Homodimerization of the human interleukin 4 receptor alpha chain induces Cepsilon germline transcripts in B cells in the absence of the interleukin 2 receptor gamma chain

Abstract

The cytokines interleukin (IL)-4 and IL-13 play a critical role in inducing Cepsilon germline transcripts and IgE isotype switching in human B cells. The IL-4 receptor (IL-4R) in B cells is composed of two chains, the IL-4-binding IL-4Ralpha chain, which is shared with the IL-13R, and the IL-2Rgamma (gammac) chain, which is shared with IL-7R, IL-9R, and IL-15R. IL-4 induces Cepsilon germline transcripts and IgE isotype switching in B cells from patients with gammac chain deficiency. Induction of Cepsilon germline transcripts by IL-4 in B cells that lack the gammac chain may involve signaling via the IL-13R. Alternatively, the IL-4Ralpha chain may transduce intracellular signals that lead to Cepsilon gene transcription independently of its association with other chains. We show that ligand-induced homodimerization of chimeric surface receptors consisting of the extracellular and transmembrane domains of the erythropoietin receptor and of the intracellular domain of IL-4Ralpha induces Janus kinase 1 (Jak1) activation, STAT6 activation, and Cepsilon germline transcripts in human B cell line BJAB. Disruption of the Jak1-binding proline-rich Box1 region of IL-4Ralpha abolished signaling by this chimeric receptor. Furthermore, B cells transfected with a chimeric CD8alpha/IL-4Ralpha receptor, which is expressed on the cell surface as a homodimer, constitutively expressed Cepsilon germline transcripts. These results suggest that homodimerization of the IL-4Ralpha chain is sufficient to transduce Jak1-dependent intracellular signals that lead to IgE isotype switching.

Figure 1

FACS analysis of surface EpoR expression and induction of Cɛ germline transcripts in BJAB transfectants. (A) Surface EpoR expression: BJAB cells transfected with pcDNA3 vector alone, EpoR, or EpoR/IL-4Rα constructs were subjected to G418 selection and fluorescence activated cell sorting, then analyzed for murine EpoR surface expression by FACS. Background fluorescence was determined by staining with normal rabbit serum (—). Filled fields show staining with rabbit anti-EpoR antiserum. Similar results were obtained in two independent transfections. (B) Expression of Cɛ germline transcripts: Transfectants were left unstimulated (None), or stimulated with Epo (50 units/ml) or IL-4 (200 units/ml) for five days then assessed for expression of Cɛ germline transcripts by Northern blot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was used as standard for equal loading. Similar results were obtained in three independent experiments.

Figure 2

FACS analysis of surface CD8 expression and induction of Cɛ germline transcripts in BJAB transfectants. (A) Surface CD8 expression: BJAB cells transfected with pCEP4 vector alone, CD8, or CD8/IL-4Rα constructs and selected in hygromycin were examined 13 days after transfection for surface expression of human CD8 by FACS. Background fluorescence was determined by staining with isotype control antibody (—). Filled fields show staining with anti-CD8 mAb. Similar results were obtained in two independent transfections. (B) Expression of Cɛ germline transcripts: expression of Cɛ germline transcripts and of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were examined by Northern blot analysis on the indicated days after transfection. Similar results were obtained in three independent experiments.

Figure 3

Induction of STAT6 nuclear binding activity to Iɛ oligonucleotide. (A) Electrophoretic mobility-shift assay. BJAB cells transfected with pcDNA3 vector alone, EpoR, or EpoR/IL-4Rα constructs were left unstimulated (None), or were stimulated for 30 min with Epo (50 units/ml), or IL-4 (200 units/ml). The cells were lysed and nuclear extracts were incubated with ³²P-labeled Iɛ oligonucleotide, then subjected to PAGE. (B) Specificity and identity of the Iɛ oligonucleotide binding nuclear complex. Nuclear extracts from EpoR/IL-4Rα transfectants left unstimulated or stimulated for 30 min with Epo (50 units/ml) or IL-4 (200 units/ml) were incubated with ³²P-labeled Iɛ oligonucleotide and 100-fold molar excess of the following cold competitors: unlabeled Iɛ (Iɛ), Iɛ mutant (Iɛ mut), and an AP-1 consensus sequence (AP-1). For supershift assay, nuclear extracts were pre-incubated with rabbit anti-STAT6 antiserum or with NRS. Similar results were obtained in three independent experiments.

Figure 4

Effect of homodimerization of the EpoR/IL-4Rα chimeric receptor and its Box1 mutants on Jak3 and Jak1 activities. Cells from intact EpoR/IL-4Rα transfectants (A and B) and EpoR/IL-4Rα Box1 mutants transfectants (B) were stimulated with Epo (300 units/ml) or IL-4 (1,200 units/ml) for the indicated periods of time (A) or for 10 min (B). Then cells were lysed as described in Materials and Methods. Cell lysates were immunoprecipitated with anti-Jak3 antibody (A) or anti-Jak1 antibody (B) and the immunoprecipitates were electrophoresed on SDS/PAGE, transferred to a nitrocellulose membrane, and immunoblotted with anti-phosphotyrosine (P-Tyr) mAb 4G10. The membrane was then stripped and reimmunoblotted with anti-Jak3 antibody (A) or anti-Jak1 antibody (B). Similar results were obtained in two independent experiments.

Figure 5

Disruption of the Jak1 binding proline-rich Box1 region of IL-4Rα abolishes the induction of Cɛ germline transcripts following homodimerization of the EpoR/IL-4Rα chimeric receptor . The cells transfected with empty pcDNA3 vector, EpoR/IL-4Rα Box1 deletion mutants (Box1 mut 1 or Box1 mut 2), or intact EpoR/IL-4Rα were left unstimulated (None), or stimulated with Epo (50 units/ml) or IL-4 (200 units/ml) for five days and assessed for induction of Cɛ germline transcripts and expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by Northern blot analysis. Similar results were obtained in two independent experiments.