Activation and fixation of C3 by human B cell lines is enhanced by interferon-gamma and tumor necrosis factor alpha.

Human B cell lines were tested for the capacity to convert C3 to C3b/iC3b through the alternative C pathway (ACP) and to fix the generated C3 fragments. The panel of lines included four Epstein-Barr virus (EBV)-negative Burkitt's lymphomas and their sublines converted with two EBV strains: the prototype B958 (B virus) and the nontransforming P3HR-1 (P virus). Comparison of these groups of parallel lines (derived from a single patient) showed that the EBV-carrying cells were more efficient in activation and fixation of C3. All cell lines activated C3 with higher efficiency when treated with interferon gamma or tumor necrosis factor alpha. After treatment, the comparative relationships between the sublines changed. The treated EBV-negative and the P virus converted sublines showed similar levels of ACP activation, while the B virus carrying sublines were more efficient. The amounts of bound C3 fragments on the activating cells correlated with the ACP efficiencies. In accordance with the elevated ACP activation capacity of the cells after cytokine treatment, the amounts of C3 fragments detected on their surfaces were higher. Among the lines tested, only two (BL28 and Ramos) were sensitive to C-mediated lysis. The lytic sensitivity of these two lines increased after treatment with the cytokines. The results indicate, therefore, that the cytokine-induced increase in ACP activation by Burkitt's lymphoma line is not sufficient to overcome resistance to C lysis.

Lysis of P3HR-1 cells induced to enter the viral cycle by antibody-dependent and independent immunological mechanisms.

The P3HR-1 Burkitt lymphoma line carries the Epstein-Barr virus (EBV) genome and a small proportion of the cells (1-3%) enter the lytic cycle spontaneously. Treatment with TPA and n-butyrate elevates considerably the number of virus-producing cells (25-35%). Cells which enter the lytic cycle express the EBV early antigen EA, the viral capsid antigen VCA, and the membrane antigen MA. Antibodies against these antigens are present in EBV-immune human sera. The expression of virus envelope protein on the plasma membrane renders the cells sensitive to immune effector mechanisms. These were shown to be initiated by the alternative complement pathway (ACP)-activating capacity of the cells and by their reactivity with antibodies directed to the MA. When incubated with EBV-immune or nonimmune human serum, the induced (P3HR-1-V) cells activated C3 through ACP and fixed the generated C3 fragments. The efficiency of opsonization was higher in immune serum. By varying the experimental conditions we showed the damage of the induced cells by the complement system and by blood lymphocytes, and analysed the involvement of antibodies and the activated C3 fragments in the lymphocyte-mediated lysis. P3HR-1-V cells were lysed by immune serum and also by nonimmune serum though with lower efficiency. The induced cells had elevated sensitivity to the NK effect which was potentiated if the conditions allowed their opsonization. In the presence of antibodies the lymphocyte-mediated lysis was considerably higher and the ADCC mechanism was also potentiated by opsonization. These experiments suggest that B cells which enter the virus-producing cycle may be eliminated in EBV nonimmune host by NK cells. After the antibody response against the virus develops, the attack on these cells is more efficient through complement and lymphocyte-mediated antibody-dependent mechanisms. These effector mechanisms are enhanced by opsonization which is the consequence of the C3-activating capacity of the cells. The multiple ways of the immune attack on the B cells prepared to produce EBV may explain the absence of EA and VCA positive B cells in tumor cell populations and during the acute phase of infectious mononucleosis.

Ligands of CR2 do not interfere with C3 fragment fixation or enhanced NK sensitivity of Raji cells treated with human serum.

Raji cells activate the alternative complement pathway (ACP) and fix C3 fragments when incubated in human serum (HS). Earlier experiments have shown that CR2 molecules are involved in this phenomenon and the opsonized cells have elevated sensitivity to the lytic effect of CR3-bearing NK cells. We show here that Raji cells treated with CR2 site-specific ligands, (C3d, OKB-7 and HB-5 mAbs, and a synthetic peptide which binds to CR2) generated and bound C3 fragments after exposure to HS. The elevated lytic sensitivity of HS-treated cells was not altered by the presence of the various CR2 ligands. Thus, the membrane-bound C3 fragments are not fixed at the C3dg receptor binding site.

Requirement of leukocytic cell adhesion molecules (CD11a-c/CD18) in the enhanced NK lysis of iC3b-opsonized targets.

