Human Natural Cell-mediated Cytotoxicity against Fetal Fibroblasts

The dependence of human natural killer (NK) cell activity on antibodies was investigated. Absorption of the culture medium with target cells, trypsinization of the effector cells followed by a brief recovery, and incubation of the effector cells in serum-free nutrient medium did not affect natural killer cell activity against fetal fibroblasts. No soluble mediator in the nutrient media of effector cell--fibroblast co-cultures could be demonstrated. It was therefore concluded that antibodies are not involved in the cytolytic activity of natural killer cells. However, competition data and the activity of isolated NK cells against antibody-coated target cells suggested overlapping between the effector cells mediating natural and antibody-dependent cell-mediated cytotoxicity.     

Antibody-induced Cell-mediated Cytotoxicity in an Allogeneic Human System

In a ⁵¹Cr-release assay system it is found that serum from normal women with pregnancy-induced lymphocyte allo-antibody can cooperate with normal non-sensitized peripheral human leucocytic cells in producing cytotoxic activity against target cells from the husband. Conventional complement activation is not involved. The cooperating serum factor is found to be antibody and is located in the IgG fraction. The effector cells are lymphocytes and the activity is blocked by cytochalasin B.     

Mechanisms of Cell-mediated Cytotoxicity in Mice Rejecting Xenogeneic Human Lymphoblastoid Cells

The in vivo immunization of mice with human lymphoblastoid cell line LHN13 generates direct cell-mediated cytotoxicity by spleen cells. The lytic activity appears as early as day 3 after the intraperitoneal inoculation of 7.5 × 10⁵ cells and persists at least until day 11. The killer cells do not adhere to plastic and are not retained on nylon wool columns or on Degalan heads coaled with mouse Ig plus rabbit-anti-mouse Ig The effector cells are partly inhibited by treatment with anti-Thy-1.2 serum plus complement, but this inhibition appears to be non-specific since antiserum alone or normal mouse serum plus complement have the same effects. Heat-aggregated IgG strongly inhibit cytotoxicity, indicating that the effector cells are Fc-positive and that such receptors are implicated in lysis. Altogether, these features strongly argue for an ADCC phenomenon. The involvement of antibodies is demonstrated by the fact that eluates (56°C, 30 min) from immune cells alone induce lysis in the presence of normal spleen cells as effectors The lytic activity of these eluates can be removed by specific adsorption on protein A coupled to Sepharose heads and on the human lymphoid target cells. Positive complementation between immune and non-immune spleen cells suggests that the arming process may occur in vitro during the assay, when antibodies are released by plasmacytes     

Characterization and Possible Mechanisms of Mitogen-induced Cell-mediated Cytotoxicity

Mitogen-induced cell-mediated cytotoxicity (MICC) of human peripheral blood granulocytes (PMN) and lymphocytes (PBL) was studied by using chicken erythrocytes as targets. The possible mechanisms were elicited bidirectionally by means of the MICC inhibition test and by functional analysis of lectin. In our preliminary data it was noted that PMN-mediated cytotoxicity was more potent than that of PBL. In addition, erythrophagocytosis or lysosomal enzymes released from PMN offered little contribution to cytotoxic activity of PMN. From the inhibition study it was realized that intact cytoskeletal structures, functionally preserved membrane lectin receptors, active DNA synthesis, and environmental divalent cations were mandatory for the full expression of MICC activity by both effectors. We also identified the cytotoxic activity of PHA-P as having a unique character that is thermostable and independent of the other biological activities.     

