人单核细胞共刺激分子在异种免疫反应中的表达及其作用机制

目的 揭示人单核细胞共刺激分子在异种免疫反应中的表达及其作用机制.方法 从猪的主动脉分离血管内皮细胞(PEC)并培养扩增;从人单个核细胞(PBMC)中纯化CD4+T淋巴细胞和单核细胞.建立PEC和人PBMC混合培养体系,培养后收集细胞,然后加入荧光标记的单克隆抗体,通过流式细胞术检测CD14+单核细胞表面共刺激分子表达情况.为了检测淋巴细胞增殖反应以及阻断共刺激分子对PEC免疫反应的作用,在PEC和人PBMC混合培养体系中分别加入抗CD154、CD80和CD86单克隆抗体.在培养的最后24 h加入同位素,于培养结束后收集细胞并经同位素计数仪进行检测.纯化的单核细胞经PEC刺激后与CD4+T淋巴细胞共培养来研究这些单核细胞诱导CD4+T淋巴细胞的增殖以及阻断共刺激分子的作用.结果 PEC和人PBMC混合培养后可检测到PBMC对异种PEC的高度免疫增殖反应;流式细胞术检测到PBMC中的CD14+单核细胞表面无CD40和CD80的表达,但表达CD86,经PEC刺激后,CD14+单核细胞膜表面显著上调CD40和CD80蛋白分子的表达,CD86表达上调.与未经刺激的单核细胞相比较,经PEC刺激后的单核细胞和CD4+T淋巴细胞共培养后可诱导CD4+T淋巴细胞明显增殖,抗人CD154、CD80、CD86单克隆抗体可以阻断CD4+T淋巴细胞对PEC的增殖反应.结论 人CD14+单核细胞在异种免疫反应过程的间接抗原提呈和共刺激信号传导中发挥重要作用,通过上调其共刺激分子的表达与CD4+T淋巴细胞共刺激分子CD154和CD28相互作用形成第二信号,并诱导CD4+T淋巴细胞对PEC的增殖反应;阻断共刺激分子可抑制异种细胞免疫反应.     

T淋巴细胞共刺激分子调节单核细胞与内皮细胞相互作用中CD80表达的研究

目的 研究CD4+T淋巴细胞在调节单核细胞与血管内皮细胞相互作用中CDS0的表达水平及其意义.方法 建立单核细胞一血管内皮细胞(EC)共培养和单核细胞-CD4+T淋巴细胞-EC共培养体系,于培养72 h时收集细胞.采用流式细胞术(FACS)检测CD80在CD14+单核细胞表面的表达;通过实时逆转录聚合酶链反应(RT-PCR)技术检测CDS0基因转录的表达变化.建立含有或不含抗CD86、CD28和CD154单克隆抗体的单核白细胞(PBMC)-EC混合培养体系,通过FACS检测CD14+单核细胞表面CDSO的表达,采用混合淋巴细胞-EC反应(MLER)来研究CD54和CD28封闭在抑制淋巴细胞对同种异体EC增殖中的作用.结果 经FACS分析确定,无论是活化还是未活化的EC膜均缺乏CD80、CD86和CD14的表达.RT-PCR表明在缺乏CD4+T淋巴细胞时,经同种EC刺激的单核细胞可上调CD80基因转录水平的表达.但这些CD14+单核细胞膜表面CD80的表达并未上调,当CD4+T淋巴细胞加入到共培养中时,CD80的表达上调.用抗CD28和抗CD86抗体封闭CD28和0986并不能阻止CD14+单核细胞表面CDSO表达的上调.此外,阻断CD154也不能下调CD80的高表达.MLER证明淋巴细胞对EC的增殖反应可部分的被抗CD28和抗CD154抗体所阻断;阻断CD80可抑制经EC刺激的单核细胞诱导T淋巴细胞增殖反应.结论 在缺乏T淋巴细胞的条件下,经同种EC刺激的单核细胞可在基因转录水平上调CD80,而不是在其细胞膜表面的表达.当T淋巴细胞存在时,经EC刺激的CD14+单核细胞可在其膜表面上调CD80的表达.CD14+单核细胞膜表面CD80表达的上调并不能被CD154和CD28封闭所阻止,显示其上调是通过CD154和CD86非依赖性通道.     

