PWM依赖的小鼠腹腔巨噬细胞介导的细胞毒作用的研究

本文采用间接MTT法,对凝集素美洲商陆(PWM)依赖的小鼠腹腔巨噬细胞(PEMφ)介导的细胞毒作用(PDMC)进行检测分析,发现凝集素(PWM、ConA)预处理靶细胞(Raji、U_(14)),对细胞毒作用的影响视凝集素、靶细胞的不同而异;在PWM分别预处理效、靶细胞的实验中,PWM对Raji靶细胞的作用显更重要;在PWM直接作用下,其PDMC活性可进一步提高,后者可为GlcNAc有效地抑制.观察PDMC中Raji靶细胞的形态学变化,发现坏死与凋亡现象并存.     

Effects of 1 alpha,25-dihydroxyvitamin D3 and cytokines on the expression of MHC antigens, complement receptors and other antigens on human blood monocytes and U937 cells: role in cell differentiation, activation and phagocytosis.

The effect of calcitriol/1 alpha,25-dihydroxyvitamin D3, alone and in combination with cytokines, on the expression of various antigens (Ag) on human peripheral blood monocytes and U937 cells was studied by flow cytometry. Both constitutive and interferon-gamma (IFN-gamma), interleukin-4 (IL-4), IL-6 and tumour necrosis factor-alpha (TNF-alpha)-induced human leucocyte antigen (HLA)-DR, HLA-DP and HLA-DQ Ag expression on monocytes was significantly down-regulated by calcitriol, IL-10 and transforming growth factor-beta (TGF-beta). The effects of calcitriol were concentration dependent and reached maximal inhibitory levels after 3-5 days. Modulation of HLA-DR by calcitriol and IFN-gamma at the protein level correlated with the amount of mRNA specific for the HLA-DR alpha-chain, as judged by Northern blot analysis. The basal as well as IL-4, IL-6, IFN-gamma, TNF-alpha and TGF-beta-driven levels of HLA-ABC Ag were significantly diminished by calcitriol. On U937 cells calcitriol markedly induced CD11a and CD11b expression and weakly up-regulated CD11c whereas on monocytes, constitutive CD11a, CD11b and CD11c expression was significantly down-regulated by calcitriol. The expression of CD14 Ag was strongly induced on U937 cells but only modestly on monocytes. Both the basal level of CD71 and IL-4, IFN-gamma or TNF-alpha-driven expression was diminished on calcitriol-treated U937 cells. In addition, calcitriol suppressed the expression of CD71 Ag on monocytes. The ability of monocytes to phagocytize opsonized Escherichia coli was diminished by calcitriol. Our results demonstrate that calcitriol, alone or in combination with cytokines, modulates expression of MHC, CD11b, CD11c, CD14 and CD71 Ag on both monocytes and U937 cells, and impairs the phagocytic property of monocytes.     

The role of tumor necrosis factor in the regulation of antigen presentation by human monocytes

Human peripheral blood monocytes pretreated with human recombinant tumour necrosis factor alpha (rTNF) showed an enhanced ability to present a soluble antigen, a purified protein derivate of tuberculin, to autologous T lymphocytes as assessed by their increased proliferation in vitro. This enhancing activity was due to TNF and not impurities in TNF preparations as anti-TNF antibodies abolished this phenomenon. The rTNF-treated monocytes showed an increased expression of HLA-DR molecules and enhanced co-stimulatory activity in the murine thymocyte assay. Pretreatment of monocytes before an antigen pulse with anti-TNF mAb inhibited antigen presentation, which indicated that endogenously produced TNF was involved. These studies thus suggest that TNF acts in an autocrine fashion and enhances the ability of monocytes to present protein antigen. It is unclear at present whether this effect is due to the modification in antigen processing, expression of MHC class II molecules, or other factors (IL-1, IL-6, adhesion molecules, etc.) that are important for the induction of T cell response to a nominal antigen. The enhancement of the antigen presenting capacity of monocytes/macrophages may be the additional mechanism of pro-inflammatory activity of TNF.     

Heterogeneous susceptibility of human melanoma clones to monocyte cytotoxicity: role of ICAM-1 defined by antibody blocking and gene transfer.

