Increased immunogenicity to P815 cells modified with malondialdehyde and acetaldehyde

Abstract

Aldehyde modified proteins have been associated with the development and/or progression of alcoholic liver disease (ALD). These protein adducts are capable of initiating many immunological responses that are harmful to the normal homeostasis of organism function. Previous studies have shown that malondialdehyde (MDA) and acetaldehyde (AA) synergistically form a unique adduct (MAA) with soluble proteins, which are capable of inducing cytokine release, T-cell proliferation, and antibody production. The purpose of this study was to determine whether MAA adduction can elicit similar responses to cells using a well-defined tumor model. The mouse mastocytoma P815 tumor cell line was modified with MAA (P815-MAA) or left unmodified (P815) and 10(6) irradiated cells were injected into DBA/2 mice once a week for 5 weeks. Serum was collected and tested for antibody responses to P815 cells and the MAA epitope. Immunization of MAA adducted P815 cells into syngeneic DBA/2 mice induced a strong antibody response to the MAA epitope as determined by ELISA on Alb and MAA-Alb (508 microg/ml and 1092 microg/ml, respectively). In addition, antibody to unmodified P815 cells was detected by fluorescent technique. Mice immunized with P815 cells or PBS showed little or no reactivity to the MAA epitope or P815 cells. Studies to assess IL-12 stimulation showed that peritoneal macrophages from P815 and PBS immunized animals produced modest amounts of IL-12 (20 and 35 pg/ml) when stimulated with Alb or MAA-Alb. However, macrophage from P815-MAA immunized mice responded to soluble MAA adduct (142 pg/ml). Finally, in tumor survival studies the mean survival was 14.25 days in PBS treated mice; 15.75 days with P815 immunized mice and 18.25 days with P815-MAA immunized mice. Therefore, these data strongly suggest that antibody responses are induced by P815 cells modified with MAA adducts. This may be a possible tool to begin looking at how alcohol metabolites potentially modify cells and/or cellular components making them recognizable to the immune system as foreign. It is thought that these studies define a model system that will be useful in assessing antibody and potentially T-cell responses to cells that are modified by MAA.

Figure 1

Antibody activity to the MAA epitope in DBA/2 mice immunized with PBS, P815 or P815-MAA modified cells as assayed on Alb or MAA-Alb. Serum from DBA/2 mice injected with P815-MAA modified cells contained a 2 fold increase in MAA antibody when compared to the DBA/2 mice injected with P815 cells. Mice injected with PBS had only background antibody production to Alb or MAA-Alb. Each bar is the mean SE of 5 mice. * P< 0.05, significantly different from PBS and P815 injected animals when sera was screened on MAA-Alb. # P < 0.05 significantly different from PBS and P815 injected animals when screened on Alb.

Figure 2

Antibody activity to P815 cells in DBA/2 mice immunized with PBS, P815 or P815-MAA modified cells. Serum from DBA/2 mice was incubated with P815 cells and the antibody activity determined. Mice injected with P815-MAA had a 3 fold increase in antibody activity to the P815 cells, while the mice injected with P815 cells or PBS had background levels of approximately 100 relative fluorescent units. Each bar is the mean SE of 5 mice. * P < 0.001, significantly different from PBS and P815 injected animals.

Figure 3

IL-12 release by peritoneal macrophages presensitized with P815-MAA cells. Peritoneal macrophage from DBA/2 mice injected with PBS, P815 or P815-MAA modified cells were incubated in the presence of (25 µg/ml) Alb or MAA-Alb for 3 hours. Supernatant was assayed for IL-12 production. P815-MAA injected animals showed a 3 fold increase in IL-12 over the PBS or P815 injected animals. Each bar is the mean SE of 5 mice. * P < 0.001, significantly different from PBS and P815 injected animals when MAA-Alb was used as the antigen. # P < 0.001 significantly different from PBS or P815 injected animals when Alb was used as the antigen.

Figure 4

Survival of mice immunized with PBS, P815 or P815-MAA cells modified following the administration of a lethal dose of P815 cells. Mice immunized with PBS lived 14 days, while P815 injected animals lived 14–18 days. However, mice immunized with P815-MAA modified cells lived 4 days longer than the PBS of P815 injected animals. Each point is the mean SE of 6 mice.

Figure 5

Survival of DBA/2 mice challenged with P815 cells. Data were extrapolated and expressed to show the concentration of cells needed to kill mice. Briefly, mice immunized with PBS and P815 cells require 10² and 10⁴ cells respectfully in order to induce tumor and death. However, mice pre-immunized with P815-MAA cells required 10⁵ cells to induce tumor and death. Each point is the mean SE of 6 mice.