树突状细胞与尤文肉瘤细胞融合诱导肿瘤免疫应答的体外研究

目的检测尤文肉瘤细胞A673和树突细胞(DC)融合构建的肿瘤疫苗对尤文肉瘤细胞株A673 的杀伤作用.方法应用促融合剂PEG对尤文肉瘤A673细胞和从外周血单个核细胞(PBMC)诱生的树突细胞进行融合.利用细胞因子hGM-CSF和hIL-4从 PBMC诱生DC,并对其表型进行流式细胞仪(FCM) 分析,红色荧光染料PKH-26标记A673细胞,绿色荧光染料PKH-67标记DC,应用促融合剂PEG融合后光镜及电镜观察DC细胞和融合细胞形态,FCM检测融合效率,通过同种混合淋巴细胞反应检测其免疫刺激活性.通过IFN-γ ELISA法产生细胞毒性T淋巴细胞(CTL)的量,通过51Cr细胞杀伤试验检测融合细胞对A673 细胞的杀伤作用.结果 FCM检测出从PBMC 成功诱生出CD83、CD80、CD86 及HLA-DR 高表达的成熟DC.DCs/A673融合细胞的融合效率达到23%,同种混合淋巴细胞反应显示DCs/A673融合细胞有很强的免疫刺激活性.IFN-γ分泌检测显示DCs/A673融合细胞组较对照组产生CTL水平显著增高.51Cr释放法检测融合细胞体外诱导抗原特异的CTL, 融合细胞激活的CTL对肿瘤细胞系A673细胞的杀伤作用强于DC-A673混合组、DC组、A673组的CTL(P<0.05),作用较对照各组显著增强.结论 DC与A673细胞融合体外致敏自体T 淋巴细胞能生成抗原特异CTL 对尤文肉瘤细胞A673 有一定的杀伤作用.     

人嗅鞘细胞的免疫学特性研究

目的 探讨人嗅鞘细胞的免疫学特征。方法 应用流式细胞仪分析人嗅鞘细胞的相应标记抗原的阳性表达率，用单向混合淋巴细胞反应测定人嗅鞘细胞的组织相容性和免疫耐受性。并与骨髓间充质干细胞对比。结果 人嗅鞘细胞与骨髓间充质干细胞表面与移植免疫排斥发生密切相关的标志：HLA-DR、B7-1（CD80）、B7-2（CD86）、CD40和CD40L均为阴性。单向混合淋巴细胞反应显示人嗅鞘细胞、骨髓间充质干细胞均对混合淋巴细胞反应无明显的刺激作用。结论 人嗅鞘细胞可能是一种免疫缺陷细胞，在体外有诱导细胞移植免疫耐受性的作用。     

第三方未成熟树突状细胞负载异体抗原诱导免疫耐受的实验研究

目的观察第三方未成熟树突状细胞（imDC）负载同种异体抗原后对其免疫特性的影响。方法从健康足月新生儿脐血中分离单核细胞，采用重组人粒细胞巨噬细胞集落刺激因子（rhGM-SCg）和重组人白细胞介素（rhIL）4联合培养7d，诱导其分化成imDC，并通过光学显微镜和扫描电镜观察细胞形态、检测细胞表型及混合淋巴细胞反应（MLR）；将培养的细胞与异体淋巴细胞抗原共同孵育，并给予共刺激分子阻断剂细胞毒性T淋巴细胞相关抗原4免疫球蛋白（CTLA-4Ig）处理，检测抗原负载前后的细胞表型变化，通过MLR比较致敏前后T淋巴细胞增殖的能力。结果（1）培养后细胞具有典型的imDC特征，CD1α、CD83、CD80、CD86等成熟标志呈低表达，分别为21．42％、0．59％、5．39％、3．85％；人类白细胞DR抗原（HLA-DR）的表达率为60．66％，MLR结果提示其不能刺激同种异体T淋巴细胞增殖。（2）负载抗原后的DC表现出成熟特性，CD1α、CD83、CD80、CD86及HLA-DR表达明显增高，分别为65．51％、42．20％、56．45％、38．52％、76．44％（P〈0．05）；能够刺激T淋巴细胞增殖[刺激指数（SI）〉2．00]。（3）给予共刺激阻断剂CTLA-4Ig致耐处理后，SI由2．51下降到0．39。可明显抑制T淋巴细胞增殖。结论第三方imDC负载抗原后能表现出成熟特性；经CTLA-4Ig致耐处理，不能刺激未致敏T淋巴细胞增殖。有望诱导受体针对供者抗原的特异性免疫耐受。     

