Staphylococcal protein (A (PA) and IgG anti-Forssman immunoglobulin formed complexes that behaved functionally like IgM in their ability to lyse sheep erythrocytes (E) in the presence of whole guinea pig complement (GPC) and to fix purified guinea pig C1. Concanavalin A, a plant lectin that inhibited IgM but not IgG hemolytic activity, inhibited the hemolytic activity of IgG-protein A complexes that behaved like IgM but had no effect on complexes that behaved functionally like IgG. Since Con A is known to bind specifically to glucose and mannose residues, our results suggested that the interaction of protein A with the Fc region of IgG led to exposure of sugar moieties that may participate in complement (C) binding. The production of IgM-like complexes depended on the ratio of protein A to IgG and the empirical formula of these IgM-like complexes was found to be [(IgG)2PA]n. As the ratio of PA to IgG was increased, the resulting complexes tended to behave functionally like IgG but with reduced hemolytic activity and C1 fixing ability. Furthermore, the binding of C1 to EIgG was inhibited by PA and the binding of PA to EIgG was inhibited by C1 indicating that the binding sites for C1 and PA were located near each other or were identical. Our results offer a reasonable explanation for the reported effects of PA or mixtures of PA and IgG in vitro and in vivo.

Animals↗

PubMed1978

Studies on the terminal stages of immune hemolysis. III. Distinction between the insertion of C9 and the formation of a transmembrane channel.

The intermediate product EAC1-8 released cytoplasmic components as a result of at least two sequential reactions after its interaction with C9. Binding of C9 to EAC1-8 occurred in a few minutes even at 0 degrees C. Trypsinization of EAC1-9 prepared and held at low temperature resulted in nullification of the potential hemolysis of these cells. A brief incubation at 30 or 37 degrees resulted in the formation of an intermediate whose hemolytic potential could not be nullified by trypsin. The failure of trypsin to nullify hemolysis was attributed to the insertion of C9 into the cell membrane. Studies on the effec of EDTA or low temperature suggested that the reported temperature-dependent step in E* formation described by Frank et al. was the insertion of C9. The results of the studies with 86Rb-labeled EAC1-8 indicated that a transmembrane channel was not formed until after the C9 had been inserted and a further reaction or reactions had occurred.

Animals↗

PubMed1977

Quantitation of antibody-complement mediated lysis of tumor cells.

An assay for quantitating antibody-complement mediated killing based on the release of 125I from 125IUdR labelled target cell is described. The temporal delay between antibody--complement damage and the release of nuclear material was shortened by treatment of the cells with a combination of trypsin and DNase. This treatment increased the rate of release of the labelled nuclear material from damaged cells without causing labelled nuclear material to be released from undamaged cells. The low level of spontaneous release of 125I from the target cells allows this assay to be used for experiments carried out over long time periods or in experiments involving extensive manipulations of the cells.

Animals↗

PubMed1977

125I protein A: applications to the quantitative determination of fluid phase and cell-bound IgG.

An improved method for the preparation of 125I-labelled Protein A (125I PA) of high specific and functional activity is described. 125I PA has been used in combination with purified rabbit IgG bound to a solid support to develop a competitive binding assay capable of detecting Protein A or human, rabbit and guinea pig IgG at the nanogram level. An optimal set of assay conditions was established and levels of IgG measured in normal human, rabbit and strain-2 guinea pig serum. 125I PA has also been used to detect IgG anti-Forssman antibody bound to sheep erythrocytes and to line-1 and line-10 tumor cells and as an indirect assay for tumor associated antigen in the ascitic fluid of tumor-bearing guinea pigs.

Animals↗

PubMed1977

Effect of concanavalin A on the functional activity of hemolytic antibody.

Concanavalin A (Con A), either in solution or insolubilized by covalent binding to Sepharose 4B, can inhibit the ability of fluid phase 19S, but not 7S, anti-Forssman antibody to sensitize sheep red cells (E) toward lysis by excess guinea pig complement. The efficiency of 19S antibody is unaffected when E are treated with Con A before sensitization or when antibody sensitized cells (EA) are exposed to the lectin before complement is added. Although whole complement activity is retained on a solumn of Con A-Sepharose, cell bound lectin did not act as a complement fixing antibody. Consistent with this result, there was no difference in the amount of C1 fixed by E and E-Con A, or by EA and EA-Con A.

