Effects of antigen dosage on early localization of specific antibodies in rat splenic germinal centers.

In order to identify the initial site of antibody formation in rat spleen, an investigation was made to determine the effects of different antigen dosages on the localization of specific antibodies against sheep erythrocytes (SRBCs). Sixty rats were intravenously injected with 1 ml of either 1%, 5%, or 10% suspensions of SRBCs and killed at days 1, 2, 3, and 4 after immunization. A tissue agglutination procedure in which the binding of SRBCs to cryostat sections of spleen was used to localized anti-SRBC antibodies. Sections used for determination of SRBC binding patterns, and adjacent sections were stained for histological localization or processed for the determination of acid phosphatase (ACP) activity. Spleens of non-immunized rats showing binding of SRBCs closely associated with the ACP-positive marginal metalophils and marginal zone macrophages. This binding was not inhibited by preincubating the sections with 2-mercaptoethanol. The bound SRBCs lysed when incubated with complement. The initial change that occurred after antigen injection was binding over the germinal was not associated with ACP-positive cells. In animals immunized with 1% SRBCs, these changes were seen on the third day after immunization. In animals immunized with 1% SRBCs, these changes were seen on the third day after immunization. In animals immunized with 5% or 10% suspensions of SRBCs, these changes occurred 24 hours after immunization, during dissociation of the germinal centers. In later stages there was heavy binding of SRBCs over the white pulp and over the red pulp. Binding induced by immunization was inhibited by pretreating the sections with 2-mercaptoethanol and the bound cells lysed in the presence of complement. The results obtained suggest that IgM antibody to SRBCs appears in the germinal centers at least as early as in the marginal zone or peripheral periarterial region, and support the view that germinal centers may participate in primary antibody responses.