Effect of histamine-receptor blocking on human natural and lectin-dependent cell-mediated cytotoxicity against adherent HEP-2 cells

The effect of histamine (H) and H1-, H2-receptor blocking agents was studied on natural (NCMC) and lectin-dependent cell-mediated cytotoxicity (LDCC) of peripheral blood lymphocytes (PBL) from eight healthy subjects on HEP-2 adherent human epipharynx carcinoma target cells. Cytotoxicity was measured by detachment from the monolayer of 3H-TdR-prelabelled HEp-2 cells. LDCC was evaluated in a 24 h assay with a Concanavalin A (Con A) dose of 25 micrograms/ml at 50:1 effector-target cell ratio. Under these conditions, but without Con A, considerable NCMC was not elicited by normal lymphocytes. The presence of histamine and the H2-receptor blocker cimetidine resulted in a significant NCMC to HEp-2 cells. On the contrary, histamine and cimetidine reduced LDCC. The H1-receptor blocker clemastine had no significant effect on either NCMC or LDCC to HEp-2 targets. The possible involvement of H2-receptor bearing cells in the regulation of cytotoxicity to HEp-2 cells is suggested.     

Lectin-dependent cell-mediated cytotoxicity and blastogenesis by large granular-enriched and depleted lymphocytes.

The role of larger granular-enriched and depleted lymphocytes was studied in lectin-dependent cell-mediated cytotoxicity (LDCC) against adherent HEp-2 human epipharynx carcinoma target cells. LDCC was evaluated by detachment from the monolayer of 3H-thymidine-prelabelled HEp-2 cells in a 24 h assay at effector-target cell ratios of 25:1 and 50:1 in the presence of 25 micrograms/ml concanavalin A (Con A). Under the aforementioned conditions but in the absence of Con A natural cell-mediated cytotoxicity (NCMC) was not found. However, cytotoxicity was significantly augmented in the presence of Con A (= LDCC) using human peripheral blood mononuclear cells (PBMC) as effectors. Large granular lymphocytes (LGL), which show high natural killer (NK) activity to K 562 target cells, failed to be cytotoxic against HEp-2 targets similar to large granular depleted lymphocytes (LGL-DL). On the other hand, LGL caused only a slight LDCC; whilst LGL-DL induced strong LDCC activity towards HEp-2 targets. In comparison to LDCC using LGL-DL as effector cells, LGL and LGL-DL mixed at a ratio of 1:2, and added to target cells, had no major effect on LDCC, while a lower level of LDCC was observed at LGL/LGL-DL ratios of 1:1, and 2:1, suggesting the dilution of LGL-DL, potential effectors of LDCC to HEp-2 cells, rather than a specific regulatory role of LGL in LDCC. In parallel studies, the proliferation of LGL-DL in response to Con A was less than that observed with PBMC or LGL. The response could be restored by replacing half of LGL-DL per culture with an equal number of LGL, or by the addition of 10% monocytes. Significant functional differences between LGL and LGL-DL in LDCC as well as in Con A-induced blastogenesis are suggested.     

Depressed Natural and Lectin-Dependent Cell-Mediated Cytotoxicity Against Adherent Hep-2 Cells in Patients with Systemic Lupus Erythematosus

Natural cell-mediated cytotoxicity /NCMC/ was evaluated using human adherent 3H-thymidine-prelabelled HEp-2 epipharynx carcinoma cells as targets at 50:1 effector-target cell ratio in a 24 hr assay. For lectin-dependent cell-mediated cytotoxicity /LDCC/ studies cultures contained also 25/Ug/ml concanavalin A /Con A/. Peripheral blood mononuclear cells /PBMC/ of nine patients with active systemic lupus erythematosus /SLE/ failed to exert NCMC or LDCC against HEp-2 targets. In contrast, an increased adherence /decreased detachment from the monolayer/ of HEp-2 target cells was observed in the presence of PBM3 from SLE patients that was further promoted by the addition of Con A during LDCC assay.     