Animals↗

PubMed1977

Effect of inhibiting DNA, RNA, and protein synthesis of tumor cells on their susceptibility to killing by antibody and complement.

A number of metabolic inhibitors and chemotherapeutic agents have been found to increase the sensitivity of a chemically induced guinea pig hepatoma (line 1) to killing by antibody and complement. We have investigated whether the mechanism whereby these drugs increase sensitivity to killing is attributable to their primary action of inhibiting DNA, RNA, or protein synthesis. Line 1 cells incubated for 1, 4, or 17 hr with actinomycin D (25 microng/ml), adriamycin (40 microng/ml), or puromycin (5 micron/ml) or with 5-fold lower concentrations of these drugs were maximally inhibited (greater than 90%) in their ability to synthesize DNA, RNA, and protein within 1 hr. However, only cells incubated for 17 hr with the high concentrations of drugs showed increased sensitivity to killing by antibody and complement. Line 1 cells incubated with high concentrations of these drugs of 17 hr, washed, and resuspended in drug-free medium recovered their resistance to killing by antibody and complement within 4 hr. These cells ever after culture for 24 hr in drug-free medium did not regain their ability to synthesize DNA, RNA, or protein. A similar lack of correlation between synthesis of these macromolecules and sensitivity to antibody-complement-mediated killing was observed after the cells were treated with physical agents that inhibit macromolecular synthesis. Both heat-treated and X-irradiated cells were inhibited in their ability to synthesize DNA, RNA, and protein immediately after treatment; however, only X-irradiated cells (6 and 16 hr postirradiation) were increased in their sensitivity to antibody-complement-mediated killing. Our data show that the ability of line 1 tumor cells to resist humoral immune attack does not depend solely on their ability to synthesize DNA, RNA, or protein.

Antibodies↗

PubMed1977

Effect of concanavalin A on the killing of tumor cells by antibody and complement.

Concanavalin A (Con A) was found to inhibit the killing of antibody-sensitized line-1 tumor cells (TA) by guinea pig complement (GPC) but not by human complement (HuC). Other plant lectins (wheat germ, leucoagglutinin, and pokeweed mitogen) were also tested but Con A was the only lectin found to inhibit antibody-GPC-mediated killing. The inhibitory effect of Con A was observed when the GPC was mixed with Con A or when the antibody-sensitized cells were pretreated with Con A (TA-Con A) before the addition of GPC. The effect could be reversed by treatment of such cells with alpha-D-methylglucopyranoside or by incubation at 37 degrees C for approximately 2 hr. Con A appeared to act by preventing the binding of the first component of GPC (GPC1) to antibody-sensitized tumor cells. Differences in the binding of the first component of HuC (HuC1) and GPC1 to TA-Con A suggested that a difference in the binding site for HuC1 and GPC1 might exist. There was no difference in the number of GPC1 molecules fixed to antibody-sensitized sheep erythrocytes (EA) or EA treated with Con A in experiments using the same antibody as used with the tumor cells and the same Con A preparation. It would consequently appear that the inhibitory effect of Con A on the binding of GPC1 to TA is not due solely to an interaction of Con A with the antibody.

Animals↗

PubMed1977

Effect of concanavalin A on the classical complement pathway.

Lysis of sheep erythrocytes (E) sensitized with anti-Forssman antiserum (EA) is inhibited by the action of concanavalin A (Con A) on whole guinea pig complement (GPC). The degree of inhibition observed for a given quantity of GPC was dependent on the Con A concentration. Specifically, Con A inhibits the activity of the early acting complement components C1 and C2 in the fluid phase, but has no significant effect on lysis once these components are bound to EA. Results of tmax experiments performed in the presence or absence of Con A showed that inhibition of C2 activity results from a direct interaction between Con A and C2 and not from a decreased number of effective EAC14 sites. Furthermore, since Con A pretreated or untreated EAC14 cells had the same tmax value, Con A and C2 apparently do not compete for the same binding site on the indicator cells. The lectin has no observable effect on either fluid phase or cell-bound C4 activity. Under similar conditions, wheat germ or soy bean agglutinin, leucoagglutinin or pokeweed mitogen did not inhibit hemolysis.