HLA Restriction of Dinitrophenyl-specific Cell-mediated Cytotoxicity in Vitro

Lymphocytes from dinitrochlorobenzene-sensitized individuals can be stimulated in vitro by autologous dinitrophenyl (DNP)-conjugated lymphocytes to produce cytotoxic T lymphocytes (CTLs). The activity of these CTLs is specific for DNP-conjugated target cells, and there is no cross-reaction with nitrosodimethylaniline or trinitrophenyl-conjugated target cells. Evidence is presented which makes it improbable that the cytotoxicity is caused by an antibody-dependent (ADCC-like) mechanism. Most of the DNP-specific cytotoxicity is restricted by the HLA-ABC antigens of the CTL donor, and there is only a low degree of lysis of DNP-conjugated allogeneic target cells not sharing HLA-ABC antigens with the donor. The CTLs did not lyse non-conjugated allogeneic target cells. When CTLs were tested against allogeneic DNP-conjugated targets sharing only one of the HLA-ABC antigens of the CTL donor, it was seen that the phenomenon of preferential restriction was pronounced; that is, only some of the antigens of the donor were restricting. A certain pattern has emerged: some antigens (e.g. A2) are good restricting antigens, some (e.g. B12) do not restrict, and some (e.g. B5) function well in one donor but not in another. The sero-logically cross-reacting antigens A2 and A28 did not restrict mutually. HLA-C antigens may in some donors function as restricting antigens.     

Effect of rIFN-γ on Antibody-Mediated Cytotoxicity via Human Monocyte IgG Fc Receptor II (CD32)

Human monocytes and macrophages express an isoform of IgG Fe receptor II (FcγRII), FcγRII. Two allotypic variants of this receptor could be distinguished with respect to their ability to bind murine (m)IgG1 complexes either strongly or weakly, defined as high-responder (HR) and low-responder (LR). respectively. We investigated the effect of recombinant (r)IFN-γ on the ability of freshly isolated monocytes, and those cultured for 40 h and 9 days, to mediate antibody-dependent cell-mediated cytotoxicity (ADCC), Using human erythrocytes (E) sensitized with mlgGl as target cells, FcγRII was studied selectively. Cells which had been cultured for 40 h exhibit a significantly decreased FcγRII expression, and FcγRll-mediated ADCC activity as compared with freshly isolated monocytes. Co-culture with rlFN-γ (40 h) reversed this decrease. Short-term rlFN-γ-cultured cells, and fresh cells express similar numbers of FcγRII, and exhibit comparable FcγRII-mediated ADCC activity. Phagocytic activity was not affected. Prolonged culture of monocytes for 9 days, co-cultured with rlFN-γ either from day 0 or from day 7, did not affect expression or functional activity of FcγRII. Furthermore, the effects were observed in both HR and LR individuals.
Our results show that rlFN-γ has strong effects on FcγRII-mediated responses specifically during the early stages of monocyte maturation, most likely by affecting receptor expression levels.     

Regulation of IgG Antibody-dependent Cellular Cytotoxicity in Vitro by IgM Antibodies

IgM antibodies have previously been reported to either inhibit or induce antibody-dependent lymphocyte cytotoxicity (ADCC). Here we show that human lymphocytes lyse bovine erythrocytes (E_b) in the presence of either IgM or IgG anti-E_b from rabbits. Seven out of 20 IgM preparations (Sephadex G-200) were ADCC-active. IgG-dependent ADCC was inhibited by human IgG but not by IgM. In contrast, IgM ADCC was inhibited by both IgG and IgM. The effector cells in IgM ADCC were a subpopulation of lymphocytes with distinct Fc receptors for both IgG and IgM. Most of them also had sheep erythrocyte receptors. Extensive purification of the ADCC-active IgM antibody preparations indicated that very small amounts of contaminating IgG anti-E_b were responsible for ADCC induction. When purified and ADCC-inactive IgM antibodies were mixed with suboptimal concentrations of IgG antibodies, a strong enhancement of ADCC was found. To achieve enhancement, the two antibody isotypes had to be present on the surface of the same target cells, and the IgM effect was not due to the release of soluble ADCC-enhancing factors. Thus, in this system, IgM antibodies are not capable of inducing ADCC on their own. However, they enhance ADCC by improving the contactual interaction between target cells and a special subset of effector cells.     

