Heterogeneity of rabbit IgM antibody as detected by C'1a fixation.

The C'1a-fixing properties of purified rabbit IgM anti-benzenearsonate antibody were determined. When tested with sheep erythrocytes to which hapten had been coupled by diazo linkage, the number of C'1a molecules fixed was 21% of the number of IgM antibody molecules bound to the erythrocyte surface. This was not due to loss of C'1a-fixing capacity during the purification procedure. Preparative electrophoresis of the antibody concentrated C'1a-fixing molecules in the anodal region so that antibody fractions with greater C'1a-fixing capacity were obtained. The demonstration that C'1a fixation is a property of a subpopulation of IgM molecules provides evidence for previously unrecognized micro-chain heterogeneity.

Mouse complement: the effect of sex hormones and castration on two of the late-acting components.

The titer of late-acting complement components in sera from male mice is 8-10 times higher than the titer of sera from female mice. Using assays developed to measure the serum content of two of the late-acting components, we have shown that this difference is due to the effect of androgen and estrogen on these two late-acting complement components. These two components have been tentatively identified as C'5 and C'6. Androgen and estrogen have greater effect on C'6 than on C'5. The possibility has not been excluded that still other of the late-acting complement components are affected by androgens and estrogens. The course of homograft rejection was unchanged in mice deficient in C'5 and C'6.

Synthesis of the first component of human complement in vitro.

Isolated segments of human colon and to a lesser extent ileum were capable of synthesizing hemolytically active C'1. This conclusion was based on the following evidence: After elimination of C'1 from tissue with EDTA, we found that segments of the intestinal tract in short-term organ culture showed a 50-1000-fold increase in C'1 activity. The rate of production of C'1 in human intestine was highly temperature dependent; C'1 production was reversibly inhibited by puromycin and actinomycin D. Furthermore, (14)C-labeled amino acids were incorporated into molecules which behaved like C'1. No significant C'1(hu) synthesis was observed in isolated segments of jejunum, stomach, liver, kidney, lung, spleen, lymph node, and thymus.

Efficiency of the first component of complement (C'1) in the hemolytic reaction.

With the use of the first component of guinea pig complement (C'1) labeled in vivo with (14)C-amino acids, we have obtained evidence that, under the conditions required for the assay of C'1, each molecule of C'1 capable of interaction with cell surface antigen-antibody complexes is capable of initiating the reaction sequence that leads to lysis of the cell.

Chemotherapeutic drugs increase killing of tumor cells by antibody and complement.

When the ascitic forms of two antigenically distinct guinea pig hepatomas induced by diethylnitrosamine are treated in vitro with chemotherapeutic drugs, their sensitivity to killing by xenogeneic antibody plus guinea pig complement increases. The effect is dependent on drug dose, is reversible, and does not appear to be due to increased antigen expression or fixation of the early acting components of guinea pig complement.

Mouse complement: influence of sex hormones on its activity.

Sex hormones influence the hemolytic of one or more of the late-acting components of complement measured in the presence of trisodium ethylenediaminetetraacetate. The titers of the serums of male mice, normally tenfold higher than those of females, fell after castration, becoming about the same as those of females. The titers of the serums from females rose after these mice were castrated, but castration did not affect the activities of the first, second, and fourth components of complement. Serums of normal and castrated mice of both sexes treated with testosterone showed increased late-acting component activity, whereas the estrogen caused decreased activity. Treatment in vitro of mouse serum with these hormones had no effect on the activity of late-acting components.

Neutralization of sensitized virus by the fourth component of complement.

Herpes simplex virus which had been sensitized with IgM antibody was not neutralized by the addition of the purified activated first component of complement. In the presence of an optimum concentration of the first component of complement, however, the sensitized virus was neutralized by the addition of a high concentration of the purified fourth component of complement. Under these conditions, the addition of the purified second and third components of complement failed to enhance virus neutralization. With low concentrations of the fourth component of complement, the addition of the second and third components enhanced virus neutralization.

Tumor-specific antigens detected by inhibition of macrophage migration.

Tumor-specific antigens of a guinea pig hepatoma induced by diethylnitrosamine were detected by the inhibition of migration of specifically sensitized macrophages from capillary tubes, and by the local passive transfer of delayed skin hypersensitivity and the suppression of growth of intradermally same injected tumor.

Immunotherapy of cancer: an experimental model in syngeneic guinea pigs.

Successful treatment of a solid tumor was accomplished by repeated intradermal injection of living tumor cells.

Persistence of immunoglobulin and complement components C4 and C3 bound to guinea pig tumor cells.

