| The overall goal of this study was to understand the molecular and cellular aspects of immunoglobulin (Ig) binding receptors (FcR) in the channel catfish, Ictalurus punctatus, a well-defined teleost immunological model. Previously it was demonstrated that catfish express two types of IgM-binding FcR. The first is a membrane associated receptor expressed on catfish natural killer (NK) cells, which enables them to kill antigen specific target cells via antibody-dependent cell-mediated cytotoxicity (ADCC). At present the gene encoding this receptor has yet to be identified. The second FcMR is a soluble receptor found in catfish serum termed, IpFcRI. It consists of three Ig domains, lacks transmembrane and cytoplasmic regions, and is structurally and phylogenetically related to mammalian IgG binding receptors. IpFcRI is the first FcR sequenced in any ectothermic vertebrate. In Study I the IgM binding capabilities of IpFcRI were further characterized and a linear epitope on catfish IgM was identified as the receptor docking site. Using Western blot analyses and latex bead binding assays the docking site was shown to consist of a consensus octapeptide motif FxCxVxHE located at the second cysteine that forms the intrachain disulfide bond of catfish Cmicro3 and Cmu4 Ig domains. Furthermore, molecular modeling of these domains confirmed that the octapeptide in both domains is exposed and accessible for IpFcRI interactions. In addition, because this motif is also found in other vertebrate Ig domains, IpFcRI binding to Ig heavy (H) and light (L) chains from rainbow trout, chicken, mouse, rabbit, and goat was examined by Western blot analyses and latex bead binding assays. IpFcRI readily bound reduced rainbow trout (Igmu), chicken (Igupsilon), mouse (Igmicro, Iggamma1, Iggamma2a, Iggamma2b, and Igalpha), rabbit (Igmu and Iggamma) and goat (Iggamma) IgH chains, and mouse Igkappa and Iglambda, and chicken Iglambda IgL chains. In Study II a third FcmicroR, different from the FcmicroR found on NK cells, was identified. This FcR termed FcmicroRII is expressed on catfish TS32.15 clonal cytotoxic T lymphocytes (CTL). The first indication of this FcR came from flow cytometric analyses, which demonstrated that TS32.15 grown in the presence of catfish serum stained positive for IgM and two catfish IgL chain isotypes. Since TS32.15 cells do not express mRNA for either Ig H or I, chains it was hypothesized that like in the catfish NK cells, serum IgM is acquired onto the cell surface via a putative FcmicroR. However, TS32.15-bound IgM did not mediate ADCC. In contrast, IgM cross-linking of TS32.15 cells resulted in reduced cell division and marked decrease in cytotoxicity against allogeneic targets. In addition, cell cycle analyses revealed that cross-linked TS32.15 cells were predominately arrested in the GO/G1 phase and the blockage could be completely reversed by allogeneic stimulation. Together, such results indicate that FcmicroRII functions as an inhibitory FcR. Furthermore, using anti-catfish IgM, three FcmicroRII or FcmicroRII associated proteins of 52, 40, and 38 kDa were co-immunoselected from biotinylated TS32.15 CTL and importantly, FcR cross-linking led to tyrosine phosphorylation of the 52 kDa protein. Finally, Western blots of immunoselected material demonstrated that the inositol phosphatase, SHIP was co-selected with the TS32.15 FcmicroRII.;Thus, these data strongly suggest that at least three IgM-binding FcR, one of which is inhibitory, are present in catfish. Our findings emphasize that Ig-binding receptors play important and diverse functional roles in immune responses of all vertebrates. |
