IgG inhibits the increase of platelet-associated C3 stimulated by anti-platelet antibodies

We investigated the increase of platelet-associated IgG and complement component 3 (C₃) caused by the in vitro action of anti-platelet MoAbs, and the effect of mouse and human IgG on these events. Anti-glycoprotein IIb/IIIa and anti-glycoprotein Ib MoAbs caused a slight increase of C₃, but not of platelet-associated IgG. In contrast, anti-CD9 and anti-Fcγ II receptor MoAbs caused an increase of both platelet-associated C₃ and IgG. In particular, three MoAbs which activated the complement system caused a marked increase of C₃. When platelet-rich plasma was treated with aspirin and prostaglandin E₁ before incubation with antibodies, the increase of platelet-associated IgG was inhibited in all cases. In contrast, the increase of platelet-associated C₃ was scarcely influenced. These results suggest that the binding to platelets of platelet-activating antibodies caused the increase expression of IgG molecules on the platelet surface and a possible increase of platelet-associated IgG. However, the increase of platelet-associated C₃ appeared to depend on specific characteristics of the antibodies tested, such as a complement-activating effect. In addition, intact mouse or human IgG inhibited the increase of platelet-associated C₃ caused by complement-activating antibodies, while F(ab')₂ mouse or human IgG had no such effect. This suggested that the Fc portion of IgG may block the increase of C₃ mediated by anti-platelet antibodies.     

人白细胞介素18(hIL-18)的纯化及生物学活性的鉴定

目的：在大肠杆菌中高效表达重组人IL-18(rhIL-18)蛋白，制备具有高纯度、高活性的rhIL-18。方法：用IFTG诱导重组蛋白表达载体pKK223-3／hIL-18进行蛋白表达；菌体经超声破碎分离包含体，并对包含体进行洗涤、变性和复性处理，用DEAE-Sepharose CL-6B阴离子交换柱纯化复性的rhIL-18；以人外周血单核淋巴细胞，通过T细胞增殖实验、^125I-UdR标记的细胞毒实验、ELISA方法检测细胞因子的产生量等方法，测定rhIL-18的生物学活性。结果：在大肠杆菌中成功地诱导、表达了rhIL-18，SDS-PAGE凝胶电泳分离显示在分子量大约18．3kD处有一诱导蛋白带，Western blot证明表达产物与抗IL-18单克隆抗体有特异性免疫反应；表达产物经纯化后纯度达94％；表达的rhIL-18蛋白具有促进T细胞增殖、增强NK细胞细胞毒作用及诱导外周血单核淋巴细胞合成IFN-γ的能力，基本上具有天然IL-18相同的生物学活性。结论：为rhIL-18的获得及为进一步研究其生物学功能提供了一定的条件，并为将rhIL-18用于肿瘤的免疫生物治疗奠定了基础。     

A monoclonal antibody (RH1-38) which inhibits multiple systems of cell-mediated cytotoxicity--I. Identification of a novel epitope on a killer cell surface bimolecular complex important in the cytotoxic response

This paper presents the initial characterization of a mouse monoclonal antibody (RH1-38) which blocks, in the absence of complement, three different systems of cell-mediated cytotoxicity. This monoclonal antibody markedly inhibits cytotoxicity mediated by human natural killer cells, a monocyte-like cell [phorbol myristate acetate (PMA) stimulated HL-60], and cytotoxic T-lymphocytes generated in a mixed leukocyte reaction. RH1-38 is not nonspecifically toxic to cells since antibody-dependent cellular cytotoxicity was not inhibited and viability as assessed by trypan blue exclusion was not affected. Inhibition is specific since control hybridoma culture supernatants, parent (NS-1) ascites supernatant, monoclonal anti-HLA and normal mouse IgG were not significantly inhibitory. In the NK system, the inhibitory effect appears to be due to binding of monoclonal antibody to effector cell surface since exposure of targets to antibody followed by washing yielded no inhibition of killing. Inhibition requires the antigen-binding portion of the antibody molecule and thus appears to be related to steric hindrance of an effector cell surface molecule which is important in the expression of cell-mediated cytotoxicity. Immunoprecipitation of surface-radioiodinated membranes from PMA-stimulated HL-60 cells and analysis on sodium dodecyl sulfate-polyacrylamide gels revealed a bimolecular complex (195,000 and 125,000 daltons) without significant change under reducing conditions. Control immunoprecipitates yielded no peaks of activity. This monoclonal antibody should serve as a useful probe of the function and biochemistry of a killer cell surface antigen important in the expression of cell-mediated cytotoxicity. Since RH1-38 inhibits cytotoxicity mediated by at least three apparently unrelated effector cells, the relevant antigen may be part of a common mechanistic step. As the companion paper demonstrates, this monoclonal antibody does not affect the conjugation step, but appears to block a late step in the NK cytolytic mechanism. Thus, RH1-38 recognizes either an epitope district from previously-described anti-LFA-1 antibodies or alternatively recognizes a distinct functional killer cell surface molecule.     

