title: Characterization of an immunosuppressive factor from malignant ascites that resembles a factor induced in vitro by carcinoembryonic antigen.
abstract: Oncodevelopmental antigens may cause immunologic suppression in the host through release of suppressor molecules from the host's own immunoregulatory cells. This concept has been difficult to study until recently when carcinoembryonic antigen was shown to induce the release of such molecules from normal circulating human mononuclear cells in vitro. However, the amount of the suppressor moiety generated was too small to adequately characterize, and its presence in vivo, i.e., in the cancer-bearing host, was unknown. Therefore, we sought to isolate and characterize a similar or identical macromolecule from ascites having an elevated CEA level in patients with cancer. A single malignant ascites, when precipitated at 0 to 35% ammonium sulfate saturation, was the source of suppressive factor for purposes of isolation and standardization. Suppression was quantitated by reduction of [3H]thymidine incorporation by phytohemagglutinin-stimulated normal human peripheral blood mononuclear cells. Sephadex G-200 chromatography revealed probable aggregation of the factor in isotonic buffers; aggregation was reduced in the presence of 8 M urea. Purification was achieved by precipitation with 5% trichloroacetic acid (TCA). The suppressor factor remained soluble in TCA and demonstrated a 95-fold increase in specific activity. Analytical sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated a single protein band of 50,000 daltons. Ascites from three additional cancer patients gave identical results. Physicochemical characterization of the suppressor moiety revealed stability at 70 degrees C for 30 min and at pH 2 and pH 10 for 24 hr. Delipidation by chloroform-methanol extraction, proteolytic enzyme digestion, and protamine sulfate precipitation did not affect activity, suggesting that lipid, simple peptides, and nucleic acids were not crucial. However, periodate oxidation irreversibly destroyed suppressor activity, suggesting the importance of carbohydrate to the molecule and offering one explanation for protease resistance. Similarities in m.w. (50,000 daltons), isoelectric point (pI = 3.4), physical properties (heat and acid stability and resistance to proteases), and immunologic activity of this factor with that released from lymphocytes after in vitro exposure to carcinoembryonic antigen indicates they may be identical. Our results suggest that early aberrant events induced in the immunoregulatory network by tumor-associated antigens may be relevant and may lead to better understanding of immunosuppression in the cancer-bearing host.
mesh_terms: D001201:Ascites / Q000276:immunology; D002241:Carbohydrates / Q000032:analysis; D002272:Carcinoembryonic Antigen / Q000276:immunology*; D011232:Chemical Precipitation; D004306:Dose-Response Relationship, Immunologic; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008055:Lipids / Q000032:analysis; D008213:Lymphocyte Activation / Q000187:drug effects; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000378:metabolism; D010835:Phytohemagglutinins / Q000494:pharmacology; D013491:Suppressor Factors, Immunologic / Q000302:isolation & purification* / Q000494:pharmacology; D013997:Time Factors
chemical_list: D002241:Carbohydrates; D002272:Carcinoembryonic Antigen; D008055:Lipids; D010835:Phytohemagglutinins; D013491:Suppressor Factors, Immunologic; D010447:Peptide Hydrolases