腺病毒载体转染人未成熟树突状细胞对其成熟特性的影响

目的观察重组腺病毒载体AdEASY-增强型绿色荧光蛋白(EGFP)转染人未成熟树突状细胞(imDC)后,其表型特征及免疫学功能的变化,并探讨白细胞介素(IL)10对腺病毒转染诱导imDC成熟的抑制作用。方法贴壁法分离人脐带血来源的单核细胞,利用重组粒细胞巨噬细胞集落刺激因子(GM-CSF)和IL-4诱导分化imDC。对照组为常规培养的imDC,转染组用AdEASY-EGFP转染imDC,IL-10组用IL-10处理转染后细胞。流式细胞仪检测细胞表面成熟标志[CD86、CD83和人类白细胞DR抗原(HLA-DR)],混合淋巴细胞反应(MLR)检测其刺激同种异体未致敏T淋巴细胞的增殖能力。结果腺病毒转染imDC后,转染组细胞成熟表型表达率分别为CD86:46±10、CD83: 38±7、HLA-DR:82±10,均较对照组(10±7、8±3、68±8)显著上调,且刺激T淋巴细胞增殖的能力显著加强(SI>2．0)。IL-10组处理的imDC上述表型表达率分别为CD86:8±5、CD83:9±3、HLA-DR: 63±12,与对照组比较差异无统计学意义(P>0．05),其刺激T淋巴细胞增殖的能力也明显下降。结论腺病毒能有效转染imDC,但在转染后有促进其成熟的趋势;用IL-10能有效抑制该成熟状态。     

树突状细胞的扩增及培养上清液对胰腺癌细胞凋亡的研究

目的 体外观察胰腺癌(PC)患者树突状细胞(DC)增生成熟,DC分泌的细胞因子及DC诱导免疫效应细胞分泌的细胞因子对胰腺癌细胞PC3的凋亡作用。方法 用淋巴细胞分离液分离获取外周血单核细胞(PBMC),贴壁法获取DC和去DC的单核细胞(即免疫效应细胞),分别用粒/巨噬细胞集落刺激因子(GM-CSF)1 000 U/ml,人白细胞介素-4(IL-4)500 U/ml,肿瘤坏死因子(TNF)-α 500 U/ml、PC3肿瘤相关(TAA)和人 IL-2 100 U/ml培养 DC和免疫效应细胞,观察 DC生长状况,检测DC表型(CD1a、CD80、CD83、CD86)及DC培养上清液对PC3细胞的凋亡作用。结果 体外多种细胞因子和肿瘤相关抗原能有效引起胰腺癌患者 DC增殖 0.5×10~5～1.0×10~5个/ml,高表达CD80、CD83、CD86。DC培养上清液和DC混合免疫效应细胞培养上清液均能有效地引起PC3凋亡。结论 DC在胰腺癌免疫治疗中有重要作用。     

烧伤延迟复苏对CD14+单核细胞HLA-DR表达率和外周血淋巴细胞凋亡率的影响

目的:探讨烧伤延迟复苏对CD14 单核细胞人类白细胞组织相容性抗原-DR(HLA-DR)表达率和外周血淋巴细胞凋亡率的影响及其机制。方法:50例烧伤面积大于30%的烧伤患者,按伤后是否及时有效复苏分为延迟复苏组和非延迟复苏组,于伤后1、3、7、14、28d取外周血。流式细胞术检测CD14 单核细胞HLA-DR表达率和外周血淋巴细胞凋亡率。20例健康体检者外周血检测上述指标作为对照。结果:烧伤患者外周血CD14 单核细胞HLA-DR表达率明显低于正常对照组(P均<0.01),延迟复苏患者明显低于非延迟复苏患者(P<0.05或P<0.01)。烧伤患者外周血淋巴细胞凋亡率明显高于正常对照组(P<0.05),延迟复苏患者明显高于非延迟复苏患者,伤后7和28d差异有显著性(P<0.05)。结论:烧伤患者免疫功能低下,动态检测患者的免疫功能状态对患者预后具有指导意义。     