Guinea pig hepatoma cells (line-10) growing as ascites were studied for the presence of immunoglobulin, C4, and C3 (components of complement) on their surfaces. Immunoglobulin, C4, and C3 content increased with length of time spent in the peritoneal cavity. The persistence of these factors bound in vivo or in vitro was also determined. Complement-fixing (CF) activity of cellbound antibody disappeared from the surface more rapidly at 37 degrees C than at 4 degrees C; the continued presence of cellbound immunoglobulin in non-complement-fixing form could be demonostrated at either temperature. No CF activity was released into the medium. C4 and C3 were released into the medium and could be demonstrated in the medium by immunochemical methods.

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.

Correlation between the ability of tumor cells to incorporate specific fatty acids and their sensitivity to killing by a specific antibody plus guinea pig complement.

Line-1 diethylnitrosamine-induced guinea pig tumor cells can be rendered sensitive to killing by rabbit anti-Forssman IgM antibody plus guinea pig complement (GPC) or antitumor antibody plus GPC following prolonged incubation (17 hr ) of the cells with one of several metabolic inhibitors. Compared to control cells, these cells have been shown to be inhibited in their ability to incorporate fatty acids into complex cellular lipids, which suggested that lipid synthesis is of fundamental importance for the ability of the tumor cells to resist humoral immune killing. In this study, drug-treated cells that were rendered sensitive to killing by anti-Forssman antibody plus GPC, but not antitumor antibody plus GPC, were inhibited in their incorporation of saturated (palmitic or stearic acid), but not an unsaturated, fatty acid (linoleic acid). These data suggested that the fatty acid composition of specific lipids may also be important for the resistance of these tumor cells to killing by antibody and complement.

Forssman antigen content of guinea pig hepatomas induced by diethylnitrosamine: a quantitative approach to the search for tumor-specific antibodies.

The Forssman antigen content of diethylnitrosamine-induced guinea pig hepatomas was found to be greater than that of either autogenous or allogeneic guinea pig liver. The autogenous normal liver was obtained from guinea pigs before tumor induction, and comparison of normal and neoplastic tissues was based on the capacity of these tissues to inhibit (absorb) the hemolytic activity of rabbit antitumor serum. A method is presented for distinguishing quantitative from qualitative antigenic differences in the search for tumor-specific antigens. The results of these experiments indicate that, in the absence of strict quantitation, quantitative differences may be mistaken for qualitative differences.

Regression of established tumors and induction of tumor immunity by intratumor chemotherapy.

The inoculation of a mixture of drugs and guinea pig hepatoma cells (line-10) induced tumor-specific immunity in about 20% of guinea pigs. When guinea pigs with established intradermal tumors were given various drugs ip, no cures were observed; in contrast, multiple intralesional injections of actinomycin D, 1,3-bis(2-chlorethyl)-1-nitrosourea, adriamycin, mitomycin C, and melphalan were effective in curing animals of their intradermal tumors at a time when there were tumor cells in the draining lymph nodes; dimethyl-triazenoimidazole carboxamide, methotrexate, 5-fluorouracil, and 6-mercaptopurine were not effective. More than 80% of the cured animals were immune to rechallenge with 10(6) line-10 tumor cells.

Plasma therapy of primary rat mammary carcinoma: dependence of consumption of C3 during absorption of plasma with sepharose derivatives on the anticoagulant.

A previous study demonstrated inhibition of growth of primary rat mammary carcinomas after infusion of tumor-bearer plasma absorbed against Sepharose derivatives. In this report we have quantitated changes in individual complement components that occur during absorption of rat plasma with Sepharose derivatives and defined optimal conditions for consumption of the third component of complement (C3) (other complement components defined similarly). The concentration of functionally active C1 to C9 was measured before and after absorption in plasmas from both normal rats and rats with mammary tumors. C3 activity in plasmas from normal and tumor-bearing rats was reduced (consumed) during absorption under appropriate conditions with Sepharose 4B, inactivated CNBr Sepharose, or Protein A-Sepharose. The concentration of functionally active C1 and C4 did not decrease significantly during absorption with Sepharose derivatives. Consumption of C3 in rat plasma was influenced by the anticoagulant and by the time and temperature of incubation with Sepharose derivative. C3 consumption in rat plasma anticoagulated with acid citrate dextrose solution was variable; addition of Mg2+ (5 mM) to plasma anticoagulated with acid citrate dextrose solution augmented C3 consumption. There was no C3 consumption in plasma anticoagulated with ethylenedinitrilotetraacetic acid (a chelator of calcium and magnesium). In contrast, this reduction was observed in plasma anticoagulated with [(ethylenebis(oxyethylenenitrilo)]tetraacetic acid (a chelator of calcium). The results demonstrate optimal conditions for activation of the alternative pathway of complement during absorption of rat plasma with Sepharose derivatives and suggest in vivo experiments to define the role of this pathway in inhibition of growth of mammary tumors.

