concanavalin-a and Mast-Cell-Sarcoma

Treatment with direct electric current (DC) can inhibit tumor growth in several systems. To evaluate the cellular reactions generated by this treatment, we stimulated mouse mastocytoma P815 cells with DC and examined their viability and ultrastructural characteristics, as well as the effect of DC on surface carbohydrate expression. DC treatment affected cell viability and caused marked alterations in vital structures of P815 cells. Alterations varied depending on the duration of stimulation and polarity of electrode. Anodic and cathodic treatments caused decrease in cell viability, although the latter was more effective in generating cell lysis. DC stimulation also induced changes such as membrane damage, alterations in cell shape and chromatin organization, mitochondrial swelling and condensation, cytoplasmic swelling, and matrix rarefaction. Stimulation of P815 cells without contact with electrodes produced no alterations, suggesting that this contact might be essential for the occurrence of the cellular modifications. DC treatment also altered the membrane distribution of anionic sites of P815 cells, as well as the surface carbohydrate exposition, involving a diminished binding of Concanavalin A to the cell surface after cathodic stimulation, and an increased binding of sialic acid- and fucose-specific lectins after anodic treatment. In this work we describe important cellular targets for the action of DC, which may contribute to the understanding of the mechanisms by which DC supresses several kinds of tumors.

Topics: Animals; Binding Sites; Carbohydrate Metabolism; Cell Membrane; Cell Nucleus; Cell Survival; Concanavalin A; Electric Stimulation; Mast-Cell Sarcoma; Mice; Mitochondria; Tumor Cells, Cultured

The dog mastocytoma BR cell line provides us with a permanent source of canine mast cells, allowing a characterization of secretory mediators that exert important effects in canine allergic and nonallergic diseases and in physiological processes. We studied the ultrastructural characteristics and histamine releasing activity after immunological and non-immunological stimuli of the dog mastocytoma BR cell line, and compared the cell line to normal skin mast cells enzymatically isolated from healthy dogs. The histamine content of BR cells was 0.04 +/- 0.002 pg/cell, approximately 100-fold less than that found in canine skin mast cells. Non-immunologic stimuli induced similar concentration-dependent histamine release from skin mast cells and BR cells: 29.3 +/- 0.9% vs. 12.7 +/- 0.7% (calcium ionophore A23187), 23.3 +/- 0.7% vs. 18.8 +/- 0.7% (substance P) and 12.5 +/- 0.3% vs. 12.1 +/- 0.9% (compound 48/80), respectively. Immunologic stimulation, however, was only effective on canine skin mast cells, causing 30.9 +/- 1.7%, 27.7 +/- 0.6% and 12.2 +/- 0.9% histamine release in response to anti-canine IgE, concanavalin A, and antigen Asc S 1, respectively. The absence of functional IgE receptors in BR cells was confirmed by the lack of response to anti-IgE and antigen Asc S 1 following passive sensitization with dog atopic serum and dog antigen sensitized serum. We conclude that BR cells are able to release histamine after non-immunologic stimulation in a similar manner to canine skin mast cells, but that there are morphological and functional differences possibly due to different states of maturity or differentiation. For this reason the study of the highly homogeneous BR cells could offer insights into dog mast cell biology in contexts where freshly isolated cells cannot be used because of low purity and recovery.

Topics: Animals; Calcimycin; Cell Separation; Concanavalin A; Dog Diseases; Dogs; Histamine; Histamine Release; In Vitro Techniques; Ionophores; Mast Cells; Mast-Cell Sarcoma; Microscopy, Electron; p-Methoxy-N-methylphenethylamine; Receptors, IgE; Substance P; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Concanavalin A-induced proliferation of spleen cells of C57B1/6 mice was inhibited by syngeneic normal bone marrow cells. Elimination of Ag-Eb-positive cells by panning was shown to result in markedly reduced inhibitory activity of bone marrow cells. To evaluate the role of Ag-Eb in natural suppressor activity, bone marrow cells were preincubated with different dilutions of MAE-15 monoclonal antibody and then added to spleen cells. The inhibitory effect of bone marrow cells decreased with the increasing concentration of the monoclonal antibody in a dose-dependent manner and nearly disappeared at a concentration of MAE-15 of 150 m micrograms/ml and 300 m micrograms/ml. In control experiments, bone marrow cells were preincubated with antibodies non-reactive with Ag-Eb under the same conditions. It is concluded that the decrease of natural suppressor activity after incubation of bone marrow cells with MAE-15 monoclonal antibody is specific for anti-Ag-Eb antibodies.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Antigen-Presenting Cells; Bone Marrow; Bone Marrow Cells; Cell Adhesion; Cell Division; Concanavalin A; Dose-Response Relationship, Immunologic; Erythroblasts; Immune Tolerance; Killer Cells, Natural; Mast-Cell Sarcoma; Mice; Mice, Inbred C57BL; Spleen

We investigated the efficacy of IL-2, LPS, MDP, TRA, ionomycin and contrykal on proliferation of lymphocytes treated by tumor cell immunosuppressive factors (ISF). IL-2, LPS and/or MDP did not abolish the influence of P815 and B16 ISF on Con A or alloantigen-induced lymphocyte proliferation. TPA and in less extent ionomycin and combination of the above preparations totally abrogated the suppression of Con A-induced lymphocyte proliferation. In inverted experiments Con A abrogated ISF-mediated suppression of lymphocyte proliferation induced by TPA plus ionomycin.