趋化因子RANTES对人外周血单个核细胞的免疫活化作用

目的　探讨趋化因子RANTES刺激对人外周血单个核细胞 (PMNC)的免疫活化作用及其内在机制。方法　分离人外周血 ,应用不同终浓度的人重组RANTES(rhRANTES)以及抗CD3单克隆抗体 (抗CD3mAb)进行体外刺激 ,并对增殖反应显著者给予吡咯啉烷二甲基硫脲 (PDTC)或CTLA4Ig进行干预。采用3 H 胸腺嘧啶核苷掺入法检测PMNC增殖程度 ,流式细胞仪检测淋巴细胞表型的变化。结果　rhRANTES终浓度为 10 0ng/ml和 5 0 0 0ng/ml时 ,诱导PMNC增殖反应出现两次峰值 ;rhRANTES(10 0ng/ml)刺激产生的增殖反应显著高于抗CD3mAb(5 0ng/ml)的刺激 (P <0 .0 5 ) ,但两者无拮抗或协同作用 ;PDTC和CTLA4Ig对rhRANTES(10 0ng/ml)诱导的增殖反应均能产生抑制作用 ,并呈现剂量依赖性 ;rhRANTES刺激后 ,淋巴细胞CD2 5表达率显著增加 (P <0 .0 5 ) ,而人RANTES的受体CCR5表达率显著降低 (P <0 .0 5 ) ,CD2 8表达率及CD4 /CD8比值无明显改变(P >0 .0 5 )。结论　RANTES趋化信号具有不依赖于CD3信号的独特的诱导PMNC免疫活化的功能。     

脂肪干细胞免疫学性状的初步实验观察

目的初步研究脂肪干细胞(Adiposederivedstemcells,ADSC)表面免疫分子的表达以及体外免疫调节功能,以期为组织工程提供同种异体种子细胞来源。方法体外培养人脂肪抽吸术中获取的脂肪干细胞,体外培养至第二代,流式细胞仪检测免疫分子HLA、HLA、B7-1、B7-2、CD40的表达。1×105个/孔ADSC细胞分别刺激单一异体淋巴细胞或混合双向淋巴细胞反应,观察淋巴细胞增殖情况。同时观察ADSC经IFN-γ作用后,免疫分子表达与淋巴细胞增殖的调节情况。结果ADSC表达HLA类分子,但未检测到HLA类分子阳性表达。B7-1(CD80)、B7-2(CD86)、CD28、CD40未见明显阳性表达。人IFN-γ刺激48h后,HLA类分子表达明显增高,HLAI表达未见明显增高。异体或经IFN-γ作用的ADSC均未能刺激异体淋巴细胞增殖。同样数量的ADSC可明显抑制双相混合淋巴细胞增殖,经IFN-γ作用后抑制作用未见明显减弱。结论ADSC具有一定的体外调节淋巴细胞反应的能力,有可能成为组织工程同种异体细胞来源。     

Rabbit anti-human leukocyte polyclonal antibody inhibits xenogeneic cell-mediated immune responses