Five clones derived from the same human malignant melanoma lesion were studied for their susceptibility to killing by human monocytes activated by exposure to interferon (IFN)-gamma and lipopolysaccharide. Melanoma clones were heterogeneous in their susceptibility to human monocyte cytotoxicity, with one clone (2/21) exhibiting extremely low levels of lysis. The different levels of susceptibility to monocyte cytotoxicity were not accounted for by susceptibility or resistance to monokines [tumor necrosis factor (TNF), interleukin (IL)-1 and IL-6] because: (a) these effector molecules had little (TNF) or no (IL-1 and IL-6) cytolytic activity under these conditions; and (b) anti-TNF antibodies had marginal effects on cytotoxicity. Monocytes bound less to resistant than to susceptible melanoma cells. Monocyte-resistant 2/21 melanoma cells expressed substantially lower levels of ICAM-1 and VLA-4 than susceptible cells. Anti-CD18 and, to a lesser extent, anti-ICAM-1 mAb inhibited binding and cytotoxicity of human monocytes on malignant melanoma whereas anti-VLA-4 had no inhibitory action. Transfection of the ICAM-1 gene under the control of a constitutive promotor resulted in high levels of expression of ICAM-1 in 2/21 melanoma cells and, concomitantly, in augmented susceptibility to activated monocyte cytotoxicity. The augmented killing of ICAM-1 transfected 2/21 cells was inhibited by anti-ICAM-1 mAb. These results demonstrate that the CD18-ICAM-1 adhesion pathway can play an important role in the expression of human monocyte cytotoxicity on melanoma target cells and that heterogeneity in expression of ICAM-1 can underlie differences in susceptibility to tumoricidal activity.     

M-ficolin is expressed on monocytes and is a lectin binding to N-acetyl-D-glucosamine and mediates monocyte adhesion and phagocytosis of Escherichia coli

Ficolins are a group of multimeric proteins that contain collagen-like and fibrinogen-like (FBG) sequences. Three types of ficolins have been characterized: H-, L- and M-ficolins. Both H- and L-ficolins have demonstrated lectin activities. In the present study, the FBG domain of M-ficolin was expressed and shown to bind to N-acetyl-D-glucosamine. M-ficolin mRNA was expressed in monocytes but not in the more differentiated macrophages and dendritic cells. By flow cytometry, surface biotinylation and immunoprecipitation, we showed that M-ficolin was associated with the surface of promonocytic U937 cells. M-ficolin transiently expressed in COS-7 cells was also clearly detected on the cell surface by immunoprecipitation. By flow cytometry, M-ficolin was detected on peripheral blood monocytes but not on lymphocytes or granulocytes. Immobilized rabbit anti-M-ficolin F(ab')2 mediated U937 cell adhesion, and the antibody also inhibited phagocytosis of Escherichia coli K-12 by U937 cells. Therefore, M-ficolin might act as a phagocytic receptor or adaptor on circulating monocytes for micro-organism recognition and may potentially mediate monocyte adhesion.     

Characterization and structural analysis of Fc gamma receptors of human monocytes, a monoblast cell line (U937) and a myeloblast cell line (HL-60) by a monoclonal antibody

A monoclonal antibody, FR51, raised against the IgG Fc receptor (Fc gamma R) of the human monoblast cell line U937 was used to analyze the distribution of this antigen on various human cells. This antibody inhibited the binding of human IgG to the Fc gamma R on U937 cells, HL-60 cells and human peripheral blood monocytes. In contrast, the Fc gamma R on human granulocytes (neutrophil cells) and on an Epstein-Barr virus-transformed human lymphoblastoid cell line (Raji) were not recognized, indicated by the failure of blocking the binding of human IgG ligand to the Fc gamma R on these cells. By affinity chromatography of detergent-containing cell free lysates of surface-iodinated U937 cells, HL-60 cells and monocytes, a protein of 70-kDa was isolated. This protein was identified as the Fc gamma R by rebinding the isolated protein to immobilized human IgG. Removal of the carbohydrate moiety with endo-beta-N-acetylglucosaminidase F demonstrated that the receptors consist of a 40-kDa polypeptide. Analysis of the polypeptide patterns obtained by proteolytic digestion of either mature (70-kDa) or deglycosylated (40-kDa) receptors isolated from monocytes, U937 cells and HL-60 cells strongly suggests that the Fc gamma R are identical. The monoclonal antibody FR51 specifically reacts with Fc gamma R on human monocytes, a myeloblast and a monoblast cell line but not with the receptors on a B cell line and neutrophil cells.     

