Sarkis H. Ohanian

Molecular Interactions of Cells with Antibody and Complement: Influence of Metabolic and Physical Properties of the Target on the Outcome of Humoral Immune Attack

The discovery of complement emerged from the observations of Buchner (1839a,b,c), Von Fodor (1886, 1887), and Nuttal (1888) that guinea pig serum had bacteriocidal activity. Bordet (1895) subsequently demonstrated that the bacteriocidal activity of serum required the cooperation of a thermostabile substance, antibody, and a thermolabile substance, complement (C). Since then it has been shown that C is capable of interacting with and killing protozoa (Anziano et al., 1972), viruses (Almeida et al.,1972), erythrocytes (Kabat and Mayer, 1961; Rapp and Borsos, 1970), and nucleated cells (Green et al., 1959b; Green and Goldberg, 1960; Humphrey and Dourmashkin, 1969). In addition, complement activity has been detected in the sera of many mammalian species as well as in sera of lower vertebrates (Bellow, 1977).

Physico-chemical properties of tumor cells that influence their susceptibility to humoral immune killing

Abstract Line-10 guinea pig hepatoma cells are susceptible to killing by antibody plus human complement, but resistant to killing by antibody plus guinea pig complement. Tumor cells treated with agents that reversibly increase (adriamycin), decrease (insulin or hydrocortisone), or have no effect (5-fluorouracil) on the susceptibility of the cells to antibody-complement killing were tested for their lipid and fatty acid composition. Hormone-treated (resistant) cells showed an increased total lipid content, an increased cholesterol: phospholipid ratio, and a depressed level of unsaturated fatty acids in the cellular neutral and phospholipids compared to control untreated cells. Adriamycin-treated (sensitive) cells showed exactly the opposite effects. The lipid composition of both hormone- and adriamycin-treated cells returned to control levels when the cells reverted to control levels of susceptibility to antibody-complement killing. 5-fluorouracil-treated cells were indistinguishable from untreated controls in their lipid and fatty acid composition. The chemical composition of the cell, and its effects on the physical properties of the cellular membranes, therefore appears to be fundamental for the ability of these tumor cells to resist humoral immune attack.

Effect of lipids, structural precursors of lipids and fatty acids on complement-mediated killing of antibody-sensitized nucleated cells

Abstract The cytotoxic activity of limiting concentrations of human complement against antibody-sensitized line-10 guinea-pig hepatoma cells can be inhibited and/or enhanced by pretreatment of the complement with lipids and/or fatty acids. Cardiolipin (CL), phosphatidylglycerol (PG), phosphatidylcholine (PC), phosphatidylserine (PS), sphingomyelin (SPHMY), cholesterol (CHOL), cholesteryl oleate (CHOL-O), phosphatidic acid (PA) or mammalian phosphatidylethanolamine (PE m ) were effective in inhibiting C activity. Bacterial phosphatidylethanolamine (PE b ) had no effect. Several cholesteryl esters and triglycerides of known fatty acid composition were tested. CHOL-O and glyceryl-1, 2-stearate-3-oleate were effective, but cholesteryl palmitate, stearate, arachidonate and glyceryl-1, 3-stearate-2-oleate were not effective in inhibiting HuC action. Structural precursors of phosphoJipids effective in inhibiting HuC activity (e.g. l -α-glycerophosphoryl esters of choline, serine and ethanolamine) did not modify the action of HuC on antibody-sensitized line-10 cells under any conditions tested. In contrast to the larger mol. wt lipid, conditions were found in which fatty acid could inhibit or enhance the cytotoxic action of C. The modification of C activity was dependent upon the specificity of the antibody used to sensitize the cells, the chain length, and the concentration of the fatty acid. The results suggest that the composition and possible subtle structural characteristics of lipids may play a role in influencing the functional activity of complement.