Topics: Acetylmuramyl-Alanyl-Isoglutamine; Animals; Aprotinin; Concanavalin A; Immune Tolerance; Interleukin-2; Ionomycin; Lipopolysaccharides; Lymphocyte Activation; Mast-Cell Sarcoma; Melanoma, Experimental; Mice; Sarcoma, Experimental; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Natural polyamines (putrescine, spermidine, and spermine) are ubiquitous cellular cations that play an important role in cell proliferation and differentiation. Ornithine decarboxylase is the first and a rate-limiting enzyme in the biosynthesis of polyamines. Polyamine depletion using DL-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, has been shown to suppress cell growth in a variety of settings, including those of tumor and lymphocyte proliferation. The objective of the present investigation was to examine the inhibitory effects of DFMO on a variety of murine in vitro immune responses, including lymphocyte proliferation in response to T-cell mitogen (concanavalin A), B-cell mitogen (lipopolysaccharide), and alloantigen as well as cytotoxicity. DFMO-mediated inhibition of cell proliferation in these cases correlated with depletion of intracellular polyamines. The inhibitory effects of DFMO were reversed by polyamine repletion with putrescine. Putrescine also reversed the growth-inhibitory effects of DFMO on 4 tumor cell lines that we tested: 28-13-3S, YAC-1, P-815, and K562. However, putrescine homologues exhibited a differential effect in preventing DFMO-mediated inhibition of cell growth in normal lymphocytes and cancer cell lines. Only putrescine homologues containing a shorter methylene chain were effective in preventing the growth-inhibitory action of DFMO on normal immune response. In contrast, only the longer chain homologue 1,5-diaminopentane overcame the effect of DFMO on tumor cell growth. These findings suggest that supplementation with selected polyamine homologues may sustain normal immune response in DFMO-treated individuals while effectively suppressing malignant cell growth. The potential clinical relevance of these observations is discussed.

Topics: Animals; Cell Division; Cell Line; Concanavalin A; Cytotoxicity, Immunologic; Eflornithine; Isomerism; Lymphocyte Activation; Lymphoma; Male; Mast-Cell Sarcoma; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Polyamines; Putrescine; Spermidine; Spermine

The lymphocyte signal transduction, as determined by intracellular free Ca2+ mobilization of concanavalin A-stimulated T lymphocytes and of anti-immunoglobulin mu chain antibody-stimulated B lymphocytes, was suppressed in spleen cells from mice injected with murine P1.HTR mastocytoma-induced ascites and in spleen cells treated with the ascites in vitro. The suppression was observed both at the peak level and in the reactive pattern of Ca2+ influx. In the suppression, the ascites were replaceable with tumor culture supernatants or tumor homogenates. Correspondingly, primary and secondary cytotoxic T lymphocyte (CTL) responses of DBA/2 mice to allogeneic antigen were also significantly suppressed by injection of the syngeneic P1.HTR tumor-derived ascites. This new finding suggested that the mechanism of the tumorous ascites or of the tumor-derived factor-mediated immunosuppression involves at least in part the suppression of the early event of the signal transduction for lymphocyte activation.

Topics: Animals; Ascites; B-Lymphocytes; Calcium; Concanavalin A; Immunoglobulin mu-Chains; Immunosuppression Therapy; Lymphocyte Activation; Lymphocytes; Mast-Cell Sarcoma; Mice; Mice, Inbred Strains; Neoplasms, Experimental; Signal Transduction; Suppressor Factors, Immunologic; T-Lymphocytes

Poly-L-lysine modified with mannose derivatives, the residual cationic charges of which being neutralized by N-acylation, were synthesized and used as carriers of a macrophage activator (N-acetylmuramyl dipeptide, MDP). The influence of the acylating agent on the targeting efficiency was investigated: a hydrosolubilizing group such as a gluconoyl moiety led to very efficient carrier conjugates, while an acetyl group did not. The effect of sugar and acyl content of the polymers was assessed using these compounds as inhibitors of red blood cell agglutination by Concanavalin A. The binding and specific endocytosis of poly-L-lysine substituted with several mannose derivatives and gluconoyl residues (GlcAx-, Man(y)-PLK) have been determined by a quantitative flow cytometry analysis. MDP bound to these conjugates was much more efficient in vitro than free MDP in macrophage cytostasis assays.