A comparative study of the JAM test and 51Cr-release assay to assess the cytotoxicity of dendritic cells on hematopoietic tumor cells

Dendritic cells (DCs) are potent antigen presenting cells and possess a direct anti-tumor cytotoxic ability. Nevertheless, the mechanism of anti-tumor cytotoxicity by DCs and the methods for its evaluation are not fully elucidated. In order to clarify this mechanism of cytotoxicity, we examined the ability of DCs 1) to suppress [³H] thymidine (³H-TdR) uptake by tumor cells; 2) to induce cytolysis on ⁵¹Cr-labeled tumor cells; 3) and to induce DNA fragmentation on ³H-TdR labeled tumor cells (JAM test). Cytolysis and DNA fragmentation are markers of necrotic and apoptotic mechanisms of cytotoxicity in vitro, respectively. DCs inhibited approximately 38.6% to 54.8% of the growth of B4D6, NB4, U937, and Daudi cells as evaluated by the uptake of ³H-TdR. However no cytolysis was verified by ⁵¹Cr-release assay. On the other hand, cytotoxicity rates found using the JAM test ranged from 3 to 81% depending on the cell line and the effector to target cell ratio. The discrepancy of cytotoxicity between ⁵¹Cr-release assay and the JAM test may be due to the phagocytosis of apoptotic tumor cells or the absorption of released ⁵¹Cr by DCs surrounding the target cells. In conclusion, the JAM test was more sensitive than the 4-h and the 10-h ⁵¹Cr-release assay to investigate cytotoxicity mediated by DCs toward hematopoietic tumor cell lines in vitro.     

Cell surface antigens and function of monocytes and a monocyte-like cell line before and after infection with HIV

The human immunodeficiency virus (HIV-1) preferentially infects cells that express the CD4 molecule, including monocytes and cells of the monocyte lineage. The monocyte-like cell line U937 and monocytes isolated from peripheral blood lymphocytes (PBL) were infected with HIV-1. Cell surface antigen expression was determined in infected and noninfected cells as was the ability to stimulate in mixed lymphocyte reaction. The CD4 antigen decreased in infected cells U937 and PBL monocytes. MHC class II antigens HLA-DR, HLA-DQ, and HLA-DP increased in HIV-1 infected U937 cells. In infected PBL-derived monocytes, HLA-DR increased, HLA-DQ decreased, and HLA-DP was unchanged. Infected U937 and PBL monocytes were capable of stimulating allogeneic lymphocytes, thus demonstrating retention of the alloantigen presentation function of HIV-1-infected monocytes.     

Influence of Tumour Necrosis Factor-α on the Expression of Fc IgG and IgA Receptors, and Other Markers by Cultured Human Blood Monocytes and U937 Cells

The expression of Fc receptors for IgG (FcγR) and IgA (FcαR) and of various other antigens on the human monocytic cell line U937 and peripheral blood monocytes, under stimulation with human recombinant tumour necrosis factor-α (TNF-α) and other cytokines, was investigated by flow cytomelry. TNF-α, as well as interferon-γ (IFN-γ) or interleukin-6 (IL-6) had a significant up-regulating effect on U937 expression of FcγRI/CD64. Furthermore, the action of TNF-α was augmented by IL-6, and more evidently by IFN-γ. IFN-α alone had only a marginal effect, but was able to increase the TNF-α-driven FcγRI expression. In contrast to U937 cells, TNF-α did not enhance significantly FcγRI expression on human monocytes. Interestingly, on both U937 cells and monocytes. FcαR was augmented markedly by TNF-α. Furthermore, TNF-α induced the expression of HLA-DR and HLA-DP antigens on monocytes and U937 cells. The expression of FcγRII/CD32, FcγRIII/CD16. CD14, complement receptor type 1 (CR1/CD35). CR4 (CD11c/CD18), and MHC class-I antigens, was not influenced significantly by TNF-α. The results of this study show that TNF-α may act on human mononuclear phagocytes, alone or in combination with other cytokines, by modulating the expression of various cell-surface antigens.     

Involvement of tumour necrosis factor in monocyte-mediated rapid killing of actinomycin D-pretreated WEHI 164 sarcoma cells.

