the body. Anti-microbial factors produced by the intestinal mucosa limit the translocation of both commensal and pathogenic microbes across the intestinal epithelial cell barrier. The regulation of these host defense mechanisms largely depends on the activation of innate immune receptors by microbial molecules. Under steady-state conditions, the microbiota provides constitutive signals to the innate immune system, which helps to maintain a healthy inflammatory tone within the intestinal mucosa and, thus, enhances resistance to infection with enteric pathogens. During an acute infection, the intestinal epithelial cell barrier is breached, and the detection of microbial molecules in the intestinal lamina propria rapidly stimulates innate immune signaling pathways that coordinate early defense mechanisms. Herein, we review how microbial molecules shed by both commensal and pathogenic microbes direct host defenses at the intestinal mucosa. We highlight the signaling pathways, effector molecules, and cell populations that are activated by microbial molecule recognition and, thereby, are involved in the maintenance of homeostatic levels of host defense and in the early response to acute enteric infection. Finally, we discuss how manipulation of these host defense pathways by stimulating innate immune receptors is a potential therapeutic strategy to prevent or alleviate intestinal disease.     

Protein kinase C delta stimulates antigen presentation by Class II MHC in murine dendritic cells

Maturation of dendritic cells (DCs) regulates protein sorting in endosomal compartments to promote the surface expression of molecules involved in T cell activation. MHC Class II complexes are mobilized to the surface via intracellular effector molecules that remain largely unknown. We here show that protein kinase C (PKC) stimulates Class II antigen surface expression, using knock-in mice that express a Class II-green fluorescent protein fusion protein as a read out. Selective inhibition of PKCdelta counteracts the ability of DCs to stimulate Class II MHC-restricted antigen-specific T cells. Activation of PKC does not affect antigen uptake, peptide loading and surface display of Class I MHC and transferrin receptor in DCs. We show that activation-induced Class II MHC surface expression is dependent on activation of PKCdelta and conclude that this event is pivotal for optimal CD4 T cell activation.     

The molecular makeup and function of regulatory and effector synapses

Summary:  Physical interactions between T cells and antigen-presenting cells (APCs) form the basis of any specific immune response. Upon cognate contacts, a multimolecular assembly of receptors and adhesion molecules on both cells is created, termed the immunological synapse (IS). Very diverse structures of ISs have been described, yet the functional importance for T-cell differentiation is largely unclear. Here we discuss the principal structure and function of ISs. We then focus on two characteristic T-cell–APC pairs, namely T cells contacting dendritic cells (DCs) or naive B cells, for which extremely different patterns of the IS have been observed as well as fundamentally different effects on the function of the activated T cells. We provide a model on how differences in signaling and the involvement of adhesion molecules might lead to diverse interaction kinetics and, eventually, diverse T-cell differentiation. We hypothesize that the preferred activation of the adhesion molecule leukocyte function-associated antigen-1 (LFA-1) and of the negative regulator for T-cell activation, cytotoxic T-lymphocyte antigen-4 (CTLA-4), through contact with naive B cells, lead to prolonged cell–cell contacts and the generation of T cells with regulatory capacity. In contrast, DCs might have evolved mechanisms to avoid LFA-1 overactivation and CTLA-4 triggering, thereby promoting more dynamic contacts that lead to the preferential generation of effector cells.     

Ifi202, an IFN-inducible candidate gene for lupus susceptibility in NZB/W F1 mice, is a positive regulator for NF-kappaB activation in dendritic cells

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of autoantibodies and lupus nephritis. The [New Zealand black (NZB) x New Zealand white (NZW)]F1 (BWF1) mouse has been recognized as an important animal model of human SLE. The T(h)1-prone phenotype of BWF1 mice has been shown to contribute to the development of the lupus. However, the molecular basis for T(h)1 skewing in BWF1 mice has not been clarified. We noticed that IL-6, IL-12 and other proinflammatory cytokines as well as IkappaB-zeta induction were higher in mature bone marrow-derived dendritic cells (BMDCs) from NZB and BWF1 mice than those from NZW mice. The expression of an IFN-inducible gene Ifi202, a candidate gene for lupus, was almost undetectable in NZW BMDCs. Thus, we hypothesized that Ifi202 is involved in elevated IL-12 production from BWF1 BMDCs. Overexpression of Ifi202 enhanced the LPS-induced IkappaB-zeta, IL-12p40 and NF-kappaB promoter activities, while anti-sense (AS) RNA against Ifi202 strongly suppressed them in a monocytic cell line, RAW 264.7. Furthermore, overexpression of Ifi202 enhanced LPS-induced IL-12p40 and IkappaB-zeta mRNA induction while Ifi202 AS RNA suppressed these in RAW 264.7 cells. In addition, forced expression of Ifi202 enhanced IL-12p40 mRNA induction in NZW BMDCs. Thus, Ifi202 is an important NF-kappaB activator in DCs and involved in IL-12 production, which may account for a T(h)1-prone phenotype of BWF1 mice.     

慢性乙型肝炎患者外周血来源的树突状细胞功能的研究

目的:研究慢性乙型肝炎患者外周血来源的树突状细胞(Dendritic cells, DCs)与慢性乙型肝炎的病程发展以及不同抗病毒治疗的关系.方法:采集71例慢性乙肝患者外周静脉血,抗凝,分离外周血单个核细胞(PBMCs).进行体外诱导培养,使之发育成DC,流式细胞仪检测其表面共刺激分子的表达.其中57例HBV-DNA阳性患者,分别采用拉米夫定或α-干扰素治疗,1个疗程后,再次检测DC表面分子的表达,并分析、比较治疗前后的表达水平.结果:与正常对照比较,慢性肝炎和慢性病毒携带者DC上CD1a(13.97±5.22和11.28±5.70 vs 27.25±4.32)%以及CD86(57.27±12.57和32.94±11.37 vs 82.12±13.54)%的表达,均明显降低(P＜0.05或0.01).比较血清HBV-DNA载量与DC表面分子的表达,发现DNA阴性组DC上CD1a、CD40、CD80和CD86的表达均比DNA阳性组高,但只有CD86的表达量有明显差别(P＜0.05).此外,57例抗病毒治疗后,拉米夫定治疗组CD40的表达(66.40±7.85 vs 43.48±7.93)%明显高于治疗前水平;而α-干扰素治疗组,CD40的表达(78.71±6.31)%高于治疗前水平外,CD86的表达(87.47±16.07 vs 44.18±10.07)%也明显高于治疗前水平(P＜0.05).结论:慢性乙型肝炎病毒感染与DC功能缺陷密切相关,血清中HBV-DNA载量越高,DC功能的缺陷越明显.拉米夫定和α-干扰素抗病毒治疗均能使患者外周血来源的DC表面的共刺激分子有不同程度的升高,促使DC成熟和功能的恢复.