Depressed Natural and Lectin-Dependent Cell-Mediated Cytotoxicity Against Adherent Hep-2 Cells in Patients with Systemic Lupus Erythematosus

Natural cell-mediated cytotoxicity /NCMC/ was evaluated using human adherent 3H-thymidine-prelabelled HEp-2 epipharynx carcinoma cells as targets at 50:1 effector-target cell ratio in a 24 hr assay. For lectin-dependent cell-mediated cytotoxicity /LDCC/ studies cultures contained also 25/Ug/ml concanavalin A /Con A/. Peripheral blood mononuclear cells /PBMC/ of nine patients with active systemic lupus erythematosus /SLE/ failed to exert NCMC or LDCC against HEp-2 targets. In contrast, an increased adherence /decreased detachment from the monolayer/ of HEp-2 target cells was observed in the presence of PBM3 from SLE patients that was further promoted by the addition of Con A during LDCC assay.     

Stimulation of lectin-dependent cell-mediated cytotoxicity against adherent HEp-2 cells by carrageenan

The effect of carrageenan (CGN) was studied on lectin-dependent cell-mediated cytotoxicity (LDCC) against adherent HEp-2 human epipharynx carcinoma target cells. Cytotoxicity was measured by detachment from the monolayer of 3H-TdR pre-labelled HEp-2 cells. LDCC was evaluated in a 24 h assay at 50:1 effector-target cell ratio in the presence of 25 micrograms/ml concanavalin A (Con A). Under these conditions but without Con A considerable cytotoxicity was not elicited by peripheral blood mononuclear cells (PBMC). Addition of CGN to the system significantly increased LDCC to HEp-2 targets. Monocyte depletion of effector cells had no major influence to the effect of CGN on LDCC activity. In parallel studies CGN also enhanced the Con A-induced proliferation of PBMC.     

Effects of Lentinan on Cytotoxic Functions of Human Lymphocytes

The in vitro effects of lentinan on natural killer (NK), antibody-dependent cell-mediated cytotoxicity (ADCC), lectin-dependent cell-mediated cytotoxicity (LDCC) and mitogen-induced blast transformation were studied in patients with solid tumors and chroyic lymphocytic leukemia (CLL). NK activity was measured against Cr-labelled K-562 targets, ADCC against antibody-coated chicken red cells. LDCC and natural cell-mediated cytotoxicity (NCMC) was assessed using ³H-thymidine prelabelled HEp-2 targets. Mitogen (PHA-and Con A-) induced blast transformation was measured by thymidine incorporation.

Blastogenesis and LDCC was not influenced by lentinan. 1 μg/ml lentinan increased NCMC of tumor-bearing subjects. The most prominent enhancement of NK and ADCC activity was seen in CLL patients, where a dose-related increase was seen (from 0.01 to 1 μg/ml).     

Independence of depressed lectin-dependent cell-mediated cytotoxicity from interleukin 2 production in patients with systemic lupus erythematosus.

The relationship of lectin-dependent cell-mediated cytotoxicity (LDCC) to interleukin-2 (IL-2) production was studied in healthy subjects and in patients with systemic lupus erythematosus (SLE). Profoundly depressed levels of LDCC were elicited by peripheral blood mononuclear cells (PBMC) from nine patients with active SLE in comparison to LDCC from seven controls, and eleven inactive SLE donors, using 3H-TdR-prelabelled adherent HEP-2 cells as targets in a 24 h assay with 25 micrograms/ml Con A. In parallel experiments, no individual correlation was found between LDCC activity and IL-2 production for healthy or SLE subjects. Further, no major differences were detected in IL-2 release when the three groups of donors were compared, a tendency observed at the Con A doses (5 and 25 micrograms/ml) and incubation times (24, 48, and 72 h) used to induce IL-2 production. In additional studies, impaired Con A-induced blastogenesis was noted for PBMC from active SLE patients in comparison to the PBMC from the controls or patients with inactive SLE. While strong individual correlation was obtained between blastogenesis and IL-2 secretion in controls and patients with inactive SLE, no such relationship was found in patients with active SLE. While addition of exogenous IL-2 to the cytotoxicity assay considerably enhanced LDCC by healthy donors it failed to improve LDCC by patients with active SLE. These data suggest that depressed LDCC and Con A-induced blastogenesis of patients with active SLE may not be related to impaired IL-2 production but rather to an inherent dysfunction of the effector lymphocytes, including their unresponsiveness to IL-2.     