Treatment of Raji or Daudi cells with human serum under conditions which allow the alternative pathway of C activation results in their C3-opsonization and enhanced sensitivity to NK-mediated lysis. The effector lymphocytes have low buoyant density, carry CD16 and HNK1 markers as well as the CD11a-c/CD18 leukocytic cell adhesion molecules. One of these molecules, made up of CD11b-CD18 (alpha- and beta-chains), is also the receptor for iC3b. We studied the role of the cell adhesion molecules in the NK effect on targets with and without C3-fragments. We focused on the E/T interaction of opsonized cells in the presence of anti CD18 mAb. mAb directed to the CD11a molecule caused 0 to 30% inhibition of the lysis of both non-opsonized and opsonized cells whereas the mAb antibody directed to the CD11c molecule had no effect. Reagents reactive with the iC3b binding site of CD11b (alpha-chain of the CR3) molecule did not alter the lysis of non-opsonized targets whereas they abrogated the C3-mediated increment of the Nk effect on opsonized cells. Two mAb preparations, 60.3 and IB4, directed to the CD18 chain shared by the three cell adhesion molecules abrogated in a dose-dependent way the lysis of both non-opsonized and opsonized targets. The 60.3 mAb inhibited the iC3b binding site of CR3 (despite its localization on the alpha-chain) and in accordance it inhibited the binding of lymphocytes to the opsonized target also. The IB4 did not affect this site and in accordance it inhibited only partially the binding of effectors to the C3 fragment carrying Raji, nevertheless it inhibited their lysis. This result indicates that the iC3b-CR3 bridge is insufficient for triggering the lysis in absence of the contact through the adhesion molecules.

Elevated NK-mediated lysis of Raji and Daudi cells carrying fixed iC3b fragments.

Raji and Daudi cells were opsonized with C3b, iC3b, and C3d fragments by using purified complement components. The sensitivity of C3-opsonized cells to lysis mediated by low density blood lymphocytes was studied. Raji and Daudi cells carrying C3b or C3d fragments were lysed with similar efficiencies as the nonopsonized cells. The presence of iC3b on the target surface imposed elevated NK sensitivity. The iC3b-mediated enhancement of NK lysis was inhibited when iC3b fragments or rabbit anti-human C3 antibodies were included into the lytic assays. These results indicate that the iC3b fragments fixed on the targets bind to the CR3 on the lymphocytes. Results obtained in immobilized conjugate-lytic assays showed that iC3b-opsonized targets interact more readily with the lymphocytes. This was reflected by the elevated proportion of lymphocytes that were bound to the iC3b-carrying targets. The proportions of conjugates in which target damage occurred were similar with the control and with the iC3b-carrying cells. It seems therefore that opsonization of targets with iC3b leads to recruitment of effector lymphocytes due to contact with their CR3. However, once the effector-target contact is established, the triggering of lytic function does not seem to be influenced by the iC3b/CR3 bridge.

Lymphocytes stimulated by allogeneic B cell lines cleave the third component of complement and fix C3 fragments. Their nonspecific lytic capacity is elevated against complement receptor type 2-carrying targets.

Human blood lymphocytes stimulated in mixed cultures by allogeneic B cell lines were shown to cleave C3 molecules. The B cell lines were derived from Burkitt lymphoma patients: 1) established from their EBV-negative lymphoma, 2) the EBV-positive sublines converted in vitro, and 3) lymphoblastoid cell lines (LCL) i.e., B lymphocytes transformed in vitro by EBV. These cell lines differed considerably in their capacity to stimulate allogeneic lymphocytes. The split products of C3 were detected in the supernatants and on the surface of the activated lymphocytes at levels which correlated with the strength of stimulation. Lymphocytes cultured with LCL had the highest levels of thymidine incorporation blast transformation, C3 cleavage, and C3 fragment fixation. Lymphocytes exposed to the EBV-negative Burkitt lymphomas were stimulated weakly and their C3-activating capacity was low. Irrespective of the efficiency of lymphocyte stimulation induced in the cultures, 60 to 70% of the blasts were found to fix C3 fragments. The majority of the lymphocytes which fixed C3 fragments were T blasts that carried the CD3 marker and expressed IL-2R (CD25). CD4 and CD8 cells were represented with equal frequency in the C3-fragment fixing and C3-fragment negative populations. Pre-exposure of the MLC-activated lymphocytes to human serum increased their cytotoxic capacity toward CR type 2-carrying targets. The enhanced lysis was abrogated by F(ab)2 rabbit anti-human C3d or rabbit anti-CR type 2 antibodies. The results suggest that the C3 fragments fixed on the lymphocytes bind to CR on the targets and elevate the avidity of binding between the two interacting cells. This was also indicated by an increase in the frequency of lymphocyte-target conjugates.

The elevated natural killer sensitivity of targets carrying surface-attached C3 fragments require the availability of the iC3b receptor (CR3) on the effectors.