Human and murine monocyte-macrophages kill actinomycin D (ActD)-treated WEHI 164 sarcoma cells in a 6-hr 51Cr-release assay (drug-dependent cellular cytotoxicity, DDCC). In this study, we have investigated the cytotoxic activity of human recombinant tumour necrosis factor (hrTNF) against untreated and ActD-treated WEHI 164 sarcoma cells. Human recombinant TNF when added to the 6-hr 51Cr-release assay killed ActD-treated targets at doses ranging from 33 to 0.33 ng/ml, whereas untreated targets were resistant to lysis. The kinetics of lysis of ActD-treated targets was similar for hrTNF and blood monocytes. The protease inhibitors phenyl-methyl-sulphonyl-fluoride (PMSF) and N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) reduced the DDCC activity of monocytes, monocyte supernatants and hrTNF. Killing of drug-sensitized target cells by monocyte supernatants was totally inhibited by a rabbit anti-TNF serum. These, as well as previous data on the physicochemical properties of the soluble cytotoxic factor released by monocytes, suggest that rapid monocyte-mediated killing of ActD-pretreated WEHI 164 sarcoma cells involves TNF or TNF-like molecules.     

抗人DR5抗体mDRA- 6细胞毒作用机制分析

目的:探讨鼠抗人DR5单克隆抗体(mAb)mDRA-6对Jurkat细胞的细胞毒作用及其机制。方法:以流式细胞术测定mAbmDRA-6对Jurkat细胞的细胞毒作用和细胞凋亡作用,以及caspase8、9的抑制剂对mAbmDRA-6诱导的Jurkat细胞凋亡的影响。在荧光显微镜下,观察mAbmDRA-6对Jurkat细胞形态的影响。以琼脂糖凝胶电泳检测Jurkat细胞中的DNA片段化。结果:mAbmDRA-6对Jurkat细胞具有显著的细胞毒作用,并呈剂量和时间依赖性。经mAbmDRA-6处理后,Jurkat细胞可出现典型的细胞凋亡的形态特征:细胞膜皱缩,出泡,染色质浓缩,形成凋亡小体等。经mAbmDRA-6处理后,Jurkat细胞膜表面高表达丝氨酸磷脂,并可导致Jurkat细胞中的DNA片段化。caspase8的抑制剂可明显抑制mAbmDRA-6诱导的Jurkat细胞凋亡,caspase9的抑制剂的影响很小。结论:mAbmDRA-6可通过死亡受体信号传导途径诱导Jurakt细胞凋亡,对Jurkat细胞产生细胞毒作用,其在以TRAIL/DR5系统进行的肿瘤治疗和探讨DR5功能结构域方面具有广阔的应用前景。     

Effects of Heat Shock on Cytolysis Mediated by NK Cells, LAK Cells, Activated Monocytes and TNFs α and β

We have recently shown that a heat treatment of a murine target cell line, WEHI 164, induces resistance to lysis mediated by tumour necrosis factors alpha (TNF-α) and beta (TNF-β). In the present study the effect of the heat shock of target cells on cytotoxicity mediated by natural killer cells (NK cells), lymphocyte-activated killer cells (LAK cells), activated monocytes, TNF-α, and TNF-β was investigated, First, WEHI 164 cell line and six human cell lines (ME 180, K 562, U 937, HeLa, MCF 7, and SK-OV 3) were screened for their sensitivity to different forms of lysis, and then sensitive cell lines were heat-treated. Pretreatment of target cells at 42° C for 45-60 min also rendered human target cell lines more resistant to lysis by rTNFs, and the acquired resistance was accompanied by an increased resistance to activated monocytes, but not to NK cells or LAK cells. Thus, the heat-induced resistance mechanisms capable of protecting target cells from lysis by rTNFs and by activated monocytes do not elicit resistance to lysis by NK cells and LAK cells, supporting the hypothesis that mediators other than TNFs are involved in NK cell-and LAK cellmediated killing.     