Topics: Acetylmuramyl-Alanyl-Isoglutamine; Animals; Cell Line; Cells, Cultured; Concanavalin A; Gluconates; Glycoproteins; Hemagglutination; Kinetics; Leukemia L1210; Macrophage Activation; Macrophages; Macrophages, Alveolar; Mannose; Mast-Cell Sarcoma; Mice; Mice, Inbred Strains; Polylysine; Rats; Rats, Inbred Strains; Tumor Cells, Cultured

Cytotoxic T lymphocyte effector cells specific for a defined class I antigen can kill target cells displaying a wide range of different class I proteins in the presence of certain lectins and oxidizing agents. However, optimal lysis of the target cell (TC) still requires interaction of the CTL with the TC class I proteins. This raises the question of how the lectin or oxidizing agent alters the system in such a way that an "inappropriate" CTL-TC interaction takes place, in a class I-dependent manner. In this study we show that if papain-sensitive molecules are cleared from the TC surface and are allowed to regenerate in the presence of tunicamycin, the cells still serve as targets in direct, class I antigen-specific CTL killing, but not in LDCC or ODCC. Target cells treated in this way display N-linked carbohydrate-less class I proteins, and presumably other N-linked carbohydrate-less, papain-sensitive molecules as well. We present data showing that both types of molecules are important in nonspecific lytic reactions.

Topics: Animals; Antigens, Surface; Cell Line; Concanavalin A; Cytotoxicity, Immunologic; H-2 Antigens; Mast-Cell Sarcoma; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Neuraminidase; Papain; Receptors, Concanavalin A; T-Lymphocytes, Cytotoxic

Plasminogen activator (PA) production from mouse mastocytoma P815 was investigated. A trace level of PA was detected in the culture supernatant of P815, but addition of Con A (5 micrograms/ml) stimulated the PA production to the 20- to 50-fold of that of the unstimulated culture. Time course studies suggest that PA is actively synthesized and secreted during the culture period with Con A. Furthermore, pretreatment of P815 with sodium butyrate, which is known to differentiate interleukin-3-dependent bone marrow mast cells or P815 cells, increased the PA production from P815 cells in the presence or absence of Con A.

Topics: Animals; Butyrates; Butyric Acid; Cell Line; Concanavalin A; Graft Rejection; Histamine Release; Mast-Cell Sarcoma; Mice; Plasminogen Activators

We have studied early transmembrane events in mouse alloimmune cytotoxic T-lymphocyte (LC7, H-2b) activation by specific target cells (mouse mastocytoma P815, H-2d) and a mitogenic lectin, Con A, by using stopped-flow fluorometry with three different fluorescent probes. After binding to target cells (P815), cytotoxic T lymphocytes (LC7) first increased their membrane fluidity and, then, calcium was released from intracellular stores. After that, there was a calcium influx from the external medium into the T lymphocytes. This calcium influx was blocked by calcium antagonists (verapamil or diltiazem). The same sequence of events was also observed in the activation of T lymphocytes (LC7) by Con A and in the response of specific target cells (P815) after cytotoxic T lymphocytes (LC7) binding. Nonspecific (syngeneic) target cells (mouse lymphoma EL-4, H-2b) did not cause any early transmembrane events in cytotoxic T lymphocytes (LC7, H-2b).

Topics: Animals; Biological Transport; Calcium; Cell Line; Concanavalin A; Cytotoxicity Tests, Immunologic; Diltiazem; Fluorescent Dyes; Fluorometry; Lymphocyte Activation; Lymphoma; Mast-Cell Sarcoma; Membrane Fluidity; Mice; T-Lymphocytes, Cytotoxic; Verapamil

Two-dimensional gel electrophoresis with the 125I-Con A overlay and affinity purification with Con A-agarose revealed the presence of an abundant ubiquitous 100-kDa glycoprotein (GP100) in nucleated mammalin cells. The amount in cultured human and murine cells varies from 3 to 20 X 10(6) molecules per cell making GP100 the most abundant glycoprotein in nucleated cells. Peptide mapping shows that it is different from erythrocyte Band III protein. Several properties of GP100 suggest that it could play a structural role in nucleated cell membranes.

Topics: Animals; Anion Exchange Protein 1, Erythrocyte; Cell Line; Cell Nucleus; Cells; Chromatography, Affinity; Concanavalin A; Glycoproteins; HeLa Cells; Humans; Isoelectric Point; Mast-Cell Sarcoma; Mice; Molecular Weight; Multiple Myeloma

A monoclonal antibody, AN-18.17.24, specific for murine interferon-gamma (IFN-gamma) was produced by immunizing Wistar rats with IFN-gamma secreted by a T-cell lymphoma, L12-R4, upon stimulation with phorbol myristic acetate (PMA). Antiviral activity as well as tumoricidal activation induced by PMA-stimulated L12-R4 cell supernatant or by Con A-stimulated normal spleen cells were neutralized at the same extent by AN-18 monoclonal antibody. Moreover, depletion experiments showed that inhibition of tumoricidal macrophage activation must be ascribed to the direct binding of the IFN-gamma molecule by AN-18 MAb and not to the interference of the monoclonal antibody with the cell surface IFN-gamma receptor. These studies conclusively demonstrate that in supernatants of T lymphocytes stimulated with polyclonal activators IFN-gamma was the only molecule responsible for macrophage activation in tumor cell killing.