We studied the interactions between human monocytes and porcine endothelial cells (PEC) as well as the effects of a new generation of rabbit anti-human leukocyte polyclonal antibody (newRALG) to inhibit xenogeneic cell-mediated immune responses. Human peripheral blood mononuclear cells (PBMC) were cocultured with the florescent dye PKH-26 labeled-PEC, which showed membrane uptake by monocytes detected by florescence activated cell scanning (FACS). Scavenger receptor (SR) ligand poly-(G) or the newRALG or Thymoglobulin was added into the cocultures followed by FACS. Lymphocyte proliferation upon exposure to PEC with or without newRALG or thymoglobulin was evaluated by a xenogeneic mixed-lymphocyte-endothelial cell reaction (xMLER). FACS analysis demonstrated that CD14⁺ monocytes became positive for PKH-26 following their interaction with PKH-26-labeled PEC. These PKH-26⁺ monocytes displayed up-regulated CD40 and CD80 expression during the PBMC-PEC interaction. Furthermore, SR blockade with poly-(G) prevented PEC membrane uptake by CD14⁺ monocytes. The newRALG from rabbits immunized with activated human monocytes and lymphocytes greatly reduced SR-mediated PEC membrane uptake. xMLER demonstrated strong lymphocyte proliferation in response to PEC. Lymphocyte proliferation was dramatically inhibited in dose-dependent manner by the newRALG. In summary, monocytes up-regulate costimulatory molecules during xenogeneic interactions, indicating that they may serve as a source of T-cell costimulation during xenogeneic reactions, enguling PEC membranes. This phenomenon was inhibited by poly (G), suggesting that PEC membrane uptake was via SR. The newRALG inhibited monocyte SR-mediated PEC membrane uptake and lymphocyte proliferation in response to PEC suggesting that this new polyclonal preparation may impair the initiation of xeno-specific immune responses.     

The interleukin-2 receptor α chain (CD25) plays an important role in regulating monocyte-derived CD40 expression during anti-porcine cellular responses

Long-term xenograft survival is limited by delayed xenograft rejection, and monocytes are thought to play an important role in this process. Although typically considered a T cell surface marker, interleukin 2 the receptor chain CD25 is also functional on monocytes. We hypothesized that CD25 expression on monocytes functions to augment monocyte activation in xeno-specific cellular responses. Xenogeneic mixed lymphocyte-endothelial cell reactions were used to study the role of CD25 in facilitating xenogeneic cell-mediated immune responses an in vitro. We also tested the effect of the anti-CD25 antibody daclizumab on monocyte-mediated T cell activation during xeno-specific cellular responses. Co-culture with porcine endothelial cells (PEC) elicited a pronounced proliferative response by human peripheral blood mononuclear cells (PBMC) that was accompanied by upregulation of CD25 and CD40 on CD14⁺ monocytes. CD4⁺ cells proliferated in response to PEC-conditioned monocytes, while blockade of CD25 with daclizumab reduced CD4⁺ cell proliferation in the presence of PEC-conditioned monocytes. In addition, daclizumab inhibited proliferation of PBMC in responses to PEC. Analysis of monocytes from PBMC-PEC cocultures by flow cytometry indicated that daclizumab inhibited CD40 upregulation on PEC-activated monocytes. These data demonstrate that CD25 blockade prevents xenogeneic cellular responses by directly blocking CD25 expression on both activated T cells and monocytes. CD25 blockade on T cells or monocytes may indirectly affect upregulation of CD40 on xenoreactive monocytes. Our data strengthen the rationale for incorporating CD25 directed therapy in discordant xenotransplantation.     

CD4+ T-cell and monocyte interdependence during discordant xenoimmune responses

This study was designed to examine our hypothesis that human monocytes provide missing constimulatory signals to host CD4⁺ cells during interactions with porcine endothelial cells (PECs). PECs were isolated from the aorta. Human CD4⁺ T cells and monocytes were purified from peripheral blood mononuclear cells (PBMCs). A xenogeneic mixed lymphocyte-PEC reaction (xMLER) was performed to determine the proliferation of PBMCs or CD4⁺ cells in response to PEC. Monocyte-PEC cocultures with or without CD4⁺ cells were followed by analysis using fluorescence-activated cell scanning (FACS). We evaluated the CD4⁺ cells proliferation induced by PEC-conditioned monocytes with or without costimulation blockade. xMLER demonstrated strong lymphocyte proliferation in response to PECs. However, purified CD4⁺ cells showed reduced proliferative responses to PECs when compared with PBMCs. FACS analysis found that CD14⁺ monocytes up-regulated CD40 and CD80 expressions in the presence of CD4⁺ cells. PEC-activated but not resting monocytes induced CD4⁺-cell proliferation, which was inhibited by anti-CD154, anti-CD80, or anti-CD86 antibodies. In summary, human monocytes exposed to PECs are conditioned to up-regulate costimulatory molecules upon exposure to T cells. PEC-conditioned monocytes induced T-cell proliferation by indirect presentation. Costimulation blockade inhibited T-cell proliferation induced by PEC-conditioned monocytes. Our findings suggested that monocytes play an important role in indirect xenoantigen presentation, providing costimulation to T cells. This interaction can occur distant from the initial site of xenoantigen, but monocytes remaide void of costimulatory signals until their interaction with T cells.     