Antibodies↗

PubMed1976

The destruction of allogeneic tumour cells by antibody and adherent cells from peritoneal cavities of mice.

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Animals↗

PubMed1976

Detection of complement-dependent antibody to tumor cells in sera of strain-2 guinea pigs cured of their tumors by BCG Treatment.

Sera of strain-2 guinea pigs (cured of line-10 tumor by BCG therapy) were tested for complement-dependent, cytotoxic antibody. About 30% of the sera tested contained significant cytotoxic acitivity with the addition of human, but not syngeneic, complement. Using papain pretreated line-10 cells, we detected antibody in about 50% of the sera with syngeneic sera as the source of complement. Antibody to line-10 was also demonstrated in selected sera by indirect fluorescence and the C1 fixation and transfer test.

Animals↗

PubMed1976

Destruction of allogeneic tumour cells by peritoneal macrophages.

The population of peritoneal macrophages from mice immunised with allogeneic tumor cells contains two types of cytotoxic cell. One lyses the specific target cell, the other initiates activation of macrophages and hence leads to inhibition of growth of the specific target cell. The yield of lytic effector macrophages was found to depend on the route and the nature of the cell used for immunisation and the condition of the mice. The yield correlated with the yield of complement-dependent cytotoxic antibodies. In contrast, production of specific "activator" macrophages did not depend critically on these factors. The results underline the difference between the two types of cell and suggest that they are produced independently of one another.

Animals↗

PubMed1976

Lysis of tumor cells by antibody and complement. VI. Enhanced killing of enzyme-pretreated tumor cells.

The ascites form of a chemically induced guinea pig hepatoma, line-10, was resistant to killing in vitro by xenogeneic antibody and guinea pig complement. Pretreatment of line-10 cells with certain proteolytic enzymes rendered tham susceptible to the killing action of antibody and guinea pig complement. The effects of enzyme pretreatment were dependent on enzyme concentration, temperature, and could be blocked by addition of competitive or non-competitive inhibitors. The effect of the enzyme treatment could reversed by incubating the treated cells at 37 degrees C (but not at 0 degrees C), in the absence of the enzyme. Effective enzymes included ficin, bromelain, pronase, elastase, papain, trypsin, collagenase, lipases type I and type VI, and the neuraminidase preparation isolated from Clostridium perfringens. The activity of the lipase preparations and the neuraminidase preparation isolated from Clostridium perfringens appeared to be caused by proteolytic enzyme contamination. Enzyme preparations that proved ineffecitve in rendering the line-10 cells sensitive to killing by antibody and guinea pig complement included DNase, RNase, beta-glucuronidase type 6A or type B10, hyaluronidase type V or type VI, and pectinesterase.

Animals↗

PubMed1976

Studies on the terminal stages of antibody-complement-mediated killing of a tumor cell. I. Evidence for the existence of an intermediate, T.

The mechanism of the terminal steps in the lysis of antibody-sensitized tumor cells by complement (TAC) was studied. It was shown that once complement has reacted, lysis proceeded even in the absence of fluid-phase complement. Transformation of TAC to dead cells was found to be at least a two-step process: one of the steps was temperature dependent whereas the other was reversibly inhibited by EDTA. In analogy to the hemolytic system, TAC has been designated T.

Animals↗

PubMed1976

Studies on the terminal stages of antibody-complement-mediated killing of a tumor cell. II. Inhibition of transformation of T to dead cells by 3'5' cAMP.

Transformation of T to dead cells was prevented by 3'5' cAMP. The effect of 3'5' cAMP was dose, time, and temperature dependent. T washed free of 3'5' cAMP after short-term incubation proceeded to die to the same extent as control cells. After 3 hr of incubation of T with 3'5' cAMP the level of killing was significantly reduced. The 3'5' cyclic nucleotides of uridine, guanine, cytosine, and thymidine and the 2'3' cyclic adenosine nucleotide were not effective. It was concluded that prolonged treatment of T with 3'5' cAMP either irreversibly blocked the damage-producing process or facilitated the reapir of damaged sites.

Animals↗