The expression and biologic effects of ovine interleukin-4 on T and B cellproliferation

Using the reverse-transcriptase polymerase chain reaction (RT-PCR), cDNA en coding ovine (Ov) interleukin-4 (OvIL-4) was generated from mitogen-stimula ted peripheral blood mononuclear cells (PBMC), Two identical clones generat ed from separate RT-PCR reactions differed from a published OvIL-4 sequence , although they had a high degree of identity with the bovine and human hom ologs, We show by sequence analysis that the OvIL-4 cDNA retained the four alpha-helix structure and disulfide bonds identified in human IL-4 (HuIL-4) . Moreover, the cDNA encoding OvIL-4 was expressed in insect cells using th e baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) as a vector. Supernatants from insect cells infected with the recombinant vir us secreted an additional protein with a relative molecular mass of 17,000, This protein was recognized by an anticervine IL-4 monoclonal antibody (mA b) in a Western blot and did not react with any proteins in supernatants fr om uninfected insect cells or cells infected with the wild-type AcMNPV, Sup ernatants from insect cells infected with the recombinant virus induced the proliferation of activated B cells in a dose-dependent manner and typicall y demonstrated 5 x 10(5) dilution U/ml of activity. However, OvIL-4 had no effect on the proliferation of resting T cells isolated from efferent lymph and actually inhibited the ability of a mitogen to stimulate these resting lymphocytes. In contrast, OvIL-4 induced the proliferation of mitogen-acti vated lymphoblast, demonstrating the complex role(s) OvIL-4 plays in the re gulation of B and T cells.