Serotherapy of primary rat mammary carcinoma: inhibition by ethylenedinitrilotetraacetic acid but not by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid.

We reported inhibition of growth of primary rat mammary carcinomas after infusions of tumor-bearer plasma absorbed with Protein A-Sepharose or inactivated CNBr Sepharose. Absorbed plasmas were depleted of the third component of complement (C3) (other complement components defined similarly) and C5 but not C1, C4, or C2. These results suggested that activation of the alternative pathway of complement might be involved in the observed antitumor effects. To test this concept sera were treated with ethylenedinitrilotetraacetic acid or [ethylenebis(oxyethylenenitrilo)]tetraacetic acid before absorption with Protein A-Sepharose. Ethylenedinitrilotetraacetic acid, by chelating calcium and magnesium, prevents activation of both the alternative and classical complement pathways. [Ethylenebis(oxyethylenenitrilo)]tetraacetic acid, by chelating calcium but not magnesium, permits activation of the alternative pathway but inhibits activation of the classical complement pathway. Sera in the presence or absence of chelating agent were absorbed with Protein A-Sepharose twice at room temperature. After absorption calcium was added to the sera. Rats were treated by i.v. injection of sera twice a week for 2 weeks. Measurements of tumor size were made weekly for 5-7 weeks and then tumor weight was determined. Groups were compared both for size of index and total tumors. The results can be summarized as follows: tumor-bearer sera before absorption did not inhibit the growth of rat primary mammary carcinomas; tumor-bearer sera after absorption with Protein A-Sepharose showed significant consumption of C3 and did inhibit tumor growth; tumor-bearer sera absorbed in the presence of ethylenedinitrilotetraacetic acid did not show a decrease in C3 functional activity and did not inhibit tumor growth; tumor-bearer sera absorbed in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid did show a decrease in C3 functional activity and did inhibit tumor growth; sera from normal adult female rats after absorption with Protein A-Sepharose did inhibit tumor growth. The results are consistent with a role for the alternative pathway of complement in the inhibition of growth of rat primary mammary carcinomas observed after treatment with absorbed sera.

Inhibition of antibody-complement-mediated killing of tumor cells by hormones.

Line 1, a chemically induced guinea pig hepatoma, is susceptible to killing by anti-Forssman immunoglobulin M antibody and guinea pig complement. When these tumor cells are pretreated at 37 degrees with 10(-4) to 10(-11) M concentrations of the polypeptide hormone insulin, with the catecholamine L-epinephrine-HCl, or with the glucocorticoid steroids hydrocortisone sodium succinate or prednisolone sodium succinate, the cells show a marked reduction in their suseptibility to killing by antibody and guinea pig complement; pretreatment at 0 degrees is ineffective. Similar results were obtained with another antigenically distinct guinea pig hepatoma (line 10) when tested with anti-Forssman immuno-globulin M or specific antitumor antibodies and human complement. The ability of the hormones to render the cells resistant is dependent on time, temperature, and hormone concentration. The effect of hormone treatment is maximal between 30 and 60 min and is reversible within 4 hr even in the continued presence of hormone. Treatment of line 1 cells with up to 10,000-fold greater concentrations of the less biologically active or inactive analogs, DL-epinephrine, beta-estradiol, testosterone, or proinsulin has no effect on the susceptibility of the cells to killing by antibody and guinea pig complement. The effect of hormone treatment is not due to a direct inactivation of bound or fluid-phase complement components by the hormones or to a decrease in the ability of the cells to bind complement-fixing antibody.

