Avidin-binding to peripheral blood B lymphocytes and monocytes.

Avidin-coated magnetic beads bind peripheral blood B lymphocytes and monocytes. This unwanted reactivity is not due to the membrane expression of avidin target molecules since beads coated with a biotin-binding analogue are non-reactive and binding occurs even when cellular carbohydrate-binding sites are not active, in the absence of Mg2+ and Ca2+ cations, or when they are blocked by a alpha-D-glucose or alpha-D-mannose in presence of Ca2+ and Mg2+. The non-polar residues of avidin appear not to be engaged in a hydrophobic bond with the membrane molecule since suroptimal quantities of serum albumin do not prevent the avidin binding. It is suggested that ionic interactions explain the binding of avidin-coated beads to B lymphocytes and monocytes and that these can be inhibited with high molecular weight serum molecules or with 0.4 M NaCl.     

DR framework antigens and 63D3 antigens on human peripheral blood monocytes and alveolar macrophages

The expression of surface antigens on human peripheral blood monocytes and alveolar macrophages was compared using the monoclonal antibodies 63D3, which react with monocytes, and OKIa, which reacts with DR framework antigens. Fluorescence-activated cell-sorter analysis revealed that 71.5 +/- 4.6% of peripheral blood monocytes reacted with 63D3 and showed a uniform distribution of binding. Alveolar macrophages also reacted with 63D3 and displayed uniform binding. Furthermore, the relative fluorescence intensity was very similar to that of monocytes. These data suggest that 63D3 antigen expression is similar on peripheral blood monocytes and alveolar macrophages and may be a stable marker in the differentiation of monocytes to macrophages. Analysis showed that 45.8 +/- 4.9% of peripheral blood monocytes reacted with OKIa and showed a nonuniform distribution of binding, while 74 +/- 8.4% of alveolar macrophages reacted with OKIa and exhibited uniform binding. The relative fluorescence intensity of alveolar macrophages which were reacted with OKIa was significantly greater than that of blood monocytes (P less than 0.001). Size analysis suggested that alveolar macrophages express approximately five times more DR antigens per unit surface area than do peripheral blood monocytes. The expression of DR antigens on the alveolar macrophage surface suggests an important role for macrophages in antigen presentation in the lung.     

Heterogeneity of human peripheral blood mononuclear cells detected by monoclonal antibodies to monomorphic determinants of human Ia antigens

The reactivity patterns of several monoclonal antibodies specific for monomorphic determinants of human Ia antigens were studied using flow cytometric techniques. We observed differential reactivity of these antibodies with human lymphoid cell lines, normal fresh human mononuclear cells, and lymphoblasts from PHA-activated cultures. The molecular heterogeneity of Ia antigens previously identified with immunochemical techniques was accompanied by heterogeneity of cell surface expression as identified by an immunofluorescent probe. The determinants identified by these anti-Ia monoclonal antibodies may provide useful markers in the isolation of cellular subpopulations responsive in the immune system.     

明胶法富集外周血单核细胞并刺激其成熟为树突状细胞

背景：如何简易高效分离纯化人外周血单核细胞并刺激成熟为树突状细胞,未见标准化操作流程。 目的：观察明胶法分离外周血单核细胞的效率以及将分离出的单核细胞刺激成熟为树突状细胞的表型特征,并与普通塑料黏附法对比。 方法：使用人淋巴细胞分离液分离人外周血得到单个核细胞,根据培养瓶是否进行明胶包被分为明胶包被组和普通塑料组。均分单个核细胞,按组别分离获得单核细胞并诱导刺激成熟为树突状细胞。计数各组所得单核细胞数,使用流式细胞仪检测2组单核细胞的CD14阳性率、T、B淋巴细胞污染率、树突状细胞非成熟期和成熟期CD1a,CD83的表达情况,锥虫蓝拒染法计算细胞活率,观察对比2组血小板污染情况。 结果与结论：明胶包被组单核细胞数及CD14阳性率显著高于普通塑料组(P < 0.05),普通塑料组淋巴细胞污染率显著高于明胶包被组(P < 0.05)。2组细胞活率及树突状细胞表型差异无显著性意义(P > 0.05)。明胶包被组血小板污染率低于普通塑料组。提示明胶法可以简单高效分离出单核细胞并成功刺激成熟为树突状细胞。 关键词：明胶;单核细胞;树突状细胞;人外周血;单个核细胞 doi:10.3969/j.issn.1673-8225.2012.10.037     

Activation of human peripheral blood lymphocytes by concanavalin A dependence of monocytes.