T lymphocyte-mediated cytolysis. III. Delineation of mechanisms whereby mitogenic and non-mitogenic lectins mediate lymphocyte-target interaction

We have investigated the mechanism(s) by which mitogenic (concanavalin A [Con A], phytohaemagglutinin [PHA] and Lens culinaris agglutinin [LCA]) and non-mitogenic (soybean agglutinin, peanut agglutinin, wheat-germ agglutinin and pokeweed mitogen) lectins mediate, non-specifically, lectin-dependent lymphocytotoxicity (LDCC). We show that non-mitogenic lectins are ineffective mediators of LDCC, due to their inability to mediate effective binding of effector cytotoxic T cells (EC) and target cells (TC), and not to their failure to 'activate' TC-bound EC, as proposed before. Evidence is presented that in LDCC Con A and PHA exert their primary effect(s) by affecting the TC rather than the EC. Although the lectin LCA, unlike Con A and PHA, is equally reactive with either EC or TC, a direct comparison is difficult since the presence of LCA, but not Con A or PHA, during the entire assay is required for optimal kill. Furthermore, all three LDCC-supportive lectins (PHA, Con A and LCA) show a similar TC preference when tested in a EC-TC conjugation assay. Taken together, these results are inconsistent with the theory that lectins mediate LDCC by 'bridging' EC and TC through lectin-binding receptors followed by 'activation' of the TC-bound EC. We would like to suggest that the potential of mitogenic lectins to mediate EC-TC interaction is related to their modification of TC-surface constituents, possibly major histocompatibility complex determinants, rendering them recognizable, non-specifically by EC.     

Lectin-dependent cell-mediated cytotoxicity in an invertebrate model: Con A does not act as a bridge.

The plant lectin concanavalin A (Con A) has been used in an invertebrate model of lectin-dependent cell-mediated cytotoxicity (LDCC). Macrophage-like cells from the susceptible host snail Biomphalaria glabrata become cytotoxic effectors when they encounter sporocysts of the parasitic trematode Schistosoma mansoni that have been treated with Con A. The sugar alpha-methyl mannoside and rabbit anti-Con A antibodies fail to block this LDCC. Con A is effective only when the target, not the effector cell, has been exposed to it. These results constitute evidence against the molecular bridging hypothesis and support the notion that surface modulation of the target may be the stimulus that provokes cytotoxicity. Results from this invertebrate model are discussed in the context of murine T lymphocyte LDCC.     

Recognition of autologous and allogeneic lymphocytes and tumor cells by human natural killer cells

The shedding of the mobile Fc receptor (FcR1) and the depletion of the immobile Fc receptor (FcR11) bearing human lymphocytes revealed that human natural killer cells belong to the FcR11-bearing population. Anti-beta-2-microglobulin treatment of the effector cells decreased natural cytotoxicity against some target cells and the detectability of HLA antigens, indicating that histocompatibility antigens or related structures may be involved in natural cytotoxicity. Using a panel of 29 autologous and allogenic PHA-stimulated target cells and peripheral lymphocytes from the same donors as the effector cells, distinct cytotoxic responses against allogeneic and autologous target cells were observed. A computer analysis of selective natural cytotoxicity distinguished seven different groups of target cells that may represent common structures for NK recognition.     

Hydroxyl radical scavengers inhibit human lectin-dependent cellular cytotoxicity.

The role of oxygen-derived free radicals (ODFR) in lectin-dependent cellular cytotoxicity (LDCC) in humans was investigated. The hydroxyl radical traps thiourea, methanol, ethanol and phenol were effective in inhibiting LDCC, as was DABCO, a singlet oxygen quencher. The proposed pathway of hydroxyl radical production in living cells is either an iron catalysed Haber-Weiss reaction or a Fenton reaction. The effect of inhibitors of these pathways was investigated. The superoxide anion scavengers superoxide dismutase, ferricytochrome c and Tiron were without effect. It was shown that Tiron inhibits the lucigenin-amplified chemiluminescence produced by the action of xanthine oxidase, and also the lucigenin-amplified chemiluminescence produced by activated PMN, suggesting that this agent (Tiron) scavenges intracellular superoxide anion. Catalase gave slight inhibition of LDCC only. The ferric iron chelator desferrioxamine gave no protection of the target cells, while the ferrous chelator, 1,10-phenanthroline, inhibited LDCC and partially prevented the detection of hydroxyl radicals generated by the Fe2+-H2O2 system. Cibacron blue, an agent that inhibits NAD(P)H linked enzymes, also inhibited LDCC. The cyclo-oxygenase inhibitors indomethacin and salicylate were without effect, while the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) inhibited cytolysis. None of the LDCC inhibitors was cytotoxic to the effector cells or to the target cells, neither did they inhibit lymphocyte-target binding. The findings would suggest that hydroxyl radicals have a role to play in human T-cell mediated cytolysis, either as the active lytic agent or as an epiphenomenon.     