Enhanced human monocyte cytotoxicity by platelet-activating factor

The capacity of platelet-activating factor (PAF) to enhance human monocyte cytotoxicity for WEHI 164 cells was examined. Spontaneous monocyte cytotoxicity was 24 +/- 2% (mean +/- SEM, n = 9). Preincubation of monocytes with 1 pM-1 nM PAF for 18 hr significantly enhanced cytotoxicity in a dose-related manner, whereas less enhancement was observed at PAF concentrations above 1 nM. Maximal PAF-induced cytotoxicity was 68 +/- 6%, which was similar to that induced by optimal concentrations of tumour necrosis factor (TNF) and interferon-gamma. The specific PAF antagonist kadsurenone inhibited PAF-induced cytotoxicity but not TNF-induced cytotoxicity. The inactive PAF analogues lysoPAF and enantioPAF did not increase monocyte cytotoxicity. Two observations suggest that TNF mediates PAF-induced cytotoxicity: specific anti-TNF antibodies inhibited PAF-induced cytotoxicity toward WEHI 164 cells, and PAF did not enhance cytotoxicity to TNF-resistant cells. PAF represents a distinct class of phospholipid monocyte activators that increase monocyte cytotoxicity by TNF-dependent mechanisms.     

Specificity of γδ Receptor-Bearing Cytotoxic T Lymphocytes Isolated from Human Peripheral Blood

T-cell receptor (TcR)-gamma delta-bearing lymphocytes were isolated from the peripheral blood of two healthy donors by immunomagnetic separation and subsequently cultured. The cell lines generated showed two distinct patterns of cytotoxicity. One TcR-gamma delta + cell line (HG.D) lysed K562 and U937 target cells, three TcR-gamma delta + cell lines lysed Daudi cells, and one TcR-gamma delta + cell line showed a shift from the former to the latter specificity during culture. Cold target inhibition experiments showed that the HG.D effector cells which were cytotoxic against U937 cells also lysed K562 cells. The cytotoxicity against Daudi cells was strongly inhibited by monoclonal antibodies (MoAb) against the CD3 complex, whereas the cytotoxicity of the HG.D cell line against K562 and U937 was unaffected by such antibodies. The cytotoxicity against Daudi cells was also strongly inhibited in the presence of anti-TcR-gamma delta MoAb. However, in two of the Daudi-specific cell lines, strong cytotoxicity against K562 cells was induced by anti-TcR-gamma delta MoAb. Anti-LFA-1 MoAb caused only a partial inhibition of cytotoxicity, while anti-CD2 and anti-TcR-alpha beta MoAb were found to have no effect. The results indicate that human gamma delta receptor-bearing T cells demonstrate a certain degree of target cell specificity, and that recognition of some target cells may be mediated through the TcR-gamma delta.     

Role of the adhesion molecule ICAM-1 (CD54) in staphylococcal enterotoxin-mediated cytotoxicity.

Staphylococcal enterotoxin A (SEA) binds to major histocompatibility complex (MHC) class II molecules on target cells and directs human cytotoxic T lymphocytes (CTL) of irrelevant nominal specificity to mediate strong cytotoxicity against target cells. In this report we describe the importance of ICAM-1 (CD54) expression on the target cell in SEA-dependent cell-mediated cytotoxicity (SDCC), utilizing murine L cells co-transfected with HLA-DR and ICAM-1. Human CTL mediated a low but significant cytotoxicity against HLA-DR2- and HLA-DR7-transfected cells after preincubation with SEA, but no reactivity towards uncoated HLA-DR2 and HLA-DR7 cells or SEA-coated ICAM-1-transfected and untransfected L cells. In contrast, a strong cytotoxic response was mediated by CTL against L cells co-transfected with HLA-DR2/ICAM-1 and HLA-DR7/ICAM-1. Similar cytotoxic activity of the CTL was seen at a 30-fold lower effector-to-target cell ratio when comparing the HLA-DR2/ICAM-1-expressing cells with the HLA-DR2-expressing cells. SEA dose-response analysis demonstrated that the HLA-DR2/ICAM-1-expressing target cells enabled the CTL to respond to a 1000-fold lower concentration of SEA in comparison to the HLA-DR2-expressing cells. CD3+CD4+ and CD3+CD8+ cytotoxic T cell lines were equally dependent on the expression of ICAM-1 on the target cell. The strong CTL activity against HLA-DR2/ICAM-1-transfected cells could be blocked by anti-CD11a or anti-CD18 monoclonal antibodies (mAb), but not by anti-CD11b, anti-CD11c, anti-CD2 or unrelated control mAb. The great sensitivity of HLA-DR2/ICAM-1 expressing target cells to SDCC was strongly reduced by preincubation with various anti-ICAM-1 mAb but not by mAb against monomorphic HLA-DR or murine MHC class I determinants. The result in this study clearly demonstrates that efficient re-targeting of human CTL by SE is dependent on a proper interaction with the heterodimer CD11a/CD18 (Leu-CAMa, LFA-1) on the CTL and its target cell ligand ICAM-1.     