Topics: Animals; Antibodies, Monoclonal; Cell Line; Concanavalin A; Cytotoxicity, Immunologic; Female; Interferon-gamma; Lymphoma; Macrophage Activation; Mast-Cell Sarcoma; Mice; Rats; T-Lymphocytes; Tetradecanoylphorbol Acetate

Plastic-adherent cells enriched for dendritic cells (AC) were found to be among the most potent stimulator cells for the activation of cytotoxic T lymphocytes (CTL) in vitro in the presence of interleukin 2 (IL 2) and a constant second set of allogeneic stimulator cells. Concanavalin A-activated nylon wool-nonadherent spleen cells ( CNWT ), concanavalin A-activated unfractionated spleen cells ( Cspl ), and some variants of the ESb T lymphoma line were equally effective as stimulator cells, however, and provoked a substantial cytotoxic response at concentrations of 10(4) cells per culture or less. In contrast, nonactivated nylon wool-nonadherent spleen cells ( NWT ) or unfractionated spleen cells (Spl) and cells of the P815 mastocytoma, the Meth A fibrosarcoma, and the T cell lymphomas Ly 5178 Eb and ESb did not stimulate cytotoxic responses at these cell concentrations. The strong stimulatory potential of the Cspl preparation was reduced by treatment with anti-Thy-1 antibody plus complement, whereas the stimulatory activity of the AC preparation was resistant to this treatment. All cell types tested expressed class I major histocompatibility antigens. Nonactivated NWT cells, in contrast to the CNWT preparation, showed no detectable staining with anti-I-E or anti-I-A antibodies and also a slightly weaker staining with class I antisera. Experiments with the tumor cell lines revealed, however, that there was no strict correlation between stimulatory potential and density of class I alloantigens or the expression of I-E determinants. Experiments on primary cytotoxic responses in vivo gave similar results. Experiments in cultures with a single set of stimulator cells and I region-compatible responder cells indicated that AC and Cspl or CNWT also have a markedly stronger capacity than NWT to induce IL 2-dependent DNA synthesis.

Topics: Animals; Antilymphocyte Serum; Cell Line; Complement System Proteins; Concanavalin A; Cytotoxicity, Immunologic; Histocompatibility Antigens; Interleukin-2; Lymphocyte Activation; Lymphocyte Cooperation; Male; Mast-Cell Sarcoma; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred DBA; Spleen; Stem Cells; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Regulatory

The effect of diethylstilbestrol, a synthetic estrogen, on mast cell secretion was investigated. The results showed that 50 microM diethylstilbestrol inhibited histamine release from rat peritoneal mast cells in the presence and absence of glucose, but did not affect 45Ca uptake stimulated by concanavalin A. Diethylstilbestrol also inhibited histamine release induced by compound 48/80, exogenous ATP, or ionophore A23187. Since estradiol benzoate, hexestrol and daidzein were not inhibitory, the inhibitory action of diethylstilbestrol must be independent of its estrogenic activity. The ATP content of mast cells decreased to less than 0.1 nmol/10(6) cells on treatment with 50 microM diethylstilbestrol at 37 degrees C for 15 min. This effect of diethylstilbestrol in decreasing the ATP content of mast cells correlated well with its inhibitory effect on histamine release. Diethylstilbestrol at 50 microM depleted the cells of ATP at 37 degrees C, but not at 0 degrees C, whereas [3H]diethylstilbestrol ( [monoethyl-3H]diethylstilbestrol) binding to rat mast cells was the same at 0 and 37 degrees C. It is concluded that diethylstilbestrol reduced the ATP content of rat mast cells by inhibiting metabolism of the cells, and consequently inhibited degranulation.

Topics: Adenosine Triphosphate; Animals; Calcimycin; Cell Line; Concanavalin A; Diethylstilbestrol; Histamine Release; Mast Cells; Mast-Cell Sarcoma; Mice; p-Methoxy-N-methylphenethylamine; Rats

Mice injected repeatedly with concanavalin A (Con A) prior to and following challenge with P 815 mastocytoma are suppressed in their cell-mediated cytotoxicity responses. Earlier studies showed that pretreatment of the animals with silica to affect macrophage (M phi) functions reversed the Con A suppression. In the present paper we have shown that peritoneal exudate cells (PEC) induced/activated by ip injection of Con A were able to transfer suppression to normal mice. Separation of the PEC populations into adherent and nonadherent cells abrogated their capacity to transfer suppression. It was further shown that Con A is not functioning in this in vivo system to block effector activity of cytotoxic cells on target cells, and PEC induced with Con A were not directly cytotoxic to target P 815 cells. Finally, we were able to show that the cytotoxicity response of Con A-suppressed mice could be enhanced by treatment with concentrated culture supernatants of normal mouse spleen cells, rich in interleukin 2 (IL 2) activity. Attempts to detect a recently described mouse serum inhibitor of IL 2 in normal or Con A-treated mice were unsuccessful and spleen cells from Con A-treated mice lost their capacity to generate IL 2 in vitro when cultured under appropriate conditions. Taken together, these results suggest that suppression of cell-mediated immune responses in Con A-treated mice results from interruption of the normal generation of IL 2 helper effects necessary for the activation of cytotoxic effector T cells in vivo.