The activation of human peripheral blood lymphocytes or isolated T lymphocytes by concanavalin A (Con A) is hightly potentiated by the presence of autologous, mitomycin C-treated monocytes. The optimal lymphocyte: monocyte ratio within a broad dose range is 1:1 when the incorporation of [14C]thymidine is expressed as total incorporation per culture tube and 1:10 when expressed per lymphocyte. A five-to-ten-fold increase of total DNA synthesis is noted in the presence of 10-90% monocytes. The data may help to explain the wide variations in Con A responsiveness of human peripheral lymphocytes which may be partly related to differences in purification which give rise to cell preparations containing varying amounts of monocytes.     

Bovine monocytes induce immunoglobulin production in peripheral blood B lymphocytes

Although a role for monocytes and monocyte-derived dendritic cells (DC) in the activation of T cells is well established, it is less clear to what extent DC and their precursors, monocytes, regulate B cell immune responses. Here we show that regulatory mechanisms similar to those in humans are in place in the bovine immune system. In vitro culture of bovine monocytes with bovine B cells activated by the anti-CD3 triggered CD4⁺ T cells or through immunoglobulin (Ig) receptor crosslinking induces B cell Ig secretion. Unlike bovine monocyte-derived DC, monocytes do not promote Ig class switching to IgG and IgA in activated peripheral blood B cells. These results suggest that bovine monocytes are capable of directly inducing Ig secretion in activated bovine peripheral blood B cells, but do not provide the signals for B cell Ig class switching.     

Demonstration of non-idiotypic variable heavy chain (VH) antigens on human peripheral blood lymphocytes

In this study we have obtained evidence for the expression of Vh-like determinants on unstimulated human T lymphocytes as well as T lymphoblasts. These determinants were detected with antisera raised against isolated VH fragments of a human IgG3 cryoglobulin (KUP). The antisera detect idiotypic, VH subgroup and HV framework determinants and behave as anti-immunoglobulin antibodies when tested against peripheral blood mononuclear cells in immunofluorescence experiments. However, sensitive radiolabelling and immunoprecipitation techniques revealed a certain reactivity against highly purified T lymphocytes. The specificity of these T-cell-reactive antibodies has not been fully established, but the results suggest that the antisera contain antibodies directed at VH fragment-specific antigens or antigens not exposed on native immunoglobulins or isolated heavy chains.     

Solid-phase radioimmunoassay for the measurement of surface antigens expressed on intact lymphocytes

A solid-phase radioimmunoassay was developed for the measurement of lymphocyte surface antigens. The assay was performed in microplates, using cells that were initially fixed to the wells by air drying. The method was used for the measurement of Thy-1, Lyt-1,2,3, IL-2-R, H-2Kb and DR antigens on the surface of mouse thymus, spleen and bone marrow cells, mouse cell lines CTLL, EL-4 and DA-1 and human thymocytes and consisted of sequential incubations with rat or mouse monoclonal antibodies directed against the above antigens, rabbit anti-rat or goat anti-mouse IgG and 125I-protein A. The assay permits the processing of large numbers of samples, is easy to perform, reliable and highly specific.     

Mitogenic actions of orthoclone OKT3 on human peripheral blood lymphocytes: Effects of monocytes and serum components

The Orthoclone monoclonal antihuman T lymphocyte antibody, OKT3, induced maximal DNA, RNA and protein synthesis in peripheral mononuclear blood cells (PMBC) at concentrations as low as 10 ng ml⁻¹. This pronounced mitogenic activity was highly dependent on the presence of monocytes: removal of these cells from PMBC suspensions by complement (C)-dependent lysis with the antimonocyte antibody OKM1, completely abrogated the proliferative responsiveness of the remaining lymphocytes. The addition of adherent cells to OKM1-treated PMBC demonstrated the strict monocyte requirement for the mitogenic activity of OKT3. Mitogenic responses to OKT3 were most marked when PMBC were cultured in media containing heat-inactivated fetal calf serum (FCS) but they were considerably weaker in cultures supplemented with heat-inactivated human serum (HS). Moreover, aggregated human IgG and its Fc fragments (but not monomeric IgG and its Fab fragments) inhibited the mitogenicity of OKT3: their inhibition could be explained by stimulation of monocytes, resulting in increased prostaglandin E release, since (a) prostaglandin E₂ itself strongly suppressed OKT3 activity and (b) indomethacin blocked the inhibitory effects of aggregated HuIgG.The present data demonstrate that OKT3 shows a particular pattern of mitogenicity: the strict monocyte requirement, the inhibitory effects of HS, aggregated human IgG and prostaglandin E₂ were not observed for the phytomitogen PHA.     