Cytotoxic effector cells against HLA antigens in strong linkage disequilibrium: identification of a strong,new, CML determinant

Three sets of cytotoxic effector cells were generated against the A1, B8, DR3 haplotype using haptoidentical individuals in three different families. The three sets of effector cells generated against this haplotype showed excellent reproducibility testing, strong cytotoxicity against their specific targets, low autologous kill, and segregation with the sensitizing haplotype within the family. When tested against a panel of cells bearing all combinations the A1, B8. DR3 antigens, a hierarchy of contribution of the individual HLA antigens as CML target determinants was seen. A new strong target cell determinant was identified by cytotoxicity with one of the effector cells not explicable in terms of the A1, B8, DR3 antigens or known HLA cross-reactivity. A family study demonstrated that this determinant clearly segregates with HLA. The success of this approach in defining new CML determinants may result from the generation of effector cells across a single haplotype in strong linkage disequilibrium or from the presentation of CML determinants in the context of self.     

Structure and function of natural-killer-cell receptors

The function of natural-killer (NK) cells is modulated by the balance between a number of activating and inhibitory receptors. Killer immunoglobulinlike receptors (KIRs) are mostly inhibitory receptors. They play a critical role in recognizing self-class-I major histocompatibility complex (MHC) molecules and thus protect healthy host cells from NK-targeted lysis. In contrast, both NKG2D and CD16 are activating NK receptors that trigger the NK-cell lysis of various tumor and virally infected cells through either direct ligand engagement or antibody-dependent cellular cytotoxicity (ADCC). Through structural studies of members of these distinct receptor families, in particular, the structure and recognition between KIR2DL2 and HLA-Cw3, that between NKG2D and ULBP3, and that between CD16 and IgG Fc, considerable understandings have been achieved about their function and their ligand recognition.     

Cell-mediated immune responses of lambs to challenge with bovine respiratory syncytial virus.

The lamb is a good model to study the pathogenesis and immune responses to infections with respiratory syncytial virus (RSV) as lambs experimentally infected with bovine or human RSV may develop overt clinical disease. In the present study the development of cellular cytotoxic responses was studied in splenic, pulmonary and peripheral blood mononuclear cells obtained from lambs after primary and secondary infection with bovine RSV. Infection with bovine RSV was followed by the appearance of cytotoxic cells in the peripheral blood, the spleen and lung lavage fluids. These effector cells lysed virus-infected targets in a self-restricted manner. Depletion techniques revealed that cytotoxic activity was largely due to OvCD8+ cells. When effector cells obtained from primed lambs were stimulated with inactivated bovine RSV or with virus-infected cells in vitro, virus-specific cytotoxicity was significantly increased.     

不明原因自然流产与蜕膜NK细胞表面活化性受体表达的相关性研究

探讨不明原因自然流产患者蜕膜NK细胞杀伤活性与其细胞表面活化性受体NKp46、NKp44、NKp30和NKG2D表达的相关性。选取21例早孕不明原因自然流产患者为病例组,25例正常早孕人流妇女为对照组,收集两组的蜕膜组织,Ficoll密度梯度离心分离淋巴细胞,MACS磁珠分选CD3-CD56+NK细胞。以K562细胞为靶细胞,用细胞染色及流式细胞技术检测两组蜕膜NK细胞杀伤活性,用流式细胞技术检测两组蜕膜CD56brightCD16-NK和CD56dimCD16+NK细胞上活化性受体NKp46、NKp44、NKp30和NKG2D的表达,并与NK细胞杀伤活性进行相关性分析。结果:(1)早孕蜕膜NK细胞具有杀伤活性;(2)病例组蜕膜NK细胞的杀伤活性较正常对照组显著增强(P=0.014);(3)病例组蜕膜CD56brightCD16-NK细胞中NKp44的表达比正常对照组显著升高(P=0.021);病例组蜕膜CD56dimCD16+NK细胞中NKp46和NKp44的表达比正常对照组显著升高(分别P=0.026,P=0.041);其余活化性受体的表达两组未见明显差异;(4)蜕膜NK细胞杀伤功能与蜕膜CD56brightCD16-NK细胞中NKp44的表达呈显著正相关(r=0.677,P<0.05),和蜕膜CD56dimCD16+NK细胞中NKp46的表达呈显著正相关(r=0.634,P<0.05)。蜕膜NK细胞活化性受体NKp46和NKp44表达增加,从而使蜕膜NK细胞的杀伤功能增强可能在不明原因自然流产的发病中起重要作用。     