Topics: Animals; Ascitic Fluid; Concanavalin A; Cytotoxicity, Immunologic; Immunization, Passive; Immunosuppressive Agents; Interleukin-2; Lymphokines; Macrophage Activation; Macrophages; Male; Mast-Cell Sarcoma; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Spleen

Highly purified murine lymph node T cells were used to test the hypothesis that polyclonal T cell activation requires the recognition of mitogen-modified major histocompatibility complex (MHC) antigens on accessory cells (AC) by the T cells. A variety of tumor cells lines, including macrophage, B and mast cell tumors, as well as thymomas, were shown to function as AC in concanavalin A-induced T cell activation, even if they expressed only one class of MHC antigens or none at all. In contrast to antigen-specific responses, where the Lyt-2+ phenotype is reportedly associated with recognition of class I MHC antigens, T cells enriched for or depleted of Lyt-2+ cells were not preferentially activated in the presence of class I- or class II-positive AC, respectively. In addition, as shown by others in the guinea pig and in the rat systems, T cell proliferation induced by oxidation of cell surface sugars is equally effective if T cells or AC are oxidized. T cell mitogens, therefore, do not seem to act by altering MHC antigens on AC, but rather by providing T cell-AC contact via their agglutinating properties.

Topics: Animals; B-Lymphocytes; Cell Communication; Cell Line; Concanavalin A; Female; Galactose Oxidase; H-2 Antigens; Histocompatibility Antigens; Lymphocyte Activation; Macrophages; Mast-Cell Sarcoma; Mice; Mice, Inbred BALB C; Neuraminidase; Phenotype; T-Lymphocytes

C57Bl/6 (B6) mice allogeneic to the P 815 mastocytoma tumor cell line when treated with concanavalin A prior to and at frequent intervals following challenge intraperitoneally with 10(7) tumor cells showed a significant suppression of their cell-mediated immune response at 9-10 days when compared with untreated animals. Suppression of the immune response of mice syngeneic (DBA/2) or hybrid (BDF1) to the tumor was also evidenced by increased mortality rates in concanavalin A-treated animals. The suppression of cell-mediated cytotoxicity observed in B6 mice treated with concanavalin A could be reversed by pretreatment with 20 mg silica injected intraperitoneally 7 days prior to challenge. These results suggest that macrophages play a significant role in the concanavalin A-induced immune suppression observed in this in vivo tumor-host system.

Topics: Animals; Concanavalin A; Cytotoxicity, Immunologic; Dose-Response Relationship, Drug; Immunity, Cellular; Male; Mast-Cell Sarcoma; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Silicon Dioxide

The growth of the P815 mastocytoma in syngeneic DBA/2 mice led to an activation of Ly1+2- T cells. These T cells produced a soluble factor or factors in culture which, when added to normal spleen cells or B cells in the presence of syngeneic Ly1 cells, caused a genetically unrestricted augmentation of the plaque-forming response toward sheep red blood cells (SRBC). The culture supernatant of the activated T cells did not support the proliferation of an interleukin-2 (IL-2)-dependent cell, nor exhibit properties of late-acting TRF. Active supernatants appeared to affect directly B cells during the first 48 hr of culture with SRBC in such a way as to make them more responsive to antigen-specific Ly1-cell help.

Topics: Animals; Antibody-Producing Cells; B-Lymphocytes; Blood Proteins; Concanavalin A; Hemolytic Plaque Technique; Interleukin-2; Interleukin-5; Lymphocyte Activation; Lymphocyte Cooperation; Lymphokines; Male; Mast-Cell Sarcoma; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Nude; Neoplasms, Experimental; Rats; Rats, Inbred Strains; T-Lymphocytes

Culture supernatants from mitogen-stimulated splenocytes were found to stimulate protease production in P815Y mastocytoma cells. Such supernatants increased cell-associated plasminogen activator levels in a dose-dependent fashion, and under serum-free conditions. In contrast to peritoneal exudate cells, tumor-cell plasminogen activator was not enhanced by the mitogen ConA alone. The tumor cell line P815Y may, thus, be used as a homogeneous cell source for the quantitation of lymphocyte factors which activate or inhibit plasminogen activator activity.

Topics: Animals; Cell Line; Concanavalin A; Dose-Response Relationship, Immunologic; Lymphokines; Mast-Cell Sarcoma; Mice; Mice, Inbred BALB C; Plasminogen Activators

The combination of two-dimensional gel electrophoresis and post-electrophoretic staining with 125I-labelled concanavalin A was used to compare the glycoproteins of murine tumour cell lines. Comparison between different cell lines showed that there were about eight common glycoproteins. The rest of the glycoproteins were generally unique to particular cells. Thus the P815 cell could be distinguished from 13 other murine cell lines by its glycoprotein pattern. The specific glycoproteins of each cell line were unaffected by culture in vivo, virus infection or hybridisation. Different clones from the same cell line gave identical patterns. Crude membrane preparations and glycoproteins purified from cell lysates by affinity chromatography on concanavalin/agarose gave the same patterns as whole cells. Thus the glycoproteins of murine tumour cells appear to be a stable characteristic which can provide specific markers for the identification of tumour cell lines.