人外周血淋巴细胞化学发光测定的最佳实验条件探讨

本文采用正交设计对人外周血淋巴细胞(PBL)化学发光(CL)测定系统中的PBL、Luminol及ConA浓度进行分析比较,并探讨了新生牛血清(NCS)、牛血清白蛋白(BSA)、细胞保存温度与时间等对淋巴细胞化学发光(Ly-CL)的影响。结果表明,Ly-CL测定的最优实验条件为:(1)测定系统含1.0ml PBL悬液(1.4×10°/ml)、0.2ml Lumionl溶液(7×10~(-4)M)及0.2ml ConA溶液(700μg/ml);(2)分离的PBL悬于0.1％BSA-无酚红Hanks液中,4℃冰浴保存,2小时内完成测定。     

Electrophoretic study of immunoglobulins and immunoglobulin sub-units on the surface of human peripheral blood lymphocytes

Treatment of human peripheral blood lymphocytes with a polyvalent antiserum to human immunoglobulins causes a reduction in the electrophoretic mobility of the cells. Treatment of the lymphocytes with antisera to κ and λ chains has a similar effect, but antisera to α, γ and μ chains are only effective in this way when all antisera are mixed together. These findings point to the presence of immunoglobulin molecules on the surface of peripheral blood lymphocytes and the results are discussed in terms of the possible representation of immunoglobulin sub-units on the lymphocyte surface.     

Regulation of expression of class II major histocompatibility antigens on human peripheral blood monocytes and Langerhans cells by interferon

Although normal peripheral blood monocytes from different individuals are primarily DR+ (L243), they vary in the mean expression of L243 and the percentage of cells with detectable Leu-10 (DC/DS) and L03 (D). All three species of human recombinant IFNs enhance Ia expression on normal peripheral blood monocytes; however, r-IFN-gamma is much more effective in enhancing the expression of these three class II antigens in vitro than r-IFN-beta or r-IFN-alpha A. In addition, r-IFN-gamma has a more profound effect on the expression of Leu-10 (DC/DS), an antigen critical for presentation in autologous MLR, than on L243 (DR). Adherent monocytes cultured for 5 days develop macrophage characteristics and become strongly positive for all three of these class II antigens without further manipulation. Isolated skin Langerhans cells which are thought to be antigen presenting cells in the epidermis are also strongly positive for all three of these Ia antigens and are unaffected by IFN treatment. Therefore, this early interferon effect on cell surface expression may be the result of enhancing the maturation of monocytes to mature antigen presenting cells.     

Activation of human peripheral blood monocytes by lipoproteins

Activation of human peripheral blood monocytes could enhance their attachment and or migration into the arterial intima and their various secretory and other functions, thus influencing the pathogenesis of atherosclerosis. In these experiments the authors have explored the role of lipoproteins in the activation of human blood monocytes. Monocytes were purified from citrated blood by Histopaque density gradient centrifugation and countercurrent centrifugal elutriation and cultured in DMEM in the presence of 20% acid-treated autologous serum or 100 micrograms/ml each of VLDL, LDL, Ac-LDL, and HDL. Secretion of beta-glucuronidase activity into the media was measured as a marker of activation. All of the lipoprotein density classes as well as serum stimulated secretion of beta-glucuronidase activity, with LDL and Ac-LDL having a greater influence than serum, VLDL, or HDL. Serum and LDL also stimulated secretion of prostaglandin E into the culture medium. Incubation of monocytes with serum or LDL in the presence of inhibitors of arachidonate metabolism (NDGA and indomethacin) resulted in a significant decrease in secreted and intracellular beta-glucuronidase activity, indicating a role for products of arachidonate metabolism in the activation of monocytes by lipoproteins.     

Gamma interferon production by combinations of human peripheral blood lymphocytes, monocytes, and cultured macrophages.

Mitogen-induced interferon (IFN) production was studied using human peripheral blood mononuclear cells and subpopulations of lymphocytes, monocytes, and cultured macrophages. Cell populations were prepared in suspension to permit quantitative analysis of the interactions among different cell types. After stimulation by staphylococcal enterotoxin A, nylon column-purified lymphocytes produced only 5% as much IFN as the peripheral blood mononuclear cells from which they were prepared. When lymphocytes were supplemented with as little as 2% monocytes, IFN production increased two- to eightfold; with the addition of up to 20% monocytes, IFN production increased further, to levels approximating those of peripheral blood mononuclear cells. Monocytes alone produced no or very little IFN. Macrophages were derived from monocytes by culturing in vitro for 7 days. The addition of 2 to 5% autologous macrophages augmented IFN production to the same extent as 2 to 5% monocytes. However, more macrophages consistently resulted in less, rather than more, IFN, so that lymphocytes with 20% monocytes produced three- to eightfold more IFN than did lymphocytes with 20% macrophages. Thus, whereas the addition of monocytes over a broad dose-response range (2 to 20%) progressively augmented IFN production, macrophages showed an optimal effect at 2 to 5%, with higher percentages being inhibitory. The IFN induced by stimulation with staphylococcal enterotoxin A was characterized as IFN-gamma by its resistance to neutralization by antibody to IFN- alpha and its inability to induce antiviral protection in embryonic bovine trachea cells.     

Age-related changes in surface antigens on peripheral lymphocytes of healthy children.

