Conversion of acetaldehyde-protein adduct epitopes from a nonreduced to a reduced phenotype by antigen processing cells

Many investigators have suggested that an immune reaction to acetaldehyde-p rotein adducts may be involved in the development and/or progression of alc ohol liver disease. The most often reported acetaldehyde adduct is the redu ced adduct prepared in vitro in the presence of strong reducing agents. How ever, the production of this adduct in vivo has been difficult to prove. Ne vertheless, the detection of serum antibodies to this reduced adduct follow ing alcohol exposure in animals and humans has been used to support the for mation of this adduct in vivo. We have recently observed that when acetalde hyde-protein adducts prepared under nonreducing conditions are used to immu nize animals, antibody to the reduced protein adduct is detected. Therefore , it was the purpose of this study to demonstrate that nonreduced (NR) addu ct epitopes can be modified by intact cells to express reduced (R) adduct e pitopes. This was accomplished using the monoclonal antibody RT1.1 that has been previously characterized by this laboratory and has been shown to rec ognize only R and not NR acetaldehyde adducts. In these studies, Balb/c mic e were injected intraperitoneally (500 mu g/animal) with either keyhole lim pet hemocyanin (KLH)-NR or KLH-R adducted proteins. Immunization with KLH-N R produced significant amounts of antibodies that recognized both NR and R epitopes. In contrast, immunization with KLH-R produced antibodies to only R and not NR epitopes. Isolated peritoneal macrophages from nonimmunized mi ce were incubated in vitro with either KLH-NR, KLH-R, or unmodified KLH pro teins, and the cell surface expression of the reduced epitope (RT1.1) and t he activated macrophage marker (MAC-3) determined by double immunofluoresce nt staining. Activated macrophages incubated with KLH-NR expressed the R ad duct on 11.5% of the cells, compared with 3.8% following incubation with un modified KLH, and 19.4% following incubation with KLH-R. These data suggest that the NR adduct and/or the carrier protein are modified by peritoneal m acrophages in vivo and present an epitope that is detected asa reduced addu ct (RT1.1 positive). These observations may explain the presence of circula ting antibodies to the reduced adduct that has been reported in human and a nimal studies.