Topics: Animals; Cell Line; Concanavalin A; Electrophoresis; Glycoproteins; Iodine Radioisotopes; Isotope Labeling; Mast-Cell Sarcoma; Mice; Neoplasm Proteins; Neoplasms, Experimental; Thymoma

Activation of spleen cell cultures by T-cell mitogens appeared enhanced or suppressed by the presence of some nonsteroid anti-inflammatory drugs such as indomethacin and indomethacin esters in a dose-dependent fashion. However blastogenesis of B cells by bacterial lipopolysaccharide was inhibited by the same drugs at every concentration tested. In vivo treatment of mice with indomethacin led to an inhibition of both T and B lymphocyte responses to mitogens. Addition of indomethacin to Concanavalin A-stimulated spleen cells from indomethacin-treated mice further inhibited the response to the mitogen. Indomethacin and indomethacin esters induced only a slight inhibition of cell-mediated cytotoxic reactions when added to cultures, whereas these reactions were markedly inhibited in spleen cells from indomethacin-treated mice.

Topics: Animals; B-Lymphocytes; Concanavalin A; Cytotoxicity, Immunologic; Esters; In Vitro Techniques; Indomethacin; Lipopolysaccharides; Lymphocyte Activation; Male; Mast-Cell Sarcoma; Mice; Mice, Inbred Strains; Prostaglandins; Sarcoma, Experimental; T-Lymphocytes

Concanavalin A (Con A)-activated cytotoxic lymphocytes have been investigated, mapping the genetic differences between the P815 target and the effector cells required for cell-mediated lympholysis to occur. The target antigens recognized during the effector phase and the phenotype of the killer cell population(s) were also determined. It was found that Con A could activate a population of primed cytotoxic lymphocytes capable of killing target cells that were identical at the major histocompatibility complex (MHC) but differed at other background genes. Thus, after in vivo priming with DBA/2, B10.D2 lymphocytes cultured with Con A were capable of killing the P815 target. Unprimed B10.D2 cells, however, would not. Studies on the involvement of the MHC indicated that differences in the H-2K through H-2S, as well as differences in H-2D and H-2L alone could cause lysis. This killing could not be accounted for by additional differences at Qa-2, a MHC-linked locus. However, the contribution of other similar non-MHC linked loci could not be excluded. Cold target competition experiments indicated that MHC encoded alloantigens were involved as recognition structures on the target cell surface. Antisera plus complement depletion of cytotoxic effector function demonstrated that the cytotoxic cells had the cell surface phenotypes Thy 1.2+, Lyt 2.2+ and natural killer (NK) 1.1-. We conclude that Con A polyclonally activates population(s) of T cells that express antigen-specific cytotoxicity through clonally distributed recognition receptors intrinsic to their membranes when lectin is omitted from the cytotoxic assay.

Topics: Animals; Concanavalin A; Cytotoxicity, Immunologic; Lymphocyte Activation; Lymphocytes; Major Histocompatibility Complex; Mast-Cell Sarcoma; Mice; Mice, Inbred BALB C; Mice, Inbred DBA; Phenotype; Sarcoma, Experimental; Spleen

Topics: Animals; Antigens, Surface; Cell Line; Cell Survival; Concanavalin A; Edetic Acid; Lymphoma; Mast-Cell Sarcoma; Mice; Mice, Inbred Strains; Spleen; T-Lymphocytes; Time Factors

Topics: Animals; Cell Adhesion; Cell Line; Concanavalin A; Cycloheximide; Cytotoxicity, Immunologic; H-2 Antigens; Major Histocompatibility Complex; Mast-Cell Sarcoma; Mice; Protein Biosynthesis; Sarcoma, Experimental; T-Lymphocytes

Topics: Animals; Cell Division; Concanavalin A; Cytotoxicity, Immunologic; Epitopes; In Vitro Techniques; Lymphoma; Male; Mast-Cell Sarcoma; Mice; Mice, Inbred Strains; Neoplasm Transplantation; Neoplasms, Experimental; Protein Biosynthesis; T-Lymphocytes; Transplantation, Homologous

Topics: Animals; Antigens; B-Lymphocytes; Cells, Cultured; Chromatography, Gel; Chromatography, Ion Exchange; Concanavalin A; Isoelectric Focusing; Mast-Cell Sarcoma; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Nude; Spleen; T-Lymphocytes

Topics: Adult; Aged; Aging; Animals; B-Lymphocytes; Concanavalin A; Cytotoxicity, Immunologic; Humans; Lymphocyte Activation; Mast-Cell Sarcoma; Mice; Mice, Inbred DBA; Middle Aged; Phytohemagglutinins; T-Lymphocytes

The average frequency of cytotoxic precursor cells in DBA/2 lymph node cell preparations reactive to the syngeneic tumour P815 has been determined as 1 in 2000. This frequency is similar to the precursor frequency for an allogeneic tumour EL-4. The normal lack of response of DBA/2 lymph node cells to the syngeneic tumour P815 in vitro cannot be attributed to a lack of cytotoxic precursor cells. We conclude that in this tumour-host system non-immunogenicity reflects a defect at the inductive step.