In vitro combined effects of human interferons and interleukin-2 on natural cell-mediated cytotoxicity

In vitro modulation of natural cell-mediated cytotoxicity (NCMC), following sequential treatment of human mononuclear cells (MNC) with cytokines was investigated. Recombinant Interleukin-2 (IL2) used in combination with interferons (IFNs) induced variable effects on the cytolytic function of different MNC preparations obtained from 16 healthy donors. When MNC were treated with IFNs on day 4, after IL2 induction of LAK cells, increase or no change in cytotoxic activity was found. On the other hand, either no change or decrease in LAK activity occured when MNC were treated with IFNs on day 0 before exposure to IL2. In this case the effect of IFNs on NCMC did not correlate with their activity on cell proliferation or on TAC antigen expression. In conclusion the present study points out that the NCMC of MNC of healthy donors, subjected to IL2 treatment in vitro, can be significantly increased by IFNs. However this effect is largely schedule-dependent (i.e. detectable with IL2-IFNs but not with IFNs-IL2 sequence), and can be obtained in a relatively limited number of cases. Moreover it is suggested that these in vitro studies could provide preclinical bases for a rational approach to in vivo treatment with cytokine cascade in a clinical setting.     

Tumor‐induced CD11b+ Gr‐1+ myeloid‐derived suppressor cells exacerbate immune‐mediated hepatitis in mice in a CD40‐dependent manner

Immunosuppressive CD11b⁺Gr‐1⁺ myeloid‐derived suppressor cells (MDSCs) accumulate in the livers of tumor‐bearing (TB) mice. We studied hepatic MDSCs in two murine models of immune‐mediated hepatitis. Unexpectedly, treatment of TB mice with Concanavalin A (Con A) or α‐galactosylceramide resulted in increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum levels in comparison to tumor‐free mice. Adoptive transfer of hepatic MDSCs into naïve mice exacerbated Con A induced liver damage. Hepatic CD11b⁺Gr‐1⁺ cells revealed a polarized proinflammatory gene signature after Con A treatment. An IFN‐γ‐dependent upregulation of CD40 on hepatic CD11b⁺Gr‐1⁺ cells along with an upregulation of CD80, CD86, and CD1d after Con A treatment was observed. Con A treatment resulted in a loss of suppressor function by tumor‐induced CD11b⁺Gr‐1⁺ MDSCs as well as enhanced reactive oxygen species (ROS)‐mediated hepatotoxicity. CD40 knockdown in hepatic MDSCs led to increased arginase activity upon Con A treatment and lower ALT/AST serum levels. Finally, blockade of arginase activity in Cd40^?/? tumor‐induced myeloid cells resulted in exacerbation of hepatitis and increased ROS production in vivo. Our findings indicate that in a setting of acute hepatitis, tumor‐induced hepatic MDSCs act as proinflammatory immune effector cells capable of killing hepatocytes in a CD40‐dependent manner.     