Pokeweed Mitogen Induces p55 Interleukin-2 Receptor Expression on Human Monocytes and its Effect on Interleukin-2 Activated Monocyte Cytotoxicity

Activation of human peripheral blood monocytes by pokeweed mitogen (PWM) results in the expression of interleukin 2 receptor(IL-2R) p55 chains, which are absent on resting monocytes. By dual-fluorescence flow cytometry, we found PWM induced detectable numbers of IL-2R+ cell which were further identified as LeuM3+ monocytes (22.91% of the LeuM3+ cells were IL-2R+ within 24h, and 32.17% in 48h). In addition, LPS can also induce IL-2R on 15.39% of LeuM3+ cells. On the contrary, other mitogens such as PHA or Con A, and cytokines as IFN-γ, IL-2, M-CSF, TNF-α and IL-1ß showed no influence on the IL-2R expression on monocytes. It was also noticed that PWM itself had no direct effect on HLA-DR antigen expression on LeuM3+ cells. The addition of IL-2 to PWM-pretreated IL-2R positive monocytes significantly augmented their tumoricidal activity. Thus monocytes when activated underwent a series of phenotypic and functional changes including the expression of IL-2R which may provide an important immunoregulatory pathway.     

Inhibitory Effect of Calcium Antagonists on Histamine Release from Human Leukocytes

A study was made of the influence of calcium antagonists on human basophil histamine release induced in vitro by specific antigen, anti-IgE or the calcium ionophore A23187. Both verapamil, nifedipine, and nimodipine were found to inhibit the release, and a similar effect was also observed after peroral administration of verapamil and nifedipine. The inhibitory effect of the drugs on histamine release seems to depend on interaction with calcium at different sites. The anti-allergic effect might explain the improvement found with calcium antagonists in exercise-induced asthma.     

Blocking Effect of Rheumatoid Factor on the In Vitro Cytotoxicity of Lymphoid Cells from Carcinoma Patients

Human cryo-IgM rheumatoid factor (RF) preparations blocked the tumor-specific in vitro cytotoxicity of ovarian or bladder carcinoma patients' lymphoid cells in microcytotoxicity assays. The effect was mediated by pretreatment of the effector cells. Cryo-IgM RF free of detectable IgG blocked in a dilution-dependent manner, and immunosorbent purification of contaminating IgG from another preparation did not abrogate the blocking effect. Control IgM preparations lacking RF activity did not block the cytotoxicity, and normal human serum preincubation of the RF preparations rendered them inactive, indicating that the blocking effect was due to the anti-IgG activity of the RF.     

组胺对皮肤肿瘤坏死因子受体分布的影响的实验研究

目的　探讨人皮肤角质细胞TNFR的分布和炎症介质组胺对其分布的影响。方法　依据Tel lides成熟实验模型 ,将人皮肤移植于免疫缺陷小鼠上 ,分别注入等量 10ml组胺或生理盐水 ,然后制成共聚焦荧光显微镜和电镜标本观察。结果　正常皮肤TNFR2阳性反应细胞较少 ,TNFR1阴性细胞数量较多 ,阳性反应主要见于角质细胞的胞质和膜 ;组胺刺激后角质细胞质和膜的TNFR1消失 ,主要分布在角质细胞间隙。结论　组胺可使角质细胞质和膜上TNFR1消失 ,移位于细胞间隙     

Effect of Storage on Insulin Receptor Binding in Human Erythrocytes

The authors established the specificity, reliability, and precision of human erythrocyte insulin radioreceptor assay. On the basis of insulin binding, cell viability, and degree of hemolysis, heparin sodium was found to be a more suitable anticoagulant than sodium fluoride, ethylenediaminetetraacetic acid, sodium oxalate, or sodium citrate. In two sets of experiments carried out at 4°C and 23°C, human erythrocytes were stored as whole blood or isolated erythrocytes suspended in Tris-{4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid} buffer. The effect of storage under these conditions was evaluated by erythrocytespecific insulin binding. Human erythrocytes can be stored for 72 hours at 4°C without any change in insulin binding, insulin receptor sites per cell, or average affinity constant at the empty sites. Isolated erythrocytes can also be stored in plasma for 72 hours or in buffer G for 24 hours at 4°C without any change in insulin binding. It is not advisable to store human erythrocytes in plasma or as whole blood for more than 24 hours at 23°. These findings are useful in preserving insulin receptor activity when storage of erythrocytes is unavoidable.     