Topics: Animals; Cell Line; Cells, Cultured; Concanavalin A; Cytotoxicity, Immunologic; Hematopoietic Stem Cells; Lymph Nodes; Lymphocyte Activation; Lymphocytes; Male; Mast-Cell Sarcoma; Mice; Mice, Inbred DBA; Neoplasm Transplantation; Neoplasms, Experimental; Sarcoma, Experimental; Transplantation, Isogeneic

Topics: Animals; Antibodies; Concanavalin A; Cytotoxicity, Immunologic; Female; H-2 Antigens; Immune Sera; Immunologic Capping; Lymphoma; Male; Mast-Cell Sarcoma; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred DBA; Parainfluenza Virus 1, Human; T-Lymphocytes

Primary cytotoxic responses of DBA/2 lymph node cells to a syngeneic tumour (the mastocytoma P815) have been generated in vitro. The development of these responses is dependent on the addition of a soluble factor (CSCS) which is produced by concanavalin A-activated spleen cells. The response is mediated by T lymphocytes, can be detected at low effector to target cell ratios and is directed against P815 tumour-associated antigens.

Topics: Animals; Concanavalin A; Cytotoxicity, Immunologic; In Vitro Techniques; Lymph Nodes; Mast-Cell Sarcoma; Mice; Mice, Inbred DBA; Sarcoma, Experimental; Spleen; T-Lymphocytes

Lymphokine-containing supernatants derived from seven different human lymphoid cell lines and lymphokine-containing supernatants from concanavalin A-stimulated murine lymphocytes were found to be capable of reversibly inhibiting the migration of tumor cells in vitro. The tumor cell lines used in these studies were the P815 mastocytoma, Ehrlich ascites, Walker carcinosarcoma, Hepatoma 129, and Sarcoma 37. Preliminary physiochemical evidence suggests that the mediator, here termed TMIF, is distinct from MIF. In any case, these results suggest the possibility that lymphokines other than lymphotoxin or macrophage-activating factors may play a role in tumor immunity.

Topics: Animals; Cell Line; Cell Migration Inhibition; Chromatography, Gel; Concanavalin A; Humans; Lymphokines; Mast-Cell Sarcoma; Mice; Rats; Spleen; Ultrafiltration

Topics: Animals; Cell-Free System; Concanavalin A; Cytotoxicity Tests, Immunologic; Immunosuppression Therapy; Lectins; Lipopolysaccharides; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; Male; Mast-Cell Sarcoma; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Spleen

The response of normal murine bone marrow and mastocytoma cells to the cytotoxic effect of the four lectins WGA, PHA, SBA and Con A was studied. It was found that the two types of cells had similar sensitivities to each of the four lectins, as judged by decreased ability to develop colony growth in soft agar. They differed, however, in their sensitivity to particular lectins. The cells were most sensitive to WGA, less sensitive to PHA and SBA, and least sensitive to Con A. In further experiments, carried out on bone marrow cells, it was found that pluripotent stem cells (CFU-S) and committed stem cells (CFU-C) were equally sensitive to the cytotoxic effect of WGA. Incubation of bone marrow cells with 12 mug/ml WGA at 37 degrees C for 30 minutes had no effect on CFU-C; incubation for 60 minutes produced a 70% decrease in CFU-C. Additional periods of incubation up to 120 minutes did not further affect CFU-C, suggesting that some particular phase of the cell cycle may be sensitive to the cytotoxic effect of lectins.

Topics: Animals; Cell Division; Cell Line; Concanavalin A; Lectins; Male; Mast-Cell Sarcoma; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred ICR

The cellular basis of the cross-priming observed with minor histocompatibility antigens on H-2 different cells was investigated. Cytotoxic T cells induced against minor alloantigens show absolute H-2 restriction at the effector level ([51Cr]-release). That is, F1 (BALB/c X BALB.B) (H-2d/b) cytotoxic cells induced by immunization with B10(H-2b) cells are not able to lyse B10.D2(H-2d) targets. But an injection of B10 cells does prime F1 mice for a secondary cytotoxic response to B10.D2. The technique of inducing cytotoxic effector function polyclonally with Con A in the absence of alloantigen was used here to establish that such cross-priming reflects what happens at the cytotoxic cell level. It is shown that an F1 animal previously injected with B10 cells has expanded pools of memory cytotoxic cells reactive with B10 and B10.D2. From this it is concluded that: a) minor H structures on B10.D2 and B10 do cross-react at the cytotoxic T cell level during in vivo priming, and b) because normal cells cross-prime whereas tumor cells do not, then the F1 cytotoxic precursors are probably committed to respond to antigen on cells bearing either the maternal or paternal H-2 haplotype before they encounter antigen. Cross-priming may be explained by foreign minor H antigens being presented to F1 host T cells on the surface of host macrophages. Therefore priming is not restricted to the H-2 type of the injected cells but to both H-2 types of the F1 host.

Topics: Animals; Concanavalin A; Cross Reactions; Cytotoxicity Tests, Immunologic; Female; Histocompatibility Antigens; Immunity, Cellular; Immunologic Memory; Lymphocyte Activation; Mast-Cell Sarcoma; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred DBA; Spleen; T-Lymphocytes

Treatment of mouse spleen cells with periodate (NaIO4) or with neuraminidase and galactose oxidase (NAGO) induces blastogenesis and renders the cells cytotoxic to mastocytoma (P815) target cells. Treatment of target cells (P815 cells and turkey erythrocytes) with NaIO4 or with NAGO renders them susceptible to cytolysis by untreated mouse spleen cells. The cytotoxicity induced by NaIO4 is reduced upon reacting the NaIO4-treated, effector or target cells with borohydride or hydroxylamine. Thus the formation of free surface aldehydes on either the effector or target cell induced a cytotoxic effect. It is postulated that cross-linkage via a Schiff base between effector and target cell initiates the cytotoxic effect. Cytotoxicity induced by NaIO4 or NAGO is immunologically nonspecific and is independent of major antigenic differences between effector and target cells. Phagocytic cells are not involved in NaIO4-or NAGO-induced cytotoxicity toward P815 target cells.