Role of oxidative damage and IL-1{beta}-converting enzyme-like proteases in Fas-based cytotoxicity exerted by effector T cells

The Implication of oxidative damage and/or intact mitochondrialfunction in physiological Fas-based cytotoxicity has been testedusing the cytolytic hybridoma d11S and the CD8⁺ CTL clone KB5.C20,previously stimulated to express Fas ligand (FasL) on theirsurface, as effectors and U937 or U937-p^° cells (depletedof mitochondrial DNA) as targets. Immobilized anti-Fas mAb,which induced death of U937 cells, Inhibited the growth of U937-p°cells but without inducing cell death. By contrast, FasL-expressingeffectors readily killed both targets, with induction of DNAfragmentation, in 20 h assays. These results demonstrate thelack of involvement of mitochondrial-derived free radicals and/orIntact mitochondrial function in physiological Fas-based cytotoxicity.Supplementation of Fas-sensitive cells (Jurkat, U937, L1210Fas)with a polyunsaturated fatty acid, which induces cell deaththrough the generation of lipid free radicals, resulted in thepotentiation of Fas-based cytotoxicity. This potentiating effect,but not Fas-based cytotoxicity Itself, was eliminated by thephysiological antioxidant vitamin E. On the other hand, theIL-1ß-converting enzyme (ICE)-like protease tetrapeptideinhibitor Ac-YVAD-cmk partially Inhibited Fas-based cytotoxicity,while the specific inhibitor of CPP32/Yama Ac-DEVD-CHO was amuch more effective inhibitor of Fas-Induced apoptosis. It wasconcluded that Fas-Induced cytotoxicity was clearly dependenton ICE-LIke protease activation, and especially on that of CPP32in Fas-sensitive cells, including mitochondrial DNA-depletedones.     

Molecular characterization of U937-dependent T-cell co-stimulation

U937 cells provide a co-stimulatory signal for CD3-mediated T-cell activation which is independent of the CD28/CD80/CD86 interaction. This study set out to identify which molecules contribute to this co-stimulatory activity. Monoclonal antibodies (mAb) to the known accessory molecules CD11a, CD18, CD54 and CD45, all inhibited T-cell proliferation. Although CD11a/18 mAb inhibited U937/T-cell cluster formation as well as proliferation, CD45 enhanced the size of the clusters formed, suggesting that this was not the only mechanism of inhibition. The alternative co-stimulatory pathway provided by U937 cells preferentially stimulated a response in the CD18+ T-cell population, and this reflected the reduced sensitivity of CD8+ T cells to CD28-mediated activation. Monoclonal antibodies to three molecules, CD53, CD98 and CD147, also inhibited U937-dependent T-cell proliferation. The mAb to CD98 and CD147 were inhibitory when prepulsed on to the U937 cells, suggesting an effect mediated by these molecules on the antigen-presenting cell.     

Eosinophil cytotoxicity enhancing factor: purification, characterization and immunocytochemical localization on the monocyte surface

The monokine eosinophil cytotoxicity enhancing factor (ECEF) increases antibody-dependent cytotoxicity of eosinophils towards helminth larvae. A monokine biochemically indistinguishable from ECEF increases the release of leukotriene C4 and other arachidonic acid metabolites by eosinophils. We have developed monoclonal antibodies (mAb) to these monokines by immunizing mice with ECEF made by the U-937 histiocytic lymphoma cell line. mAb 81.10.C9 (IgG2b) and 9A6G (IgG1) inhibit the effect of the monokine on release of AA products. Both mAb bind ECEF, which appears after affinity chromatography purification as a major 13-14-kDa and a minor 62-kDa component (13-14 kDa and 52 kDa after reduction) in silver-stained gels. An additional component of 30 kDa is detectable after radioiodination of the immunopurified material. The specificity of both mAb was studied in several ways. In immunoprecipitation, both recognize the 13-14-kDa and the 30-kDa components, while the 62-(52)-kDa protein is not significantly precipitated. Both mAb react in enzyme-linked immunosorbent assay with products secreted by peripheral blood mononuclear cells and monocytes, as well as with those secreted by phorbol 12-myristate 13-acetate and lipopolysaccharide-stimulated U-937 cells and with the immunopurified proteins. These were separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroeluted and assayed for ECEF activity. Activity was associated with the 13-14-kDa and the 30-kDa fractions, as seen by increased eosinophil antibody-dependent adherence to schistosomula and cytotoxicity. Granulocyte-monocyte-colony-stimulating factor and interleukin 1, but not tumor necrosis factor, could be detected in crude U-937 supernatants. However, active immunopurified ECEF has no activity in assays for granulocyte-monocyte-colony-stimulating factor, interleukin 1 or tumor necrosis factor. Immunocytochemical localization of ECEF employing the mAb shows strong surface staining of viable monocytes and U-937 cells, suggesting that ECEF is associated to the cell surface. These properties distinguish ECEF from other monokines previously reported to activate eosinophils.     