Increased monocyte-dependent suppression of polyclonal activation of B lymphocytes from cystinotic children

In infantile cystinosis the amino acid cystine preferentially accumulates in phagocytic cells, polymorphonuclear leucocytes (PMN) and monocytes, rather than in lymphocytes. We previously described functional abnormalities in the oxidative metabolism and locomotion of cystinotic PMN and monocytes. The present study shows an abnormal lymphocyte polyclonal activation as evidenced by a decreased immunoglobulin (Ig) production and generation of Ig-containing cells (ICC) in cultures of peripheral blood mononuclear cells (PBMC) from cystinotic children upon stimultion with pokeweed mitogen andStaphylococcus aureus Cowan I. However, monocyte depletion from cystinotic PBMC fully reconstituted Ig production and ICC generation, indicating: (1) the presence of an increased monocyte-dependent suppression on lymphocyte polyclonal activation, and (2) that the intrinsic ability of cystinotic lymphocytes to respond to polyclonal stimulation was preserved. The increased cystinotic monocytedependent suppressive effect was not mediated by prostaglandin E₂ (PGE₂) since its production by cystinotic PBMC upon polyclonal activation was not different from that of controls. In addition, the sensitivity of cystinotic lymphocytes to the immunosuppressive effect of varying concentrations of exogenous PGE₂ was similar to that of controls. Finally, indomethacin and 2-mercaptoethanol, two agents able to scavenge hydroxyl (OH) radicals, restored Ig production by cystinotic PBMC, suggesting a role for reactive oxygen species in the increased cystinotic monocyte-dependent suppression.     

Deviation of xenogeneic immune response and bystander suppression in rats fed porcine blood mononuclear cells

Reducing or deviating xenogeneic immune response prior to xenotransplantation may enhance the efficacy of conventional immunosuppressive therapies in prolonging xenograft survival. The potential to suppress or steer immune responses by oral administration of xenoantigens was evaluated. Based on knowledge of oral tolerance, hypotheses tested were that feeding xenoantigens would inhibit cell-mediated immune response (CMIR) and production of antibodies associated with graft rejection and induce bystander suppression. DA and LEW rats, high and low responders to xenoantigens, respectively, were fed dead porcine blood mononuclear cells (PBMC) and subsequently received live PBMC and hen egg-white lysozyme (HEWL, a third-party antigen) by subcutaneous injection. Delayed-type hypersensitivity (DTH) to PBMC was an indicator of CMIR. Quantification of T(H)1 (IgG(2b)) and T(H)2 (IgG(1))-associated antibodies and their ratio measured magnitude and bias of the antibody-mediated response to PBMC and HEWL. Feeding PBMC reduced IgG(2b) antibody production by 90% (DA) and 71% (LEW) and increased IgG(1) antibodies by 116% in DA but not LEW rats (p相似文献   