Topics: Alcohol Oxidoreductases; Animals; Borohydrides; Cell Separation; Chromium Radioisotopes; Concanavalin A; Cytotoxicity Tests, Immunologic; Galactose; Hydroxylamines; Lymphocytes; Mast-Cell Sarcoma; Mice; Mice, Inbred DBA; Neuraminidase; Periodic Acid; Phagocytosis; Spleen; Thymidine; Tritium

Topics: Animals; Antibody Specificity; Binding Sites; Cell Membrane; Cells, Cultured; Concanavalin A; Cytotoxicity Tests, Immunologic; Dose-Response Relationship, Drug; Erythrocytes; Female; Fluorescent Antibody Technique; Idoxuridine; Immunity, Cellular; Iodine Radioisotopes; Leukemia, Experimental; Lipopolysaccharides; Lymphocytes; Male; Mast-Cell Sarcoma; Mice; Moloney murine leukemia virus; Spleen

The effects of soluble concanavalin A (Con A) or Con A-activated spleen cells on the generation of cytotoxic lymphocytes (CL) in mixed leukocyte cultures (MLC) were examined. Mitogenic concentrations of soluble Con A or small numbers of Con A-activated spleen cells substantially inhibited CL responses. The suppression was partial rather than absolute and was critically dependent upon the concentration and time of addition of soluble Con A or Con A-activated spleen cells to the MLC. Suppressive effects of Con-A activated spleen cells were mediated by T cells since suppressor cell activity was abrogated by treatment of spleen cells with anti-theta serum and complement before or after Con A activation. X irradiation of spleen cells before Con A treatment also abrogated generation of suppressor cell activity. After activation by Con A, however, the function of suppressor cells was radioresistant. Although the precise mechanism(s) of suppression is, as yet, unknown, the precursors of CL must be exposed to Con A-activated cells during the early phases of the immune response for suppression to occur. Kinetic studies revealed that suppression of CL responses was not due to a failure to initiate an immune response, but represented a response which developed initially, but subsequently aborted. The relevance of these observations to the concepts of T-cell-T-cell interaction and regulatory control of immune responses by T cells is discussed.

Topics: Animals; Antigen-Antibody Reactions; Antilymphocyte Serum; Cell Line; Chromium Radioisotopes; Concanavalin A; Cytotoxicity Tests, Immunologic; Female; Immunity, Cellular; Immunosuppression Therapy; Leukemia, Experimental; Lymphocyte Culture Test, Mixed; Mast-Cell Sarcoma; Mice; Mice, Inbred Strains; Mitomycins; Neoplasm Transplantation; Neoplasms, Experimental; Radiation Effects; Spleen; T-Lymphocytes

Topics: Animals; Cell Line; Chromium Radioisotopes; Concanavalin A; Cytotoxicity Tests, Immunologic; Female; Lectins; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; Lymphocyte Depletion; Male; Mast-Cell Sarcoma; Mice; Mice, Inbred CBA; Mice, Inbred DBA; Spleen; T-Lymphocytes; Thymectomy; Thymus Gland

Topics: Animals; Antilymphocyte Serum; Cell Count; Cells, Cultured; Chromium Radioisotopes; Concanavalin A; Cytotoxicity Tests, Immunologic; DNA; Female; Lectins; Lipopolysaccharides; Lymphocyte Activation; Mast-Cell Sarcoma; Mice; Mitogens; Plant Extracts; Polysaccharides, Bacterial; Spleen; T-Lymphocytes; Thymidine; Thymus Gland; Tritium

Topics: Animals; Antilymphocyte Serum; Cells, Cultured; Chromium Isotopes; Concanavalin A; Lectins; Lymphocyte Culture Test, Mixed; Mast-Cell Sarcoma; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Perissodactyla; Spleen; T-Lymphocytes; Thymidine; Tritium

Topics: Agglutination Tests; Animals; Cell Count; Cell Division; Cell Line; Concanavalin A; Cyclic AMP; Epinephrine; Histamine; In Vitro Techniques; Isoproterenol; Mast-Cell Sarcoma; Mice; Mitosis; Neoplasm Transplantation; Radioimmunoassay; Serotonin; Theophylline; Thymidine; Tritium

Topics: Cell Adhesion; Cell Line; Cells, Cultured; Concanavalin A; Cycloheximide; Dactinomycin; Edetic Acid; Galactose; Glass; Glycosides; Humans; Lectins; Leukemia, Myeloid; Lymphoma; Mannose; Mast-Cell Sarcoma; Microscopy, Phase-Contrast; Plant Lectins; Temperature; Trypsin; Vegetables