Recombinant Mycobacterium bovis bacillus Calmette-Guérin (BCG) expressing mouse IL-18 augments Th1 immunity and macrophage cytotoxicity

Interleukin-18 (IL-18) has been demonstrated to synergize with BCG for induction of a T-helper-type 1 (Th1) immune response. Since successful treatment of superficial bladder cancer with BCG requires proper induction of Th1 immunity, we have developed a recombinant (r) BCG strain that functionally secretes mouse (m) IL-18. This rBCG-mIL-18 strain significantly increased production of the major Th1 cytokine IFN-gamma in splenocyte cultures, at levels comparable to that elicited by control BCG plus exogenous rIL-18. IFN-gamma production by splenocytes was eliminated by addition of neutralizing anti-IL-18 antibody. Endogenous IL-12 played a favourable role whereas IL-10 played an adverse role in rBCG-mIL-18-induced IFN-gamma production. Enhanced host antimycobacterial immunity was observed in mice infected with rBCG-mIL-18 which showed less splenic enlargement and reduced bacterial load compared to control mice infected with BCG. Further, splenocytes from rBCG-mIL-18-infected mice, in response to BCG antigen, displayed increased production of IFN-gamma and GMCSF, decreased production of IL-10, elevated cellular proliferation and higher differentiation of IFN-gamma-secreting cells. rBCG-mIL-18 also enhanced BCG-induced macrophage cytotoxicity against bladder cancer MBT-2 cells in a dose-dependent manner. Neutralizing all endogenous macrophage-derived cytokines tested (IL-12, IL-18 and TNF-alpha) as well as IFN-gamma severely diminished the rBCG-mIL-18-induced macrophage cytolytic activity, indicating a critical role for these cytokines in this process. Cytokine analysis for supernatants of macrophage-BCG mixture cultures manifested higher levels of IFN-gamma and TNF-alpha in rBCG-mIL-18 cultures than in control BCG cultures. Taken together, this rBCG-mIL-18 strain augments BCG's immunostimulatory property and may serve as a better agent for bladder cancer immunotherapy and antimycobacterial immunization.     

IL-10 down-regulates costimulatory molecules on Mycobacterium tuberculosis-pulsed macrophages and impairs the lytic activity of CD4 and CD8 CTL in tuberculosis patients

Activation of T cells requires both TCR-specific ligation and costimulation through accessory molecules during T cell priming. IFNgamma is a key cytokine responsible for macrophage activation during Mycobacterium tuberculosis (Mtb) infection while IL-10 is associated with suppression of cell mediated immunity in intracellular infection. In this paper we evaluated the role of IFNgamma and IL-10 on the function of cytotoxic T cells (CTL) and on the modulation of costimulatory molecules in healthy controls and patients with active tuberculosis (TB). gamma-irradiated-Mtb (i-Mtb) induced IL-10 production from CD14(+) cells from TB patients. Moreover, CD3(+) T cells of patients with advanced disease also produced IL-10 after i-Mtb stimulation. In healthy donors, IL-10 decreased the lytic activity of CD4(+) and CD8(+) T cells whereas it increased gammadelta-mediated cytotoxicity. Furthermore, we found that the presence of IL-10 induced a loss of the alternative processing pathways of antigen presentation along with a down-regulation of the expression of costimulatory molecule expression on monocytes and macrophages from healthy individuals. Conversely, neutralization of endogenous IL-10 or addition of IFNgamma to either effector or target cells from TB patients induced a strong lytic activity mediated by CD8(+) CTL together with an up-regulation of CD54 and CD86 expression on target cells. Moreover, we observed that macrophages from TB patients could use alternative pathways for i-Mtb presentation. Taken together, our results demonstrate that the presence of IL-10 during Mtb infection might contribute to mycobacteria persistence inside host macrophages through a mechanism that involved inhibition of MHC-restricted cytotoxicity against infected macrophages.