Stimulation of B cell differentiation by adherent mononuclear cells in systemic lupus erythematosus

We studied B cell proliferation and differentiation in response to factors released by adherent monocytes in patients with systemic lupus erythematosus (SLE). Adherent cell supernatants (ACS) were added to peripheral blood mononuclear cells, and the effects on IgG synthesis, the number of Ig-secreting cells (ISC), and proliferation were determined. Exposure of SLE mononuclear cells to autologous ACS caused an increase (approximately two-fold) in IgG production and ISC numbers. In contrast, exposure of normal mononuclear cells to autologous ACS did not significantly increase IgG production or ISC numbers. Addition of SLE ACS to cultures of normal mononuclear cells did not stimulate ISC production. There was no significant level of 3H-thymidine uptake by cultures of SLE or normal mononuclear cells in response to either SLE or normal ACS. In the presence of an excess number of autologous T cells, ACS stimulation of IgG synthesis was further enhanced. These findings indicate that adherent monocytes contribute to B cell hyperactivity in SLE by stimulating B cell differentiation. SLE mononuclear cells appear to be more responsive to ACS stimulation than are normal mononuclear cells.     

Treatment of murine lupus using nucleosomal T cell epitopes identified by bone marrow-derived dendritic cells

OBJECTIVE: Systemic lupus erythematosus (SLE) is characterized by the existence of a heterogeneous group of autoantibodies directed against intact nuclear structures, such as nucleosomes. The most prominent of these autoantibodies are those directed against double-stranded DNA (dsDNA) and histones. The majority are of the IgG isotype and show affinity maturation, both of which are known hallmarks of T cell help. Much evidence suggests that the nucleosome is a major candidate autoantigen in SLE. In this study, a novel strategy was used to identify the critical CD4+ T cell autoepitopes in nucleosomes. In addition, peptide-based therapy was then performed in a lupus animal model. METHODS: Bone marrow (BM)-derived dendritic cells (DCs) were used to examine the self-T cell responses against nucleosomes and to characterize the T cell epitope(s) of nucleosomes in (NZB x NZW)F1 (BWF1) mice. RESULTS: Several potential auto-T cell epitopes of core histone proteins (H2A, H2B, H3, and H4) were identified. Nucleosome-pulsed BM-derived DCs elicited release of interleukin-4 and interferon-gamma, representing a Th0 (i.e., mixed Th1 and Th2) pattern of cytokine production. In addition, intradermal immunization of BWF1 mice with the H3(111-130) peptide not only suppressed the level of anti-dsDNA and anti-single-stranded DNA IgG, but also significantly delayed the progress of glomerulonephritis in lupus-prone BWF1 mice. CONCLUSION: These results will help in understanding how pathogenic autoimmune responses develop in spontaneous SLE. This may potentially open the way to T cell-based immunotherapy for lupus.     

Antigen-specific immature dendritic cell vaccine ameliorates anti-dsDNA antibody-induced renal damage in a mouse model

OBJECTIVES: Dendritic cells (DCs) can inhibit immune response by clonal anergy when immature. Recent studies have shown that immature DCs (iDCs) may serve as a live cell vaccine after specific antigen pulse based on its potential of blocking antibody production. In this study, we aimed to investigate the effects of nuclear antigen-pulsed iDCs in the treatment of lupus-like renal damages induced by anti-dsDNA antibodies. METHODS: iDCs were generated from haemopoietic stem cells in bone marrow and then pulsed in vitro with nuclear antigen. The iDC vaccine and corresponding controls were injected into mice with lupus-like renal damages. The evaluation of disease was monitored by biochemical parameters and histological scores. Anti-dsDNA antibody isotypes and T-lymphocyte-produced cytokines were analysed for elucidating therapeutic mechanisms. RESULTS; The mice treated with antigen-pulsed iDCs had a sustained remission of renal damage compared with those injected with non-pulsed iDCs or other controls, including decreased anti-dsDNA antibody level, less proteinuria, lower blood urea nitrogen and serum creatinine values, and improved histological evaluation. Analysis on isotypes of anti-dsDNA antibody showed that iDC vaccine preferentially inhibited the production of IgG3, IgG2b and IgG2a. Furthermore, administration of antigen-treated iDCs to mice resulted in significantly reduced IL-2, IL-4 and IL-12 and IFN-γ produced by T-memory cells. Conversely, the vaccination of antigen-pulsed mature DCs led to increased anti-dsDNA antibody production and an aggravation of lupus-like disease in the model. CONCLUSIONS; These results suggested the high potency of iDC vaccine in preventing lupus-like renal injuries induced by pathogenic autoantibodies.     