Human serum-treated Raji and Daudi cells were shown to bind C3 fragments on their surface as a consequence of their capacity to activate C via the alternative pathway. C3 molecules were detectable on the cell surfaces up to 24 h after serum exposure. The C3 fragment-coated cells showed increased sensitivity to spontaneous lymphocyte-mediated cytotoxicity. The effector lymphocytes involved in the enhanced cytotoxicity were NK cells with low buoyant density, carrying both CR3 and FcR. Blocking of the FcR and CR3 with F(ab)2 fragments from Leu-11c or Leu-15 mAb, respectively, did not influence the lysis of targets that did not carry C3 fragments. In contrast, the accessibility of CR3 on the effector lymphocytes was essential for the C3 fragment-mediated enhancement of cytotoxicity. In addition to the Leu-15 antibody, N-acetyl-D-Glucosamine, a compound known to block iC3b binding to CR3, also abrogated the C3 fragment-imposed effect. Our previous experiments showed that the C3 fragments bind to acceptor sites on target cells. The present experiments show that the C3 fragments fixed onto the target bind to CR3 on effector cells. These data substantiate the hypothesis that the bivalent C3 fragments, which are fixed on the targets, promote their interaction with lytic lymphocytes by bridging the two cells.

Alternative pathway of complement activation by stimulated T lymphocytes. I. Binding of C3 fragments.

Human blood lymphocytes cultured for 3 days with concanavalin A (Con A), phytohemagglutinin or pokeweed mitogen, in mixed lymphocyte culture with added interleukin 2 and stimulated by a lymphoblastoid cell line were found to activate and bind C3 molecules when exposed to human serum. The split products of C3 were detected in the supernatants and on the surface of the activated cells. The surface-attached C3 fragment on the Con A blast was identified as C3b by immune adherence i.e. binding of CR1 carrying human erythrocytes. In the Con A-stimulated population the majority of cells that activated and bound C3 were CD3 and Fc gamma receptor (CD16)-positive but complement receptor-negative blasts. In this cell subset both CD4 and CD8-positive cells were detected but their frequency suggested that a proportion of them carried both markers.

Alternative pathway of complement activation by stimulated T lymphocytes. II. Elevation of cytotoxic potential against complement receptor-carrying cell lines.

Exposure of lectin-stimulated (concanavalin A, phytohemagglutinin and pokeweed mitogen) blood lymphocytes to human serum or to purified C3 increased their cytotoxic capacity towards complement receptor positive targets such as Raji and Daudi cells. The lysis of complement receptor-negative lymphoblastoid cell lines was not influenced. The lytic capacity of lymphocytes exposed to 12-O-tetradecanoylphorbol 13-acetate was not elevated by human serum. Lectin-stimulated lymphocytes were previously shown to activate and bind C3. The results using lymphocytes activated in different ways and targets with or without complement receptor expression suggest that the C3b deposited on lymphocytes binds to the complement receptor on the targets. This contact elevates the avidity between the two cells as indicated also by the increased frequency of the lymphocyte-target conjugates. On the basis of immune adherence the C3 fragment bound on the lymphocytes was identified as C3b. The increase of the conjugate formation and cytotoxicity was abrogated when the target cells, Raji, were pre-exposed to purified C3d which occupy the CR2 receptor. The majority of lymphocytes responsible for the cytotoxicity were CD8+.

Complement-dependent cellular cytotoxicity: lymphoblastoid lines that activate complement component 3 (C3) and express C3 receptors have increased sensitivity to lymphocyte-mediated lysis in the presence of fresh human serum.

Lymphocyte-mediated lysis of cells of the Raji, Daudi, Jijoye, and Bjab lines was elevated when fresh human serum was added to the assay. A higher proportion of effector-target conjugates was observed in the presence of human serum. In similar experiments lysis of 1301, Rael, and P3HR-1 cells was unaltered. All cell lines activated the alternative pathway of complement but they varied in the expression of receptors for complement component 3 (C3) and in the ability to fix the C3 cleavage products on their membrane. The enhancement of lysis in the presence of human serum occurred only with those cells that bound C3. This characteristic was correlated to the expression of C3 receptors. Analysis of the nature of the deposited C3 was performed with Raji cells. Raji cells exposed to human serum bound C3b as indicated by the immunoadherence test. The C3b was further processed to C3bi, because the immunoadherence declined with time and conjugate formation increased with Daudi cells, which carry the C3 receptors CR2 and CR3. This suggests that in the lytic assay lymphocytes with C3bi receptors are recruited in the presence of human serum. We assume that the bridge of C3 molecules between targets and effectors increases the avidity of their interaction.

Modulation of human blood lymphocyte cytotoxicity by the phorbol ester tumor promoter P(Bu)2: increase of target binding, impairment of effector recycling, and activation of lytic potential which is independent of IL-2.