The age-related changes in proportion of various subsets within lymphocytes were investigated in cord blood and peripheral blood from healthy children and adults. The percentages of T and B cells did not show age-related changes, whereas natural killer (NK) cells increased significantly with age. Within lymphocytes or the CD3+ T cell population the proportion of CD45RAbright+ lymphocytes decreased and that of CD45RO+ cells increased, while that of CD45RAdim+ cells showed no age-related change. Within lymphocytes, the percentage of CD45RAbright+ CD4+ cells decreased, together with a decline of that of CD4+ cells. The proportions of CD45RAbright+ CD8+ cells and S6F1bright+ CD8+ cells increased with age, and the age-dependent increase of the proportion of CD8+ cells seems to be mainly attributable to the increases in these subsets. The CD45RAdim+ CD4+ and CD45RAdim+ CD8+ cells co-expressing CD45RO at a low level nevertheless showed no age-related changes. In gamma delta T cells, both delta TCS1+ and delta TCS1- T cells increased with age, but the delta TCS1- gamma delta T cells increased more than the delta TCS1+ subset. Among lymphocytes, the percentages of CD20+, CD21+ and CD22+ cells remained similar, with no age-related changes, but the proportion of CD5+ cells within lymphocytes or B cells decreased. The proportions of CD16+ NK cells among lymphocytes increased with age, and this change was attributable to the increase of CD56+ cells.     

人外周血T淋巴细胞胀亡现象的观察

目的观察人外周血T淋巴细胞的胀亡现象,探讨建立T细胞胀亡检测方法.方法密度梯度离心法及尼龙棉柱法分离健康成年人外周血T淋巴细胞,分空白组及地塞米松组,培养后观察细胞光镜、荧光镜及电镜形态学,并用流式细胞仪检测胀亡细胞比例变化.结果①人外周血T淋巴细胞经96小时体外培养,可自然出现典型细胞胀亡形态学改变.②经72小时培养,不同浓度地塞米松组(1×10-6、1×10-5、1×10-4、1×10-3 mol/L)T细胞的胀亡率分别为(3.49±0.42)%、(5.17±0.48)%、(8.44±0.72)%、17.93±1.50)%.③在1×10-5mol/L地塞米松作用下,不同培养时间(48、72、96、120小时)T淋巴细胞的胀亡率分别为(0.53±0.10)%、(6.36±0.80)%、(20.60±1.59)%、25.56±1.76)%.结论人外周血T淋巴细胞存在胀亡现象,地塞米松可诱导人外周血T淋巴细胞胀亡.     

Separation of human blood monocytes and lymphocytes on a continuous percoll gradient

A method for separation of human blood monocytes and lymphocytes is described. Mononuclear leukocytes are centrifuged on a continuous gradient of colloidal silica particles (Percoll) in phosphate-buffered saline. This leads to formation of 4 bands: a layer containing dead material (if present) which did not enter the gradient; a layer near the bottom of the tube containing granulocytes and red cells, and two other bands in between. The upper one is enriched for monocytes (av. 78%) and the lower one for lymphocytes (av. 98%). The final yields of these cell types are 73% and 79%, respectively, and their viability is greater than 95%. No functional impairments could be detected by different criteria including the ability of B lymphocytes to produce immunoglobulins when stimulated with pokeweed mitogen and the ability of monocytes to phagocytize opsonized red cells and latex particles.     

Differential binding and internalization of Clostridium difficile toxin A by human peripheral blood monocytes, neutrophils and lymphocytes

Colitis due to Clostridium difficile infection is mediated by secreted toxins A and B and is characterized by infiltration by cells from the systemic circulation. The aim of our study was to investigate interactions between fluorescently labelled toxin A and peripheral blood monocytes, neutrophils and lymphocytes. Purified toxin A was labelled with Alexa Fluor^® 488 (toxin A⁴⁸⁸) and incubated with isolated human peripheral blood mononuclear cells or washed whole blood cells for varying time intervals at either 37 or 4 °C/ice. The ability of trypan blue to quench cell surface–associated (but not cytoplasmic) fluorescence was also investigated. At 37 °C, toxin A⁴⁸⁸‐associated fluorescence in monocytes peaked at 1 h (majority internalized), with subsequent loss associated with cell death. In contrast to monocytes, binding of toxin A⁴⁸⁸ in neutrophils was greater on ice than at 37 °C. Studies using trypan blue suggested that over 3 h at 37 °C, most of the toxin A⁴⁸⁸‐associated fluorescence in neutrophils remained at the cell surface. Over 48 h (37 °C and ice/4 °C), there was minimal toxin A⁴⁸⁸‐associated fluorescence in lymphocytes. These studies suggest major differences in interactions between toxin A and circulating cells that infiltrate the mucosa during colonic inflammation in C. difficile infection.