内皮细胞穿越模型中系统性红斑狼疮患者树突状细胞分化及功能状态的初步研究

目的利用已经成功构建的内皮细胞穿越模型研究系统性红斑狼疮(systemiclupusery-thematosus,SLE)患者的单核细胞起源的树突状细胞的分化和功能状态(膜表面分子和刺激功能及细胞因子分泌等)。方法构建内皮细胞穿越模型;分别诱导正常人单核细胞和SLE患者单核细胞在模型中分化为树突状细胞(dendriticcells,DCs);电子显微镜观察其形态特征;流式细胞术检测其表型[人类白细胞抗原(HLA)-DR、CD80、CD86],混合淋巴细胞反应检测DCs刺激异源T细胞活化的能力,酶联免疫吸附试验(ELISA)检测DCs分泌的干扰素(IFN)-!。结果正常人和SLE患者单核细胞在内皮细胞穿越模型中能分化成树突状细胞形态。正常人单核细胞来源的DCs(normalMonocyte-derivedDCs,NMDCs)和SLE患者单核细胞来源的DCs(SLEMonocyte-derivedDCs,SMDCs)都高表达HLA-DR;SMDCs的协同刺激分子CD80和CD86的表达水平明显高于NMDCs;刺激异源T淋巴细胞增殖的能力,SMDCs和NMDCs差异无统计学意义;SMDCs分泌IFN-!的水平明显低于NMDCs。结论SLE患者单核细胞异常分化为树突状细胞,这种异常涉及树突状细胞协同刺激分子CD80和CD86的表达升高以及细胞因子IFN-!分泌降低。     

B cells lacking RP105, a novel B cell antigen, in systemic lupus erythematosus

OBJECTIVE: RP105 is a leucine-rich repeat (LRR) protein found on all mature mouse B cells. Its function is poorly defined, although it has been suggested that RP105 activates B cells to make them resistant to apoptosis. The human homolog of RP105 has been reported, but knowledge of its function is limited. We explored the expression and the function of the human homolog of murine RP105 on B cells in patients with systemic lupus erythematosus (SLE). METHODS: The expression of RP105 and various markers on B cells in patients with SLE was analyzed using monoclonal antibodies and flow cytometry. Susceptibility to corticosteroid-induced apoptosis was examined by annexin V binding, and the production of immunoglobulin by RP105-negative B cells was examined by intracellular staining of IgG. RESULTS: As in mice, virtually all B cells in the peripheral blood of normal humans expressed the RP105 molecule. However, a significant proportion of circulating B cells (15.9%) in SLE patients were RP105 negative. Serial analyses of B cells in 7 SLE patients revealed that RP105-negative B cells markedly decreased in parallel with a reduction in disease activity (from 35.2% to 3.3%; P = 0.000003). The SLE Disease Activity Index and serum levels of IgG also correlated with the percentage of RP105-negative B cells. The phenotype of RP105-negative B cells was defined as CD95-positive, CD86-positive, CD38-bright, IgD-negative, IgM-dull, indicating that the cells were highly activated, as further suggested by the detection of intracellular IgG. RP105-negative B cells were clearly distinct from CD5-positive B1 cells. In vitro experiments indicated that RP105-negative B cells were susceptible to corticosteroid-induced apoptosis. CONCLUSION: These findings suggest that loss of RP105 is associated with B cell activation and increased disease activity in SLE patients.     

