ABSTRACT
Cytotoxicity of anti-DNA autoantibodies from sera of SLE and CLL patients was assayed on permanent cell lines L929, HL-60, Raji, and K562. L929 cells appeared to be the most sensitive to antibody treatment. DNA-hydrolyzing properties of the same autoantibody preparations were analyzed in parallel. The data obtained outlined the correlation between cytotoxicity and DNA-hydrolyzing properties of these autoantibodies. It was shown that treatment of the cells with cytotoxic anti-DNA autoantibodies induced internucleosomal DNA fragmentation and Annexin V binding to the cell surface characteristic of apoptotic pathway of cell death. A time-dependent profile of antibody-mediated toxicity to L929 cells suggested recruitment of at least two distinct mechanisms of cell death. The first peak of cell death observed in 3 h of incubation was completely inhibited by preincubation of cells with caspase inhibitor YVAD-CHO, while the second increase in cell mortality (18-30 h) persisted. Possible mechanisms for anti-DNA autoantibody cytotoxicity are discussed.
Subject(s)
Antibodies, Antinuclear/immunology , Antibodies, Antinuclear/toxicity , DNA/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Lupus Erythematosus, Systemic/immunology , Caspase Inhibitors , Caspases/metabolism , Cell Death/drug effects , DNA/metabolism , DNA Fragmentation/drug effects , Flow Cytometry , Humans , Hydrolysis/drug effects , Immune Sera/immunology , Immune Sera/toxicity , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fab Fragments/toxicity , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Lupus Erythematosus, Systemic/genetics , Time Factors , Tumor Cells, CulturedABSTRACT
Gangliosides induce apoptosis in the cells of the IL-2-dependent cytotoxic mouse line CTLL-2. Upon incubation with gangliosides for 24 h, their effect resulting in appearance of apoptotic cells, falls in a series GM2 > GM3 > GM1 > GD1a > GD1b > GT1b. In the presence of rIL-2, apoptosis induced by GM1 is suppressed, whereas that induced by GM2 is enhanced (the effect of intracellular agent C2-Cer is independent of this cytokine). The GM1-induced apoptosis is cancelled by the caspase I inhibitor. The gangliosides under study are not able to induce apoptosis in the promyelocyte leukemia cell line HL-60. Physiological aspects of the phenomenon found are discussed.
Subject(s)
Apoptosis , Gangliosides/physiology , T-Lymphocytes, Cytotoxic/physiology , Animals , Apoptosis/drug effects , Caspase Inhibitors , Cell Line , Flow Cytometry , Gangliosides/classification , HL-60 Cells , Humans , Interleukin-2/pharmacology , Mice , Oligopeptides/pharmacology , Signal Transduction , T-Lymphocytes, Cytotoxic/drug effectsABSTRACT
The cytotoxicity of DNA-specific autoantibodies from sera of patients with systemic lupus erythematosis (SLE) and with lymphoproliferative diseases, and from blood of healthy donors was examined on tumor-cell lines L929 and HL-60. DNA-binding IgG fractions from SLE and chronic lymphocytic leukemia (CLL) sera were cytotoxic at concentrations of up to 10(-10) M. No detectable changes in cell viability were observed after incubation with antibodies devoid of DNA-binding activity and DNA-specific antibodies isolated from blood of healthy donors and patients with T-cell lymphoma, B-cell lymphosarcoma, and acute B-cell leukemia. There was good correlation between the cytotoxic activity and DNA-hydrolyzing activity of anti-DNA antibodies. The cytotoxic effect of DNA-binding antibodies presumably was complement-independent, because it was attributed only to the Fab fragment. The cytotoxic effect was completely inhibited by preincubation with double-stranded DNA (dsDNA). Both the cytotoxic effect and the DNA-hydrolyzing activity of anti-DNA antibodies were significantly increased in the antibody fraction that displayed cross-reactivity with nuclear matrix proteins. Possible mechanisms for the formation and pathogenicity of cytotoxic anti-DNA antibodies are discussed in this article.
Subject(s)
Antibodies, Antinuclear/metabolism , Antibodies, Catalytic/metabolism , Cytotoxicity, Immunologic , DNA/immunology , DNA/metabolism , Antibodies, Antinuclear/blood , Antibodies, Catalytic/blood , Cross Reactions , HL-60 Cells , Humans , Hydrolysis , Immunoglobulin Fragments/blood , Immunoglobulin Fragments/metabolism , Immunoglobulin G/blood , Immunoglobulin G/metabolism , In Vitro Techniques , Lupus Erythematosus, Systemic/immunology , Lymphoproliferative Disorders/immunology , Nuclear Envelope/immunology , Nuclear Matrix/immunology , Tumor Cells, CulturedABSTRACT
Gangliosides have been shown to inhibit proliferation of the interleukin-4 (IL-4) responsive cell line CT.4R. Kinetic analysis has revealed that ganglioside GT1b is a competitive inhibitor of proliferation, while GM and GM3 show a mixed pattern of inhibition, i.e., exhibit more than one inhibition type. Contribution of the competitive cell inhibition for GM1 and GM3 depends on serum factors added: the higher is the percentage of FCS, the larger is the contribution of competitive inhibition. The pattern of proliferation inhibition shown for GT1b does not depend on the FCS content. We have also studied the interaction of the recombinant IL-4 with fluorescent (anthrylvinyl-labelled) gangliosides GM1 and GM3 and lactosylceramide incorporated into liposomes. Dissociation constants of the IL-4-ganglioside complexes have been determined; lactosylceramide does not interact with rIL-4. The K(d) values for the lymphokine complexes with gangliosides support the conclusion based on the kinetic analysis that IL-4 has a higher affinity for GM3 (K(d) = 5 nM) than for GM1 (K(d) = 0.28 microM).