Killing of Nucleated Cells by Antibody and Complement

The ability of antibody-antigen complexes to fix complement (C) has been known since the end of the last century. Up to and including the recent past, complement has been used mainly as a very sensitive indicator to detect the interaction of antibody with antigen. With the development of refined immunochemical methods it has been possible to analyze what complement is and to study its interaction with antigen-antibody complexes and with the surface of the cell (Muller-Eberhard, 1968; Rapp and Borsos, 1970; Humphrey and Dourmashkin, 1965, 1969).

Tierexperimentelle Untersuchungen zur intraarteriellen Chemotherapie mit Bleomycin

Intra-arterial chemotherapy for treatment of head and neck carcinomas is performed on the basis of clinical reports, which postulate higher effective concentrations at the tumor for this therapeutic method than after systemic administration. This assumption, which has so far not yet been confirmed experimentally, has led to the present study. The study is concerned with different modes of application of bleomycin with the aim to achieve high cytostatic concentrations at the site of action required. In sheep the tissue concentration of 57Co-bleomycin was determined in a hypothetical tumor area, the planum buccale, and in the lymph nodes draining this area, after submucous administration (hypothetical tumor area), intra-arterial administration (A. transversa faciei, A. temporalis superficialis, A. carotis externa) and i.v. administration (V. saphena). Only submucous injection led to significantly increased 57Co-bleomycin activities in the hypothetical tumor area and in the corresponding primary draining lymph nodes. The intra-arterial administration into the A. temporalis superficialis and A. carotis externa did not show any differences compared to the i.v. injection of 57Co-bleomycin. Compared to i.v. administration, injection into the isolated A. transversa faciei led to a slightly increased concentration of radioactively labeled bleomycin, restricted to the hypothetical tumor area. The findings made by animals experiments print out that the clinical application of intra-arterial is not justified and favor intratumoral treatment of head and neck carcinomas with bleomycin.SummaryThe intense clinical interest in bleomycin as an anti-tumor agent has led to different methods of administration in an attempt to administer sufficiently high concentrations of the drug to the tumor. Therefore, a study was designed to determine the distribution and the therapeutic effect of a bleomycin emulsion and aqueous bleomycin after different routes of application. The tissue distribution of radioactively labelled bleomycin emulsion and aqueous bleomycin was determined in tumor-free CF 1 and tumor-bearing (EL 4, L 1210) C 57 Bl 6 and DBA 2 mice after local (s.c., i.t.) and systemic (i.v.) injection. The distribution studies for aqueous 57Co-bleomycin showed increased activity in the injection sites and the lymph nodes draining the injection sites after s.c. and i.t. injection compared to i.v. administration of the drug. In comparison to the aqueous local administration, the application of 57Co-bleomycin emulsion resulted in a disproportional increase of the 57Co-bleomycin concentration at the injection sites and in the draining lymph nodes. To prove the therapeutic relevance of the bleomycin tissue distribution tumor-bearing (line 10) strain 2 guinea pigs were treated with different modes of bleomycin. Animals with already lymphogenously metastasized tumors have been cured by means of low i.t. doses of the bleomycin emulsion. Guinea pigs treated with i.t. administration of aqueous bleomycin need, compared to the bleomycin emulsion, five times higher doses for tumor-free survival. Intravenously treated animals died either because of progressive tumor growth or because of toxic bleomycin effects. These findings made by animal experiments favor the i.t. treatment of head and neck carcinomas with a bleomycin emulsion.ZusammenfassungExperimentelle Hinweise auf hohe Zytostatikakonzentrationen am geforderten Wirkungsort nach lokaler Applikation haben zu Verteilungs- und Therapieexperimenten mit einer Bleomycin Emulsion und wäßrigem Bleomycin geführt. Gewebeverteilungsuntersuchungen wurden mit 57Co-Bleomycin Emulsion (Sojaöl) und wäßrigem 57Co-Bleomycin nach lokaler (s.c, i.t.) und systemischer (i.v.) Gabe an tumorfreien CF 1 und tumortragenden (EL 4, L 1210) C 57 Bl 6 und DBA 2 Mäusen durchgeführt. Die lokale Applikation von 57Co-Bleomycin Emulsion führte im Vergleich zur lokalen Gabe wäßrigen 57Co-Bleomycins zu gesteigerten Zytostatikum-Konzentrationen am Injektionsort und in den dazugehörigen drainierenden Lymphknoten. Die systemische Gabe wäßrigen 57Co-Bleomycins wies gegenüber den lokalen Applikationsvarianten stark erniedrigte 57Co-Bleomycin Konzentrationen am geforderten Wirkungsort auf. Die kinetischen Ergebnisse wurden an tumortragenden (Linie 10) Stamm 2 Meerschweinen überprüft. Tiere mit bereits lymphogen metastasierten Tumoren wurden mit niedrigen intratumoralen Dosierungen der Bleomycin Emulsion geheilt. Intratumoral mit wäßrigem Bleomycin behandelte Meerschweine benötigen gegenüber der Bleomycin Emulsion fünfmal höhere Dosierungen, um tumorfrei zu überleben. Intravenös behandelte Tiere starben entweder am Tumor oder toxischen Bleomycin Wirkungen.Die erhobenen tierexperimentellen Befunde favorisieren die intratumorale Behandlung von Kopf-Hals-Karzinomen mit einer Bleomycin Emulsion.