Characteristics of intestinal lamina propria dendritic cells in a mouse model of postinfectious irritable bowel syndrome

Background and Aim: Postinfectious irritable bowel syndrome (PI‐IBS), which results from inflammation has been emphasized a lot recently. Dendritic cells (DCs) may contribute to intestinal mucosal immune activation in the pathogenesis of PI‐IBS. This study tested the hypothesis that phenotype and function of intestinal lamina propria DCs (LPDCs) changed in the development of a PI‐IBS mouse model. Methods: Mice infected with Trichinella spiralis underwent abdominal withdrawal reflex (AWR) to evaluate visceral sensitivity. LPDCs were isolated and purified by intestine digestion and magnetic label‐based technique. Surface markers were analyzed by flow cytometry. Endocytic activity, mixed lymphocyte reaction (MLR) and chemotaxis were studied. Cytokine production of the LPDCs cocultured with CD4⁺ T cells was determined. Results: Intestinal inflammation resolved after 8 weeks infection with sustained visceral hyperalgesia. Surface markers CD86 and MHCII were lower in the acute infection group, but increased in the PI‐IBS stage. Enhanced ability of endocytic activity and decreased abilities to attract and stimulate CD4⁺ T cell proliferation were in the acute infection group. However, LPDCs in the PI‐IBS stage showed weakened endocytic ability with enhanced abilities to attract and stimulate CD4⁺ T cell proliferation. Cocultured LPDCs with CD4⁺ T cells showed a predominant Th2 response in the acute infection stage, and more important roles of Th1, Th17 responses in the PI‐IBS stage. Conclusions: The hypothesis was supported that the phenotype and function of LPDCs changed in the development of PI‐IBS, which induced the maintenance of intestinal mucosal immune activation and might provide a clue for the treatment of the disease.     

Direct stimulation of T cells by membrane vesicles from antigen-presenting cells

Activation of na?ve T cells generally requires T cell receptor-mediated contact with MHC-bound peptides on viable antigen-presenting cells such as dendritic cells (DC). Here evidence is presented that dissociated cell membrane fragments from a DC line can be used as an effective substitute for viable DC. Ultracentrifuged material derived from sonicates of IFN-gamma-matured DC is enriched in small membrane vesicles that closely resemble exosomes. When complexed with MHC class I-restricted specific peptide, vesicles from DC sonicates generate strong responses by purified na?ve CD8(+) cells in vitro in the absence of normal antigen-presenting cells and can also efficiently prime T cells for tumor rejection in vivo. Both in terms of total yields from DC and relative immunogenicity, membrane vesicles from DC sonicates are much more effective than classic exosomes and may be a valuable tool for tumor immunotherapy.     

Protection of CLL B cells by a follicular dendritic cell line is dependent on induction of Mcl-1

Chronic lymphocytic leukemia (CLL) B cells have defects in apoptosis pathways and therefore accumulate in vivo. However, when removed from the patient and cultured in vitro, these malignant cells rapidly undergo apoptosis. Recent studies suggest that leukemia cell survival is influenced by interactions with nonleukemia cells in the microenvironment of lymph nodes, marrow, and other tissues. To model such cell-cell interactions in vitro, we cultured freshly isolated CLL B cells with a follicular dendritic cell line, HK. CLL B cells cocultured with HK cells were protected from apoptosis, either spontaneous or induced by treatment with anticancer drugs. Protection against spontaneous apoptosis could also be induced by coculturing the CLL B cells with normal dendritic cells (DCs) or with a CD40-ligand (CD154)-expressing fibroblast cell line. Examination of the expression of several apoptosis-regulatory proteins revealed that coculture with HK cells or DCs induced up-regulation of the antiapoptotic Bcl-2 family protein Mcl-1 in CLL B cells, whereas CD40 ligation increased expression of Bcl-X(L). Cell-cell contact was required for HK-induced protection, and introducing neutralizing antibodies against various adhesion molecules showed that CD44 was involved in HK-mediated survival, whereas CD40, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were not. Anti-CD44 antibodies also blocked Mcl-1 induction by HK cells. Mcl-1 antisense oligonucleotides reduced leukemia cell expression of Mcl-1, and significantly suppressed HK-induced protection against apoptosis, whereas control oligonucleotides had no effect. Thus, HK cells protect CLL B cells against apoptosis, at least in part through a CD44-dependent mechanism involving up-regulation of Mcl-1, and this mechanism is distinct from that achieved by CD40 ligation. Consequently, the particular antiapoptotic proteins important for CLL survival may vary depending on the microenvironment.     

