Macrophage processing of peptide antigens: identification of an antigenic complex

In this report, we present data from our examination of the fate of the octapeptide antigen angiotensin II (AII) after processing by guinea pig macrophages. Macrophages were cultured with [125I]-AII or [3H]-AII for various times, and the AII products in the culture supernatant fluid and solubilized cell lysate (macrophage-associated AII) were analyzed by chromatography on Sephadex G-25. With [3H]-AII, both the cell-associated and cellfree AII was catabolized to three distinct peptide fragments: Peak B, containing seven to eight amino acids; Peak B1, with five to six amino acids; and Peak C, containing four to five amino acids. Surprisingly, in the cells another major form of AII was obtained, Peak A, which showed an m.w. of 50,000 to 70,000. Formation of Peak A was blocked by inclusion of non-radiolabeled AII into the incubation mixture, and could be formed by repulsing fresh macrophages with isolated Peaks B and B1, and to only a minor extent with Peak C. Peak A was formed within an hour of culture of macrophages with [3H]-AII, and remained stably cell-associated through a day of culture. In a pulse-chase type of experiment, it was shown that macrophages rapidly exocytose cell-associated Peaks B and C, and Peak B1 less rapidly, whereas Peak A is retained and is the predominant cell-associated form of AII after a day in culture. Incubation of [3H]-AII with isolated macrophage plasma membranes also resulted in Peak A formation, showing that the complex can be formed by a membrane event not requiring cellular uptake of peptide. The complex of AII in Peak A was acid-stable, and approximately 20% of Peak A was recovered after boiling in SDS. Because Peak A is the primary form of AII retained by macrophages, it was important to determine if this complex was antigenic for T cells. AII-immune T cells cultured with Peak A showed significant proliferation that was specific for AII. Thus, Peak A appears to represent a stable complex of AII with macrophage membrane structures that provide an immunologically relevant form of the antigen. These results are discussed with respect to a pathway for macrophage processing of peptide antigens to form a stable membrane-bound complex that is important for T cell responses.