[Figure 1 ILLUSTRATION OMITTED]
To identify the 120-kD salivary gland protein, we electroblotted the band onto an Immobilon membrane and directly sequenced it with an Applied Biosystems 477A Protein Sequencer. The bands had a single NH2-terminal sequence. This sequence was identical to the [NH.sub.2]-terminal sequence of human [Alpha]-fodrin (nonerythroid [Alpha]-spectrin) (5): RQKLEDSYRFQFFQRDAEEL (6).
Proliferative T cell responses of spleen cells from 3d-Tx NFS/sld mice to the identified 120-kD antigen developed spontaneously at 4 weeks of age, consistent with the onset of the autoimmune lesions in the salivary gland (Fig. 1C) (7). This response increased at 8 and 12 weeks of age, then declined by week 16. In contrast, no response was detected with control antigens (fraction 40, lysozyme, and a-amylase). The spontaneous development of a proliferative T cell response to the 120-kD antigen is consistent with endogenous priming. We tested the 120-kD antigen-reactive T cells for additional properties to confirm the specific T cell responses. Splenic T cells from 8-week-old 3d-Tx NFS/sld mice challenged with the 120-kD antigen produced interleukin-2 (IL-2) and interferon-[Gamma] (IFN-[Gamma]) (Fig. 1D) (8). Thus, a potentially pathogenic T helper type 1 [(T.sup.H1)] T cell population may be spontaneously primed to the 120-kD salivary gland autoantigen early in the development of autoimmune lesions in the salivary glands of this mouse model.
To examine the organ specificity of the 120-kD [Alpha]-fodrin, we immunoblotted various tissue homogenates from 3d-Tx NFS/sld mice with a commercially available monoclonal antibody to [Alpha]-fodrin. More 120-kD [Alpha]-fodrin was detected in the salivary gland homogenate than in homogenates from other organs (Fig. 2A) (4). A very faint 120-kD band in brain, heart, and lung tissue homogenates seems to be a degradation product of intact [Alpha]-fodrin, because autoimmune lesions were not detected in these organs. In nonthymectomized NFS/sld mice, a negligible amount of 120-kD [Alpha]-fodrin was detected in the salivary gland or in other organs (Fig. 2A). These data suggest that the salivary gland expression of 120-kD [Alpha]-fodrin may be an initial response in the development of autoimmune lesions in the salivary glands. Polyclonal rabbit…