Neuroinvasion by a Creutzfeldt-Jakob disease agent in the absence of B cells and follicular dendritic cells

With the potential spread of bovine spongiform encephalopathy to people as a variant Creutzfeldt-Jakob disease (CJD), it becomes critical to identify cells in the periphery that carry infection. Initial work with scrapie agents suggested that B cells were central vectors for neuroinvasion. Subsequent studies indicated that B cells played an indirect role by promoting the development of follicular dendritic cells (FDCs) that accumulate abnormal prion protein (PrP). The mechanism for the role of FDCs, however, has not been clear. To further dissect potential B cell functions that contribute to neuroinvasion, we inoculated a CJD agent into mutant mice that (i) lacked B cells, (ii) had B cells unable to secrete Ig, or (iii) could secrete only IgM. Remarkably, all these mice developed disease with practically indistinguishable incubation times. The demonstration that neither immune complexes nor B cells were required for neuroinvasion from the periphery mandates a reanalysis of the accepted view of the essential role of B cells and FDC in these infections. Moreover, immune complexes were not required for the accumulation of pathologic PrP on the surface of FDCs, suggesting that PrP can bind to FDCs autonomously or by means of another factor. Wild-type mice had incubation times approximately 50 days less than all mutant mice at the same peripheral doses, indicating that an intact immune system may increase agent uptake and delivery, but this condition is not essential. Specifically, the evidence to date suggests that IgG may enhance pivotal agent interactions with migratory myeloid cells.     

Treatment of murine lupus using nucleosomal T cell epitopes identified by bone marrow–derived dendritic cells

Objective

Systemic lupus erythematosus (SLE) is characterized by the existence of a heterogeneous group of autoantibodies directed against intact nuclear structures, such as nucleosomes. The most prominent of these autoantibodies are those directed against double‐stranded DNA (dsDNA) and histones. The majority are of the IgG isotype and show affinity maturation, both of which are known hallmarks of T cell help. Much evidence suggests that the nucleosome is a major candidate autoantigen in SLE. In this study, a novel strategy was used to identify the critical CD4+ T cell autoepitopes in nucleosomes. In addition, peptide‐based therapy was then performed in a lupus animal model.

Methods

Bone marrow (BM)–derived dendritic cells (DCs) were used to examine the self–T cell responses against nucleosomes and to characterize the T cell epitope(s) of nucleosomes in (NZB × NZW)F₁ (BWF₁) mice.

Results

Several potential auto–T cell epitopes of core histone proteins (H2A, H2B, H3, and H4) were identified. Nucleosome‐pulsed BM‐derived DCs elicited release of interleukin‐4 and interferon‐γ, representing a Th0 (i.e., mixed Th1 and Th2) pattern of cytokine production. In addition, intradermal immunization of BWF₁ mice with the H3^111–130 peptide not only suppressed the level of anti‐dsDNA and anti–single‐stranded DNA IgG, but also significantly delayed the progress of glomerulonephritis in lupus‐prone BWF₁ mice.

Conclusion

These results will help in understanding how pathogenic autoimmune responses develop in spontaneous SLE. This may potentially open the way to T cell–based immunotherapy for lupus.

     