Effect of Cimetidine on Exogenous Histamine Inhibition of Histamine Release in Vivo

We previously reported that exogenous histamine inhibits in vivo histamine release and eosinophil accumulation in ragweed-challenged skin sites of sensitive human subjects. The mechanism(s) involved were unclear. In this study, we repeated similar approaches in four of the same subjects pretreated for 3 days with cimetidine, an H2 receptor antagonist. The pattern of exogenous histamine effects was now different in that local exogenous histamine (50 ng/ml) did not significantly alter ragweed-induced mast cell alteration, histamine release, or the degree of eosinophil accumulation in skin challenge sites. These findings suggest that the observed exogenous histamine inhibitory effects may be mediated through the H2 receptor.     

Cell-mediated Cytotoxicity during Vaccinia Virus Revaccination in Man: Influence of Antibodies and Interferon

Seven healthy human adults were revaccinated with vaccinia virus. Cell-mediated cytotoxicity to vaccinia virus-infected fibroblasts was investigated on the day of vaccination and on days with peak activity. Three donors were studied until day 30 after vaccination. The addition of interferon to cytotoxicity reactions resulted in an increase in killing. This increase was not seen when antibodies were added. When a mixture of lymphocytes from a revaccinated and a non-revaccinated donor was used as effector cells, the killing observed corresponded to the killing seen with lymphocytes from the revaccinated donor, when tested alone. This finding indicates that no antibodies or other soluble mediators capable of increasing cytotoxicity are released from the lymphocytes during the cytotoxicity assay.     

Inhibitory Effect of Cyclosporin A on Histamine Release from Human Leukocytes and Rat Mast Cells

The influence of cyclosporin A (CyA) on human basophil histamine release induced in vitro by specific antigen, anti-IgE, calcium ionophore A23187, or concanavalin A (Con A) was studied. CyA inhibited the release induced by these four stimulators. It is suggested that the drug acts directly on the target cell, since similar effect was obtained with isolated peritoneal rat mast cells. The basophil histamine release was not changed by a non-immunosuppressive cyclosporin derivate.     

Standardization of the Human in vitro Cell-mediated Lympholysis Technique

Cytotoxic lymphocytes (CTL) can be generated in mixed lymphocyte culture and cryopreserved after a 6-day sensitization phase. CTL tested after storage in liquid nitrogen do not show lysis of autologous target cells and can mediate strong cytotoxicity against other target cells. This lysis shows the same specificity as that of the corresponding fresh CTL. The ability to cryopreserve CTL enables performance of sequential CML testing. Data from separate CML experiments can be pooled using a percent relative cytotoxic response (%RCR) to normalize values within individual experiments. Together, these methods provide a standardized cell-mediated lympholysis technique.     

维生素C对培养人胚成纤维细胞LDL受体活性的影响

研究了维生素Ｃ对培养的人胚皮肤成纤维细胞低密度脂蛋白（ＬＤＬ）受体活性的影响。从ＬＤＬ受体饱和分析和Ｓｃａｔｃｈａｒｄ作图以及单点分析表明，维生素Ｃ可增加细胞ＬＤＬ受体数量，但不影响受体对ＬＤＬ的亲和力。提示维生素Ｃ可通过增加ＬＤＬ受体数量而促进胆固醇的代谢，具有一定的抗动脉粥样硬化作用。     

Histamine Release Induced by Human Leukocyte Lysates: Effect of Metabolic Inhibitors and Carbohydrates

The effects of metabolic inhibitors and carbohydrates on histamine release induced by human leukocyte lysates have been studied to gain an understanding of the mechanism of histamine release in this system. Dinitrophenol produced 80% inhibition of histamine release at a concentration of 10⁻³ M. Iodoacetic acid inhibited release to a lesser extent, and deoxyglucose had relatively little effect. Colchicine was a potent inhibitor of release and produced 88% inhibition at a 10⁻³ M concentration. Puromycin and cycloheximide had essentially no effect on histamine release. Various sugars inhibited release with mannitol and glucose, producing greater than 50% inhibition at 10⁻³ M concentrations. Other sugars inhibited histamine release to a lesser degree with the following order of potency: mannitol > glucose > sucrose > galactose > fructose. These studies suggest that intact, metabolically active cells are required for histamine release induced by human leukocyte lysates and that a secretory process is involved. The results also provide information on the possible nature of the histamine-releasing factor(s) in human leukocyte lysates.