CTLA4-Ig和IL-4诱导异种骨移植免疫耐受的体外研究

目的探讨CTLA4Ig和IL4在诱导异种骨移植免疫耐受中的作用。方法反应细胞为BALB/c小鼠脾淋巴细胞,刺激细胞为新西兰白兔血淋巴细胞,刺激抗原为兔骨上清液。采用经典的混合淋巴细胞培养法及骨上清液与淋巴细胞混合培养法作为异种骨移植的体外实验模型。在各培养液中分别加入CTLA4Ig、IL4及两者联合应用,通过测定其3HTdR掺入率,观察不同细胞因子对刺激淋巴细胞增殖的影响。结果1细胞刺激组CTLA4Ig和IL4均对淋巴细胞增殖有显著抑制作用P<0.001),CTLA4Ig与IL4联合应用并未显示出比CTLA4Ig单独应用更为明显的细胞增殖抑制作用(P>0.05)。2骨上清液刺激组:CTLA4Ig对细胞增殖无抑制作用(P>0.05),而IL4则有较为显著地细胞增殖抑制作用(P<0.05);CTLA4Ig与IL4联合应用也未产生比单独应用IL4更为明显的细胞增殖抑制作用(P>0.05)。结论CTLA4Ig对由细胞刺激产生的淋巴细胞增殖抑制效果较好,而IL4则对骨上清液刺激的细胞增殖抑制作用更好;CTLA4Ig与IL4联合应用并未产生协同抑制作用。     

A nuclear factor-kappaB inhibitor BAY11-7082 inhibits interactions between human endothelial cells, T cells, and monocytes

Costimulatory molecules play critical roles during cell-mediated immune responses. We undertook this study to determine whether CD154-CD40 interactions induced human endothelial cell (EC) activation via the nuclear factor (NF)-κB pathway, and whether the upregulation of monocyte-derived CD40 and CD80 is NF-κB pathway dependent. A CD154-expressing D1.1 cell-EC coculture with or without the NF-κB inhibitor BAY11-7082 was established to examine EC activation as indicated by CD62E expression. Peripheral blood mononuclear cell (PBMC)-EC cocultures were performed in the presence or absence of BAY11-7082; the expression of CD40 and CD80 on monocytes was analyzed by FACS. Allogeneic mixed lymphocyte-EC reaction (MLER) was performed to determine the inhibitory effects of BAY11-7082 to prevent lymphocyte proliferation. FACS demonstrated upregulation of EC-derived CD62E expression induced by CD154 expressing D1.1 cells. BAY11-7082 pretreated EC failed to upregulate CD62E after interaction with D1.1 cells. Monocytes upregulated CD40 and CD80 expression during PBMC-HEC interaction, and BAY11-7082 suppressed monocyte-derived CD40 and CD80 expression in a dose-dependent manner. The monocyte-derived CD86 expression was downregulated by NF-κB inhibitor. BAY11-7082 demonstrated inhibition of lymphocyte proliferation of allogeneic MLER. This study demonstrated that the NF-κB inhibitor BAY11-7082 prevented CD154-CD40 interaction-induced EC activation, suggesting that the activation of EC by T-cell-derived CD154 is via NF-κB pathway. The NF-κB inhibitor suppressed upregulation of monocytederived CD40 and CD80. Additionally, BAY11-7082 suppressed lymphocyte proliferation in response to allogeneic EC. These data indicated that NF-κB plays an important role in regulating costimulatory molecules in allogeneic immune responses, and strengthens the rationale for the use of NF-κB-directed therapy in allotransplantation.     

Comparison of in vivo lymphocyte proliferation between allogeneic and xenogeneic heart transplantation in mice

There are controversial in vitro data comparing the strength of the cellular immune response between allogeneic and xenogeneic stimulator/responder combinations. The present study therefore compares in vivo lymphocyte proliferation using heart transplantation (HTx) models in mice. Heterotopic HTx into BALB/c mice was performed using donor organs from mice (BALB/c and C57BL/6) or Lewis rats. Intraperitoneally given bromodeoxyuridine (BrdU) was incorporated into the DNA and was subsequently analyzed by flow cytometry. On postoperative days 3 and 5, proliferation of splenocytes, CD4(+) T-lymphocytes, and CD19(+) B-lymphocytes was significantly higher after xenogeneic than after allogeneic and isogeneic HTx. No significant difference was observed when proliferation of CD8(+) lymphocytes was determined. The increased in vivo proliferation after xenotransplantation may reflect an earlier and probably stronger cellular immune response compared to allogeneic transplantation. The higher CD4(+) lymphocyte proliferation underscores the importance of this cell population in xenograft rejection.