免疫治疗对支气管哮喘小鼠树突细胞共刺激分子的影响

目的应用卵白蛋白(OVA)建立特异性免疫治疗小鼠模型,探讨特异性免疫治疗对支气管哮喘(简称哮喘)小鼠树突细胞(DC)表面分子CD80、CD86表达的影响。方法120只BALB/c小鼠按随机数字表法分为哮喘模型组(A组)40只:用0.1%OVA 10μg连续腹腔注射(共70μg)及1%OVA雾化吸入(共300 mg);免疫治疗模型组(B组)40只:用与A组同剂量的OVA致敏和激发,同时连续尾根部皮下注射OVA 1 mg;对照组(C组)40只:以磷酸盐缓冲液代替OVA,其余同A组。留取各组肺组织切片,经苏木精-伊红(HE)染色观察炎症反应;用酶联免疫吸附测定(ELISA)法检测血清OVA特异性免疫球蛋白IgE(sIgE)及脾脏T淋巴细胞中白细胞介素2(IL-2)和IL-4的分泌。分离各组小鼠脾脏DC,用流式细胞仪检测其表面CD80、CD86分子表达。分离正常小鼠脾脏T淋巴细胞,与上述各组DC共培养;用ELISA法检测T淋巴细胞IL-4、IL-5的分泌量;用3H-胸腺嘧啶核苷(3H-TdR)掺入法检测其增殖反应。结果(1)B组小鼠肺组织中支气管及血管周围以大量淋巴细胞和嗜酸粒细胞为主的炎性细胞浸润明显轻于A组,但并未完全消失;A组血清sIgE吸光度(A)值为712±129,B组为124±59,C组为20±13,A、C组间比较差异有统计学意义(P<0.05),B、C组比较差异无统计学意义(P>0.05)。B组T淋巴细胞分泌IL-2、IL-4水平分别为(8±3)、(8.4±4.3)pg/m l,A组分别为(22±8)、(32.4±12.1)pg/m l,C组分别为(6±4)、(5.1±1.1)pg/m l,A、B两组比较差异有统计学意义(P<0.05),B、C组间比较差异无统计学意义(P>0.05);(2)B组DC表面CD86、CD80阳性表达率分别为58.23%、95.63%,A组分别为77.59%、96.98%,C组分别为77.37%、77.84%;(3)与B组DC共培养的正常小鼠T淋巴细胞体外经OVA刺激后,IL-4、IL-5水平分别为(10.8±2.3)、(18.8±3.8)pg/m l,A组分别为(17.3±4.7)、(35.7±7.9)pg/m l,C组分别为(5.7±2.7)、(11.0±2.2)pg/m l,A、B两组比较差异有统计学意义(P<0.05),B、C组间比较差异无统计学意义(P>0.05);B组DC与正常小鼠T淋巴细胞共培养时,刺激指数(SI)为3.8±0.7,A组为11.5±3.2,C组为5.8±1.5,A、B组间比较差异有统计学意义(P<0.05);B、C组间比较差异无统计学意义(P>0.05)。结论建立了OVA特异性免疫治疗小鼠模型;DC表面CD86分子表达的下调可能是OVA特异性免疫治疗诱导T淋巴细胞功能丧失的机制之一。     

Human macrophage and dendritic cell-specific silencing of high-mobility group protein B1 ameliorates sepsis in a humanized mouse model