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.

Effect of protease treatment on the sensitivity of tumor cells to antibody-GPC killing

Abstract Treatment of the guinea pig hepatoma designated line-10 with a variety of proteolytic enzymes increased their sensitivity to killing by antibody and guinea pig complement. This increase in sensitivity could not be attributed to the quantity of complement-fixing antibody bound by the cells or to a direct effect on fluid phase complement. Anticomplementary material was removed from the tumor cells by proteolytic enzyme treatment but this material was not solely responsible for the observed increase in sensitivity of the cells to antibody-guinea pig complement killing.

Effect of hormones on the killing by antibody and complement of tumor cells treated with proteolytic enzymes

Line-10 guinea pig hepatoma cells are normally resistant to killing by antibody plus GPC but they are susceptible to killing by antibody plus HuC. Pretreatment of the cells with selected proteolytic enzymes renders the cells susceptible to killing by antibody plus GPC, whereas pretreatment with polypeptide, catecholamine, or steroid hormones renders the cells more resistant to killing by antibody plus HuC. Hormone pretreated tumor cells incubated with proteolytic enzymes remained resistant to antibody-GPC-mediated killing. Enzyme-pretreated cells incubated with hormones were either sensitive or resistant to antibody-GPC-mediated killing depending on the enzyme used to pretreat the cells, the enzyme concentration, the hormone class, and the specificity of the antibody used to sensitize the cells. The enzyme pretreated cells were able to bind amounts of hormone sufficient to render the cells resistant to humoral immune killing. These results suggest that the hormones exert their effects on certain areas of the tumor cell membrane rather than having a generalized effect on the cells. It is postulated that proteins, along with complex lipids, participate in the mechanism whereby tumor cells resist humoral immune killing.

Antigenic distinction between human C9 and guinea pig C9

Abstract Immunization of rabbits with the functionally purified ninth component (C9) of human (Hu) or guinea pig (GP) complement resulted in the production of antibodies which specifically inhibited the functional activity of fluid-phase but not bound HuC9 and GPC9, respectively. There was no detectable immunochemical cross-reactivity between the two C9 species as measured by immunodiffusion, immunoelectrophoresis, and cross-neutralization. Despite these differences, HuC9 and GPC9 were found to be interchangeable for the hemolysis of antibody-sensitized sheep erythrocyte EAC1-8 hu or EAC1-7 hu 8 gp cells. Immunization of the rabbits with either species of C9 resulted in a 5- to 10-fold depletion of the number of hemolytically effective C9 molecules in their serum.