Concanavalin A Inhibits the Effector Phase of Specific Cytotoxicity

The effects of concanavalin A (Con A) on the effector phase of specific and nonspecific cytotoxicity were studied. The addition of the lectin to the cytotoxicity assay resulted in inhibition of specific cytotoxicity and induced the lysis of nonspecific targets only when the lectin was added after the target cells. Preincubation of the effector cells with the ligand strongly inhibited specific cytotoxicity and did not induce nonspecific cytotoxicity. However, preincubation of the target cells with Con A before addition to the assay had no effect on the specific lysis and strongly facilitated the lysis of nonspecific targets. The inhibitory effect was not due to the agglutinating property of the lectin, since another agglutinogenic and non-mitogenic lectin ( Helix pomatia ) did not inhibit cytotoxicity. Induction of effector-to-effector killing seemed unlikely, since the addition of Con A to ⁵¹Cr-labelled effector cells did not significantly enhance the release of isotope. The inhibitory effect could be reversed by a subsequent incubation of the Con-A-treated effectors with alpha-methyln-mannoside. We suggest that Con A inhibits specific alloreactive cytotoxicity by blocking the antigen-binding receptors of T cells and induces nonspecific cytotoxocity by already activated cytotoxic T lymphocytes (CTLs) by binding to the major histocompatibility complex (MHC) antigens of the targets and creating structures mimicking allogeneic MHC products that will be recognized by CTLs via the antigen-binding receptor.     

A Comparative Analysis of Cell Surface Markers on Murine NK Cells and CTL Target-Effector Conjugates

The rosetting of sheep erythrocytes (SRBC) coated with non-haemagglutinating monoclonal antibodies rather than conventional haemagglutinating antisera revealed readily detectable FcR on most splenic natural killer (NK) cells since 76% of splenic lymphocytes forming conjugates with YAC also resetted with SRBC coated with high concentrations of monoclonal anti-SRBC antibody of the IgG2b subclass and since Ficoll depletion or enrichment of splenic lymphocytes rosetting with IgG2b-coated SRBC resulted in a corresponding 4-fold decrease or increase in conjugate-forming cells and a 10-fold decrease or increase in NK cytolytic acttvity. NK cells bound much less readily to monoclonal IgG2a and not at all to monoclonal IgGI or IgM, but the degree of binding was directly proportional to the amount of antibody on the erythrocytes and was not isotype-restricted. In addition, immunofluorescent studies revealed that YAC-1-conjugated lymphocytes were Lyt-1^-, Lyt-2^-, partially Thy-1⁺ (60%), asiato-GMI ⁺ (80%), Qa-4⁺ (77%), Qa-5⁺ (79%), and Ly-5⁺ (94%). In comparison, a proportion (39%) of alloimmune peritoneal exudate cells which conjugated with P815–2 also siained by immunofluorescence with anti-asialo GM1 antisera. Most (>90%) P815- conjugated cells were Thy-1⁺, Lyt-2⁺. and a subpopulation of Lyt-l⁺2⁺ conjugates was observed (25 %). Qa-5 and Ly-5 were also expressed on most (two-thirds) cytolytic T lymphocytes (CTL) conjugates, whereas Qa-4 and FcR for IgG2b were not detected. The best phenotypic distinctions between NK cells and CTL were therefore based on the presence or absence of Lyt-2, Qa-4, and FcR for IgG2b on most effector cells. Anti-asialo-GMl or monoclonal anti-Qa-4 and complement treatment greatly diminished both the frequency of NK conjugates and the percentage of conjugates with detectable IgG2b FcR or asialo-GM1. These results confirm that NK cells co-express asialo-GMI and Fc receptors, at the single-celt level, and provide a simple method for greatly enriching NK populations at least 10-fold.     

Analysis of Fcγ receptors on human peripheral blood leukocytes by flow microfluorometry. I. Receptor distributions on monocytes,Tγ cells and cells labeled with the 3A1 anti-T cell monoclonal antibody

A dual parameter flow microfluorometric technique for accurately measuring Fcγ receptor (FcR) expression on defined subsets of cells within a heterogeneous cell sample was developed. The FcR distribution of human peripheral blood mononuclear cells consists of three distinct peaks. By analyzing cells fluorescently labeled with the 3A1, an anti-T cell hybridoma antibody (using a green-emitting fluorophore) and for FcR (with a red-emitting fluorophore), and by using cell isolation procedures, it was shown that the cells lying within the peak with intermediate FcR density are mainly monocytes, while cells lying within the peaks with highest and lowest (i.e. negative) FcR densities are predominantly T cells. The FcR⁺ T cells (T_γ cells) express higher levels of the 3A1 antigen than other T cells, thus demonstrating the utility of the 3A1 hybridoma antibody as a marker for T_γ cells.     