Hypersecretion of cytokines by innate immune cells is thought to initiate multiple organ failure in murine models of sepsis. Whether human cytokine storm also plays a similar role is not clear. Here, we show that human hematopoietic cells are required to induce sepsis-induced mortality following cecal ligation and puncture (CLP) in the severely immunodeficient nonobese diabetic (NOD)/SCID/IL2Rγ^−/− mice, and siRNA treatment to inhibit HMGB1 release by human macrophages and dendritic cells dramatically reduces sepsis-induced mortality. Following CLP, compared with immunocompetent WT mice, NOD/SCID/IL2Rγ^−/− mice did not show high levels of serum HMGB1 or murine proinflammatory cytokines and were relatively resistant to sepsis-induced mortality. In contrast, NOD/SCID/IL2Rγ^−/− mice transplanted with human hematopoietic stem cells [humanized bone marrow liver thymic mice (BLT) mice] showed high serum levels of HMGB1, as well as multiple human but not murine proinflammatory cytokines, and died uniformly, suggesting human cytokines are sufficient to induce organ failure in this model. Moreover, targeted delivery of HMGB1 siRNA to human macrophages and dendritic cells using a short acetylcholine receptor (AchR)-binding peptide [rabies virus glycoprotein (RVG)-9R] effectively suppressed secretion of HMGB1, reduced the human cytokine storm, human lymphocyte apoptosis, and rescued humanized mice from CLP-induced mortality. siRNA treatment was also effective when started after the appearance of sepsis symptoms. These results show that CLP in humanized mice provides a model to study human sepsis, HMGB1 siRNA might provide a treatment strategy for human sepsis, and RVG-9R provides a tool to deliver siRNA to human macrophages and dendritic cells that could potentially be used to suppress a variety of human inflammatory diseases.Sepsis is an important cause of mortality in intensive-care units, with more than 750,000 individuals developing severe sepsis in North America annually and a mortality rate varying between 35 and 50% (1, 2). The pathogenesis of sepsis includes countless disturbances of the host immune system starting with a harmful, infection-triggered exaggerated inflammatory cascade that results in tissue injury and rapidly leads to massive apoptosis of immune cells (2, 3). This is followed by a secondary immune paralysis phase accompanied by uncontrolled growth of bacteria and tissue damage. Although therapy to suppress the immediate cytokine response, such as treatment with TNF and IL-1β antibodies, have failed in clinical trials (4–6), it has now come to be recognized that, at least in animal models, high-mobility group protein 1 (HMGB1), which is secreted from macrophages and dendritic cells (DCs) but not lymphocytes late in the disease, acts as a master regulator of late and sustained cytokine storm, up-regulating many cytokines including TNF-α, IL-6, IL-1β, and IL-8 (reviewed in refs. 7–11). In fact, injection of mice with HMGB1 is enough to induce the lethal organ damage seen in sepsis (12), whereas treatment with neutralizing HMGB1 antibody can rescue mice and rats from experimental sepsis (13, 14). However, although HMGB1 is also secreted in human sepsis (12), its role in sepsis pathogenesis or the impact of its neutralization on human cells remain unclear.RNA interference can be used to silence virtually any gene, including multiple genes, as long as a way can be found to introduce small interfering (si)RNAs into relevant cell types in vivo without toxicity. Several advances have been made in developing methods to deliver siRNA in vivo to different cell types, most successfully to the liver cells (reviewed in refs. 15–17). A lipid-like nanoparticle called C12-200, which had been developed for liver-specific delivery of siRNA, was recently also shown to deliver siRNA to murine monocytes, and silencing C-C chemokine receptor type 2 (CCR2) in monocytes using this reagent was effective in reducing atherosclerosis, islet transplantation and tumors (18). Whether this reagent also targets human DCs and monocytes/macrophages is unclear. We have reported previously that a short 29-aa peptide derived from the rabies virus glycoprotein (RVG), fused to 9R residues (RVG-9R), can deliver siRNA to murine macrophages and brain cells by specific binding to its ligand acetylcholine receptor (AchR) (19, 20). Because AchR is also expressed on human macrophages and DCs (21) and also because the acetylcholine-binding site on the α7 subunit is highly conserved, we reasoned that RVG-9R might also be used to target human macrophages and DCs. In this study, we validate this hypothesis in vitro, as well as in vivo, using human hematopoietic stem cell–engrafted nonobese diabetic NOD/SCID/IL2Rγ^−/− mice that lack mouse innate and adaptive immune systems (22). More importantly, we also show that silencing human HMGB1 using this delivery reagent in this mouse model substantially reduces human lymphocyte apoptosis and cytokine storm and protects mice from sepsis-induced mortality.     

