Pergamon 0161~5890(%)00154-9 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Molecular Immunology, Vol. 33, No. 41.5, pp. 361-370, 1996 Elsevier Science Ltd. Printed in Great Britain GENERATION OF A VARIANT OF HUMAN INTERLEUKIN-4 BY ALTERNATIVE SPLICING WILLIAM J. ALMS,* SERGEI P. ATAMAS,1_ VLADIMIR V. YUROVSKYt and BARBARA WHITE*$ *Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201, U.S.A.: tDepartment of Medicine, University of Maryland, MSTF Room 8-34, 10 South Pine Street, Baltimore, MD 21201, U.S.A. (First received 15 June 1995; accepted in revised form 16 November 1995) Abstract-A second species of interleukin4 (IL-4) mRNA was identified using both a reverse tran- scription-polymerase chain reaction and an RNase protection assay. This novel IL-4 mRNA was 48 base pairs smaller than IL-4 mRNA, which is the size of IL-4 exon 2. Sequence data of cloned cDNA demonstrated that this variant contained IL-4 exons 1, 3 and 4, with exon 1 spliced directly to exon 3 in an open reading frame. The entire protein encoding region of this variant, named IL-462, was identical to IL-4 except for the omission of exon 2. IL-462 mRNA was detected in all human peripheral blood mononuclear cells tested and in purified CD3+ T cells. Amounts of both IL-4 and IL-462 mRNAs increased upon T cell activation, although IL-4 mRNA increased to a greater extent than IL-462 mRNA did. Human IL-3, IL-5 IL-13, and granulocyte macrophage-colony stimulating factor did not use alternative splicing to delete exon 2. We speculate that IL-462 may regulate IL-4 function. Published by Elsevier Science Ltd. Key words; Interleukin-4, alternative spicing. INTRODUCTION Interleukin-4 (IL-4) is a 15 kilodalton (kDa) glycoprotein secreted by activated T cells (Howard et al., 1982), mast cells (Brown et al., 1987) and basophils (Seder et al., 1991) which regulates a wide spectrum of cellular functions in hematopoetic and non-hematopoetic cells. As examples, IL-4 co-stimulates proliferation of resting B cells with anti-immunoglobulin (Ig) M antibodies (Howard et al., 1982) rescues resting B cells from apoptosis (Illera et al., 1993), induces Ig production by activated B cells (Defiance et al., 1988) and regulates isotype switching to IgGl and IgE in mice (Vitetta et al., 1985; Coffman et al., 1986) and IgG4 and IgE in humans (Lundgren et al., 1989). Immunoglobulin M (Shields et al., 1989), CD23 (Defiance et al., 1987; Hudak ef al., 1987; Rousset ef al., 1988), major histocompatibility complex (MHC) class II molecules (Roehm et al., 1984; Rousset et al., 1988), leukocyte function antigen (LFA)- 1 and LFA-3 (Rousset et al., 1989), and IL-4 receptor (R) (Rem et al., 1991) molecules are all increased on the surface of B cells fol- lowing IL-4 exposure. In T cells, IL-4 promotes pro- liferation (Fernandez-Botran et al., 1986; Mosmann et al., 1986; Mitchell et al., 1989), generation of the T helper 2 phenotype (Fernandez-Botran et al., 1986; Le Gros et al., 1990) and expression of IL4R (Renz et al., 1991). $Author to whom correspondence should be addressed, at: University of Maryland, MSTF Room 8-34, 10 South Pine Street, Baltimore. MD 21201, U.S.A. IL-4 syngerizes with IL-3 to promote growth of mast cells (Mosmann et al., 1986). IL-4 activates macrophages to increase tumoricidal activity, MHC class II expression, and binding of IgG immune complexes (Crawford et al., 1987). Precursors of erythroid cells, megakaryocytes, and granulocytes-macrophages can be co-stimulated with IL- 4 to increase colony formation (Peschel et al., 1987). IL-4 also stimulates proliferation (Feghali et al., 1992), chemotaxis (Postlethwaite and Seyer, 199 l), extracellular matrix production (Postlethwaite et al., 1992) and inter- cellular adhesion molecule-l expression (Piela-Smith et al., 1992) by fibroblasts. Because of the widespread effects of IL-4, it is not surprising that the regulation of IL-4 activity is pivotal in determining the outcome of certain diseases (Scott et al., 1988; Heinzel et al., 1989; Yamamura et al., 1991; Zwingenberger et al., 1991). In murine leishmaniasis (Heinzel et af., 1989), human leprosy (Yamamura et al., 1991), and human schistosomiasis (Zwingenberger et al., 1991), the production of IL-4 is associated with chronic infection. Increased production of IL-4 in response to allergens characterizes human atopic responses (Wier- enga et al., 1990). Studies of the molecular regulation of IL-4 activity have focused on the effects of promoters, enhancers, and negative regulatory elements within the IL-4 gene (Henkel et al., 1992; Li-Weber et al., 1992, 1993; Abe et al., 1992; Szabo et al., 1993). In this paper, we describe a new mechanism for the potential regulation of IL-4 activity, in which a second mRNA is alternatively spliced from the IL-4 gene. We demonstrate the pro- 361