The Appearance of Cytotoxic Lymphocytes in Unstimulated and Concanavalin A-Activated Human Peripheral Mononuclear Cells

The presence of a mitogenic lectin during the cytotoxicity assay enables the detection of non-specific cytotoxicity (lectin-dependent cell-mediated cytotoxicity (LDCC]. Human mononuclear cells cultured at 37 degrees C in vitro became cytotoxic after 1 day, as detected by the LDCC. In some individuals, the presence of adherent cells during culture inhibited the development of cytotoxicity. Activation by concanavalin A (Con A) resulted in enhanced cytotoxicity after a few days in culture. There was no clear relationship between the lectin doses required for optimal induction of cytotoxicity and [3H]thymidine uptake. High Con A concentrations that induced a proliferative response often inhibited cytotoxicity in mononuclear cells depleted of adherent cells.     

Reconstitution of natural cell-mediated cytotoxicity with specific antibodies.

The apparent nonselective reactions of natural cell-mediated cytotoxicity (NCMC) are selective when tested by inhibition of cytotoxicity with competitor cells indicating a recognition of specificities by the effector cell. N cells that mediate this NCMC in humans have most of the characteristics of K cells that mediate antibody-dependent, cell-mediated cytotoxicity (ADCC) and possess Fc receptors. IgG antibodies attached loosely to N cells through their Fc region, form part of the class of lymphocytes with surface immunoglobulin. We hypothesized that ADCC and NCMS involved similar mechanisms but with the specificity of NCMC directed by the natural IgG antibodies already attached to N cells. Removal of the antibodies with trypsin and reconstitution with specific anti-HLA antibodies produced specific effector cells supporting the role of antibodies on N cells as directors of specificity in NCMC.     

Lectin-Dependent Cell-Mediated Cytotoxicity Following In Vitro Culture of Normal Lymphocytes in Medium Containing 2-Mercaptoethanol

Cell-mediated cytotoxic reactivity resulting from the in vitro incubation of normal lymphocytes was assessed using nonspecific lectin-dependent cell-mediated cytotoxicity (LDCC) as a measure of overall reactivity. Spleen cells from nonimmune C57BL/6 mice were incubated in vitro in RPM1-1640 supplemented with 10% fetal calf serum and 2-mercaptoethanol (2ME). Cytotoxicity was assayed against syngeneic Cr51-labeled EL-4 cells in the presence of Con A or PHA. Optimal LDCC was observed after 8 days of culture in the presence of 5 × 10^-5 M 2ME. Cytotoxicity was mediated by an activated T-lymphocyte population whose development did not appear to require macrophages. Usually LDCC in the presence of PHA was significantly greater than that obtained in the presence of Con A. The presence of 2ME during the initial phase of culture was crucial for the development of cytotoxicity, since early removal of 2ME after 1 or 3 days of culture did not alter the subsequent development of cytotoxicity, whereas delayed addition of 2ME on day 1 or 3 failed to produce cytotoxic reactivity. This rapid conversion from a 2ME sensitive state to a 2ME insensitive state may be related to a rapid loss of accessory cell viability during the early phase of culture. Together the results indicate that this system may provide a useful model for the investigation of the events leading to the development of CTL in vitro.     

Role of Vincristine in Immunochemotherapy of Leukemia in Mouse or Human Models

Synergistic antitumor effects between Vincristine (VCR) and allograft responses. have been found in mice bearing allogeneic retrovirus-induced leukemia. In this model VCR depressed weakly allograft reactivity if given before but not after antigen administration. In a parallel human tumor model in vitro using HTLV-1 induced MT-2 leukemia, additive but not synergistic immuno-chemotherapeutic effects were obtained with allogeneic mononuclear cells (MNC) combined with VCR at 0.1 but not at 1 mμg/ml. In this case natural immunity (NI) rather than antigen-dependent immunity (ADI) was involved in the combined effets of VCR + MNC. In the in vitro model pretreatment of effector cells with 1 or 0.1 mμg/ml of VCR depressed natural cell-mediated cytotoxicity (NCMC). However when the drug was added to the effector + target cells during the 4 h cytotoxicity assay, 1 but not 0.1 mμg/ml of the drug was capable of depressing NCMC function. These results would provide valuable information for developing in vitro immuno-chemotherapy studies in human tumor systems, including those characterized by the presence of tumor-associated oncogenic retroviruses, capable of depressing both NI and ADI functions.