支气管哮喘患者血清对内皮穿越模型中单核细胞分化为树突状细胞的作用

目的 探讨支气管哮喘(简称哮喘)患者血清和正常血清对内皮穿越模型中单核细胞向树突状细胞(DC)分化和功能的作用及其机制.方法 在江苏省哮喘联盟数据库中选择2008年5月至2009年5月轻～中度哮喘患者12例,募集匹配的健康志愿者12名,常规分离血清和外周血单个核细胞,免疫磁珠法获得单核细胞.选择健康产妇5名,胰酶消化法获得人脐静脉内皮细胞(第2代),培养融合至内皮细胞单层.在内皮细胞上方加入单核细胞及哮喘血清(哮喘组)或正常血清(对照组),构建内皮穿越模型.硝酸银染色法观察单核细胞来源的树突状细胞(MDDC)迁移行为.于48 h收集MDDC,扫描电镜观察形态学特征,电泳迁移率实验检测核因子κB(NF-κB)活性,流式细胞术、ELISA和混合淋巴细胞反应分别检测其表型和功能.结果 (1)在内皮穿越模型中,单核细胞2 h后发生正向穿越,48 h后发生逆向穿越并分化为DC.(2)与穿越前单核细胞[CD14(81±6)%、人白细胞抗原DR(HLA-DR)(24±5)%、CD80(2.8±2.0)%、CD86(14±4)%、CD83(0.9±0.8)%]比较,对照组MDDC的CDi4[(20±5)%]显著下降(F=49.01,P＜0.05)、HLA-DR、CD80、CD86和CD83[(43±4)%、(17.9±3.5)%、(43±11)%和(6.7±1.8)%]均显著增高(均P＜0.05),但CD83仍较低,为未成熟DC.(3)与对照组比较,哮喘组MDDC的HLA-DR和CD86[(55±6)%和(59±12)%]相仿(F值分别为15.29和35.97,均P＞0.05)、CD80和CD83[(49.7±10.2)%和(30.2±6.8)%]显著增高(F值分别为34.01和20.68,均P＜0.05),为成熟DC.并且,哮喘组MDDC呈典型的树枝样突起,其NF-κB活性、白细胞介素-12 P70水平和T细胞增殖作用[(100±11)%、(568±43)ng/L和(2033±198)cpm]较对照组[(12±3)%、(220±35)ng/L和(952±64)cpm]显著增高(均P＜0.05).结论 哮喘血清可诱导内皮穿越模型中单核细胞向DC异常分化成熟,并与NF-κB过度激活有关.该模型为剖析哮喘免疫学发病机制和筛选新型抗哮喘药物建立了新的体外实验平台.

Abstract：

Objective To explore the effect of asthmatic and healthy serum on differentiation and function of monocyte-derived dendritic cells (MDDC) in a transendothelial trafficking model. Methods The sera and peripheral blood mononuclear cells (PBMC) were separated from 12 asthmatic patients and 12 healthy volunteers, and monocytes were selected from PBMC using magnetic beads. The trypsin-digested human umbilical vein endothelial cells (HUVEC) at passage 2 from 5 healthy lying-in women were used to construct the transendothelial trafficking model under asthmatic or healthy serum, wherein MDDC were identified by silver nitrate staining and scanning electron microscopy. Nuclear factor κB (NF-κB) activity was determined by electrophoretic mobility shift assay. Flow cytometry, ELISA and mixed leukocyte reaction were relevantly utilized to detect the phenotype, cytokine and T cell proliferation. Results ( 1 ) Monocytes traversed through HUVEC monolayer after 2 h, and reverse-transmigrated to develop into DC 48 h later. ( 2 ) The healthy serum stimulated monocytes into immature MDDC with lower CD14 [ ( 20 ±5)%] (F=49.01, P＜0.05), and higher HLA-DR, CD80, CD86 and CD83 [(43 ±4)%, (17.9 ±3.5)%, (43 ± 11)% and (6.7 ± 1.8)%, respectively] (F= 10.35 -40.17, all P＜0.05) than monocytes did before transmigration at 0 h [ CD14(81 ±6)%, HLA-DR (24 ±5)%, CD80(2. 8 ±2. 0)%,CD86( 14 ±4)% and CD83(0. 9 ±0. 8)%, respectively]. (3) The asthmatic serum stimulated monocytes into mature MDDC, characteristic of dendrites, with similar HLA-DR and CD86 [ ( 55 ± 6 ) % and ( 59 ±12)%] (F=15.29 and 35.97, all P ＞0.05), higher CD80 and CD83 [(49.7 ± 10.2)% and (30.2 ±6. 8) % ] ( F = 4. 01 and 20. 68, all P ＜ 0. 05), accompanied by increased levels of NF-κB activity, IL-12 p70 and T cell proliferation [ ( 100 ± 11 )%, (568 ±43) ng/L and (2033 ± 198) cpm, respectively] (F=49. 23 - 350. 84, all P ＜ 0. 05 ) relative to the healthy serum-stimulated immature MDDC [ ( 12 ± 3 ) %,(220 ± 35) ng/L and (952 ± 64) cpm, respectively ]. Conclusion The asthmatic serum induces mature MDDC in association with NF-κB overactivation in the transendothelial trafficking model, which provides a promising experimental platform for both investigation of immunological mechanisms in asthma and screening of novel anti-asthma drugs in vitro.     