Human monocyte-mediated cytotoxicity towards erythrocytes induced by hybrid mouse monoclonal antibodies: effect of antibody binding valency on IgG-Fc gamma R interaction.

In this study, we describe the ability of hybrid mouse monoclonal antibody (mAb) to induce monocyte-mediated cytotoxicity towards human IgA1-coated E (HuIgA1-E), and the effect of mAb binding valency on Fc gamma RI-mediated ADCC. All hybrid monospecific (ms) anti-HuIgA1 and bispecific (bs) anti-HuIgA1/HRP mAb were capable of inducing monocyte-mediated lysis of HuIgA1-E, in spite of differences in mAb densities essential for optimal lysis. The cytotoxicity induced by hybrid mAb which consist of one or more mIgG2a H chains was predominantly mediated via Fc gamma RI, as shown by inhibition studies on monocytes with Fc gamma RI-blocking mAb TB-3 (approximately 80% inhibition). However, partial inhibition of mIgG1-2a and mIgG2a-2b-induced cytotoxicity (20-50%) was observed by using Fc gamma RII-blocking mAb IV.3 or CIKM5. For hybrid mIgG1-1 mAb the opposite was true; the cytotoxicity was predominantly mediated via Fc gamma RII (70-80%) and less via Fc gamma RI (20-30%). Comparing the hybrid ms anti-HuIgA1 mAb-induced cytotoxicity with the cytotoxicity induced by hybrid bs anti-HuIgA1/HRP mAb of the same isotype, we observed a decrease in cytotoxicity towards HuIgA1-E sensitized with univalently bound bs anti-HuIgA1/HRP mAb. This decrease was only found for Fc gamma RI-mediated ADCC (mIgG2a-2a, mIgG1-2a and mIgG2a-2b). This diminished recognition of univalently bound IgG relative to bivalently bound IgG by Fc gamma RI was also observed with U937 effector cells. In conclusion, this work shows that hybrid mAb are able to induce monocyte-mediated cytotoxicity towards E-HuIgA1 and that there appears to be an effect of Ag-IgG binding valency on Fc gamma RI-mediated cytotoxicity.     

Cytotoxic Factors Released by Dengue Virus-Infected Human Blood Monocytes

Human monocyte-derived cytotoxic factors (CF) induced by dengue virus were studied. Using several human leukemia cell lines as precursors, the biological activities of CF in conditioned medium from dengue virus-infected monocytes were demonstrated through the measurement of tumor cell growth inhibition. The conditioned medium from dengue virus infected monocytes suppressed significantly growth of CEM, HL60, K562, and U937 cells. In the presence of 10% conditioned medium (v/v) from dengue virus infected monocytes, DNA synthesis of U937 cells, as measured by [³H]thymidine incorporation, decreased by 99% in contrast to their synthesis in conditioned medium from noninfected control monocytes, which did not have any suppressive effect. Partial characterization of CF showed that it is a proteinase-K-sensitive and heat-labile protein with a molecular mass over 100 kDa. Employing a flow cytometric analysis of the cell cycle, it was found that U937 cells, treated either with conditioned medium from dengue virus infected monocytes or with CF, but not treated with conditioned medium from noninfected monocytes, showed cell-cycle arrest in G1 phase by 48 hr. This suppressive effect of CF on U937 growth was dose- and time-dependent. These results suggest that dengue virus-infected monocytes may produce CF to target myeloid cells, resulting in the hematological changes observed in patients with dengue fever. © 1995 Wiley-Liss, Inc.