PIP: Various aspects of hormone treatment of tumor cells are reported; it is shown that following treatment with certain hormones, the cells are less susceptible to killing by antibody and complement. The diethylnitrosamine-induced guinea pig hepatoma, designated Line 1, is susceptible to killing by anti-Forssman immunoglobulin M (IgM) antibody and guinea pig complement (GPC) but not by specific antitumor antibody and GPC. The antigenetically distinct Line 10 hepatoma, when sensitized with either antibody, is susceptible to killing by human complement (HUC) but not by GPC. Strain 2 of Servall-Wright male guinea pigs were used. 2 antigenetically distinct diethylnitrosamine-induced hepatic tumors (ascites form), Lines 1 and 10, passed in Strain 2 guinea pigs, were collected and suspended in RPMI 1640-20% FCS. Toxicity assays were performed in VBS-gel. The hormones used were hydrocortisone sodium succinate, prednisolone sodium succinate, NSC9151, bovine insulin, L-epinephrine methyl ether HC1, DL-epinephrine, beta-estradiol, testosterone, pork insulin, chicken insulin, pork proinsulin, pork DAA insulin, and the A and B chains of pork insulin. Tumor cells were cultured in 10-ml volumes of RPMI 1640-20% FCS in plastic Petri dishes. After incubation, cell cultures were washed 5 times in VBS-gel and tested for their susceptibility to killing by antibody and complement. Rabbit antiserum to sheep Forssman antigen was prepared and stored at -20 degrees until used. Tumor specific rabbit Antilines 1 and 10 antisera were prepared and similarly stored. Results of tests show that Line 1 tumor cells incubated in a medium containing the polypeptide hormone, insulin, the catecholamine, L-epinephrine HCl, or the glucocorticoid steroids, hydrocortisone sodium succinate, or prednisolone sodium succinate were rendered resistant to killing byanti-Forssman IgM antibody and GPC. This effect was dependent on hormone concentration, temperature, and time. Effects were reversible. Similar results were obtianed with Line 10 cells under attack by specific antitumor and HUC or anti-Forssman antibodies. Less physiologically active analogs of the hormones did not have this effect. Tumor cells showed maximum resistance within 30-60 minutes of exposure to the hormones and reverted to the sensitive state within 4 hours. Resistance of the cells to killing was observed at 37 degrees but not at 0 degrees. It is concluded that the effect of hormone treatment was not due to a direct inactivation of bound or fluid-phase complement components by the hormones or to a decrease in the ability of the cells to bind complement-fixing antibody.

Enhancing effect by metabolic inhibitors on the killing of tumor cells by antibody and complement.

Two chemically induced, antigenically distinct guinea pig hepatoma cell lines, line 1 and line 10, which are resistant to killing by rabbit anti-Forssman or specific antitumor antibody and complement, can be rendered susceptible when the cells are pretreated with metabolic inhibitors and drugs commonly used for the treatment of cancer patients. The effect appears within 7 hr after initial contact with the inhibitors and is dependent on temperature and on inhibitor concentration; the effect is reversible within 7 hr, and the process of reversion is also temperature dependent. Not all preparations of tumor cells were rendered susceptible following treatment with inhibitors. In some cases, susceptibility to killing by complement was observed with anti-Forssman antibody but not antitumor antibody. No clear correlation between known metabolic inhibitory activity of the inhibitors and conversion to the sensitive state could be made. The results suggest that properties of nucleated cells, which are under metabolic control, play an important role in the killing efficiency of antibody and complement.

Effect of metabolic inhibitors on the ability of tumor cells to express antigen and bind complement components C4 and C3.

Metabolic inhibitors commonly used in the treatment of cancer increase the ability of antibody and complement to kill guinea pig tumor cells in vitro. No correlation was found between increased killing and changes in cell surface antigen concentration or binding of complement components C4 and C3.

Kinetics of hormone-induced tumor cell resistance to killing by antibody and complement.

Line 1, a chemically induced guinea pig hepatoma, is susceptible to killing by anti-Forssman immunoglobulin M antibody and guinea pig complement. When these tumor cells are pretreated with insulin, L-epinephrine, hydrocortisone, or prednisolone, the cells show a marked reduction in their susceptibility to antibody-complement-mediated killing within 15 to 60 min; this effect reverses within 4 hr in the continued presence of hormone. Maximal binding of the hormones to the line 1 cells was observed within 60 min. However, the hormones remained bound to the cells after 4 hr of incubation, suggesting that line 1 cells incubated in the continued presence of hormone revert to the susceptible state despite the persistence of cell-bound hormone. Hormone-treated tumor cells, washed free of hormone and reincubated in hormone-free medium, lost nearly all their bound hormone within 15 to 30 min of washing. These cells, however, remained resistant to antibody-complement-mediated killing for up to 2 hr after washing. Line 1 cells, reverted in the continued presence of hormone, remained susceptible to killing by antibody and guinea pig complement after reexposure to the same, but not to a different, hormone. Hormone-treated cells reverted after prolonged incubation in hormone-free media; however, they were rendered resistant to killing after reexposure to the same hormone. The temporary refractoriness of reverted cells to further hormone stimulation was not due to an inability of the cells to bind hormone.