Treatment of lymphocytes with the tumor promotor P(Bu)2 enhanced their target-binding capacity. In the conventional 51Cr-release assay, the cytotoxic potential of lymphocytes pretreated with P(Bu)2 for a short time was reduced while after prolonged treatment their function was increased. The peak of lytic potential was attained after 15 hr of exposure. Comparison of the cytotoxic results obtained in suspension and immobilized conjugate assays suggested that P(Bu)2 treatment causes an impairment of the recycling capacity of lymphocytes. After prolonged exposure, the lytic machinery became activated as reflected by the reduced time elapsing after contact between effectors and targets and the delivery of lethal hit. The activation was also observed in the immobilized agarose assay. It was reflected by the elevated proportion of damaged targets that were bound to the treated lymphocytes. The P(Bu)2- and interferon-induced augmentation of lytic potential is achieved through different mechanisms. Combination treatment applied to the effectors in sequence (first P(Bu)2 followed by interferon), resulted in an additive effect. Similarly, simultaneous treatment with IL-2 and P(Bu)2 also gave an additive increase in cytotoxicity. Addition of antibodies directed against IL-2 did not abrogate the P(Bu)2 effect. Consequently, neither interferon nor IL-2 are involved in the phorbol ester-induced cytotoxic function.

Activation of cytotoxic activity of human blood lymphocytes by tumor-promoting compounds.

Three categories of tumor promoters and chemically related but inactive substances were tested for their effect on the cytotoxic activity of human blood lymphocytes against K562 and Daudi targets. Lymphocytes incubated overnight in the presence of phorbol esters 12-O-tetradecanoylphorbol-13-acetate and phorbol-12,13-dibutyrate [P(Bu)2] had enhanced function. Incubation with 4-alpha-phorbol-12,13-didecanoate was without effect. Enhancing activity was also exerted by the indole alkaloids, teleocidin and lyngbyatoxin A, and the polyacetates, aplysiatoxin and debromoaplysiatoxin, but not by dihydroteleocidin. Only the tumor-promoting compounds activated the cytotoxic potential. The substances acted in a dose-dependent manner with optimal activity at characteristic concentrations. Overnight incubation of lymphocytes at 4 degrees did not change their spontaneous cytotoxicity but abolished the enhancing effect of P(Bu)2. Thus, P(Bu)2-induced activation occurred only on metabolically active cells. The activation did not require DNA synthesis. Similar to controls, the P(Bu)2-treated cells required divalent cations and an intact cytoskeleton in order to perform their lytic function. Experiments with the various metabolic inhibitors indicate that phorbol ester treatment does not induce an alternative cytotoxic mechanism since, as with untreated lymphocytes, P(Bu)2-activated cells require contact with the target and intact secretory functions. The enhanced cytolytic potential was not due to induction of alpha-interferon (IFN-alpha) production, as shown by the fact that the effect was not abolished by addition of anti-IFN antibodies during the P(Bu)2 treatment of lymphocytes or during the cytotoxic assay. However, the presence of antiserum against IFN reduced the cytotoxic potential of control cells, suggesting that endogenous IFN production contributes to the maintenance of lytic function in cultured cells. If this mechanism is counteracted by addition of anti-IFN serum, the phorbol esters can provide an alternative activation signal. When P(Bu)2-activated lymphocytes were subsequently treated with IFN-alpha or IFN-gamma, their lytic capacity was further increased. These results indicate that P(Bu)2 and IFN activate cytotoxic potential through different pathways.

The tumor promoter phorbol-12,13-dibutyrate [P(Bu)2] stimulates cytotoxic activity of human blood lymphocytes.

The tumor promoters 12-13-phorbol-dibutyrate, P(Bu)2, and 12-O-tetradecanoylphorbol-13-acetate, TPA, were shown to augment the cytotoxic potential of human blood lymphocytes with low cell density. In kinetics experiments the enhancing effect was preceded by an initial suppression lasting for about 2 hours. Admixture of mononuclear adherent cells abrogated the P(Bu)2 effect in a dose dependent way. P(Bu)2 altered the sensitivity of K562 cells to the cytotoxic effect. Short term pretreatment increased the sensitivity, but after longer pretreatment the cells became resistant. The results show that tumor promoters can influence the cytolytic system at different levels. By acting directly on the lymphocytes they potentiate the lytic function. When mixed mononuclear populations are used, this effect may be counteracted via activation of the suppressive functions of monocytes. In addition, the target cell sensitivity can also be modulated. As a result, the final outcome of phorbol treatment depends on the strength, kinetics and the mode of its effects on the interactants.

Platelet function abnormalities in acute leukaemia.

Studies on platelet function in vitro were done in fourteen patients with different kinds of acute leukaemia. Quantitative clot retraction was abnormal in all patients. Aggregation was different with the various agents tested. Availability of platelet factor 3 was abnormal in six patients. All patients were studied prior to chemotherapy. Two patients were studied in complete remission, as well; they showed an almost normal platelet function. Another patient without remission after induction treatment displayed a platelet function worse than in the initial study. The results suggest that the defect in leukaemic platelets is due to the leukaemic process itself.