B cell activity and regulatory T cell function in systemic lupus erythematosus by human B cell colony formation

We have examined the ability of B cells from patients with systemic lupus erythematosus (SLE) to form colonies in vitro. Significantly more B cell colonies growing in the absence of irradiated T cells were observed in patients with active SLE. Helper T cell function of active SLE was normal. Culture supernatants of SLE T cells had significantly less activity to support the B cell colony formation. Our results suggest that the hyperactivity of B cells in SLE may reside in the lupus B cells themselves and that SLE T cells are defective in releasing the soluble factor.     

Insulin resistance and obesity in a mouse model of systemic lupus erythematosus

Accumulating data indicate that metabolic syndrome is an inflammatory condition. Systemic lupus erythematosus (SLE) is an autoimmune disorder associated with nephritis and cardiovascular disease. Evidence suggests that individuals with SLE are at risk for developing insulin resistance; however, this has not been directly examined. Using an established mouse strain with SLE (NZBWF1), we examined whether SLE is associated with increased body weight and fat deposition. Mean arterial pressure was significantly increased (140+/-4 versus 114+/-2 mm Hg; n > or = 5) in SLE mice by 36 weeks of age compared with control mice (NZW/LacJ). Body weight in SLE mice was higher at each age compared with controls by 12%, 22%, and 34% (n > 30). Visceral adipose tissue weight was increased in SLE by 44%, 74%, and 117% at 8, 20, and 36 weeks, respectively (n > or = 12). Plasma leptin was increased in SLE mice (8.6+/-1.0 versus 24.7+/-2.2 ng/mL; n = 5), and renal and adipose tissue exhibited macrophage infiltration. Fasted insulin was higher in SLE mice (0.6+/-0.1 versus 1.4+/-0.3 ng/mL; n > or = 10), but fasted glucose was not different (94+/-5 versus 80+/-9; n > or = 9). A glucose tolerance test caused a significantly greater and longer increase in blood glucose from mice with SLE compared with control mice. Food intake was not different between control and SLE mice. However, mice with SLE demonstrated lower levels of nighttime activity than controls. These data show that the NZBWF1 strain may be an important model to study the effects of obesity and insulin resistance on SLE-associated hypertension.     

经树突状细胞递呈的HBcAg特异性细胞毒T淋巴细胞的体外研究

目的　从人的外周血树突状细胞 (DC)的抗原递呈方面研究慢性乙型肝炎的发病机制 ,并诱导出针对HBcAg特异性的细胞毒T淋巴细胞 (CTL)。方法　取健康人DC和患者DC ,比较两组的抗原递呈功能是否存在差异。以HBcAg体外冲击致敏DC ,与自体T淋巴细胞共育 ,诱导出HBcAg特异性CTL ;以HepG2细胞为对照靶细胞 ,转染HBVDNA的HepG2 2 15细胞为靶细胞 ,分别测定CTL在效靶比为 2∶1、6∶1和 2 0∶1时对HepG2细胞的非特异性杀伤率及对HepG2 2 15细胞的特异性杀伤率 ,并比较患者组与正常组特异性杀伤率的差异。结果　患者DC的抗原递呈功能(10 99.2 6 7± 2 39.12 ,1374 .8± 36 4 .15 5 ,2 717.78± 15 89.72 )较健康人 (314 7.933± 72 6 .5 7,384 3.0 0 0±10 6 0 .85 ,5 4 86 .86 7± 1790 .6 4 )弱 ;健康组与患者组CTL对HepG2各效靶比的非特异性杀伤作用差异无显著性。健康组与患者组CTL对HepG2 2 15的特异性杀伤作用差异有显著性 ;患者组CTL的活性 (7.1± 4 .33,15 .6 8± 3.3,2 7.6 6± 4 .5 9)较健康组 (2 0 .76± 6 .0 8,33.97± 8.0 0 ,4 9.6 3± 9.4 8)弱。结论　用HBcAg体外负载患者DC ,能诱导出抗原特异性的CTL ,这些CTL能特异性地杀伤相应靶细胞。