IL-13R alpha 2 membrane and soluble isoforms differ in humans and mice

Abstract

Although mice have nanogram per milliliter serum levels of soluble (s) IL-13Ralpha2, humans lack sIL-13Ralpha2 in serum. Our data provide a mechanism for this biological divergence. In mice, discrete transcripts encoding soluble and membrane forms of IL-13Ralpha2 are generated by alternative splicing. We used small interfering RNA to specifically deplete the transcript encoding membrane (mem) IL-13Ralpha2 (full-length) or sIL-13Ralpha2 (DeltaEx10) in murine cells. Depletion of the full-length transcript decreased memIL-13Ralpha2 but had no effect on the level of sIL-13Ralpha2 in cell supernatants at baseline or following cytokine stimulation. Depletion of the DeltaEx10 transcript decreased sIL-13Ralpha2 in supernatants at baseline and following stimulation. In contrast to mice, we were unable to find a transcript encoding sIL-13Ralpha2 in humans and siRNA-mediated depletion of full-length IL-13Ralpha2 decreased both sIL-13Ralpha2 and memIL-13Ralpha2 in human cells. Inhibition of matrix metalloproteinases (MMP)/MMP-8 abolished production of sIL-13Ralpha2 from human cells. Thus, sIL-13Ralpha2 is derived exclusively from the memIL-13Ralpha2 transcript in humans through MMPs/MMP-8 cleavage of memIL-13Ralpha2, supporting a limited role for sIL-13Ralpha2 in humans and highlighting the potential importance of memIL-13Ralpha2 in human immunity. These observations require consideration when results of murine IL-13 studies are applied to humans.

Figure 1. sIL-13Rα2 in mouse and human plasma samples

(A) ELISA of plasma samples from BALB/c WT and IL-13Rα2 KO mice (n=5). (B) ELISA of random human plasma samples using rat anti-human IL-13Rα2 monoclonal, goat anti-human IL-13Rα2 polyclonal or normal goat IgG for capture. (C) ELISA of human plasma samples preincubated with excess normal goat IgG or goat anti-human IL-13Rα2 polyclonal antibody at room temperature for 1h. (D) U937 human IL-13Rα2 transfectants were resuspended in buffer containing 1:5 diluted human plasma samples prior to flow cytometry using goat anti-human IL-13Rα2 antibody and FITC-rabbit anti-goat IgG antibody. Shaded area: unstained; Solid thin line: stained with normal goat IgG; Solid thick line: stained with anti-human IL-13Rα2; Dotted, dashed and long dashed lines: stained with anti-human IL-13Rα2 in the presence of three different human plasma samples. (E) ELISA of human IL-13Rα2-Fc fusion protein diluted in 1% BSA PBS or 1:20 human plasma samples. (F) ELISA of human IL-13Rα2-Fc fusion protein preincubated with IL-13 (1, 10 or 50 ng/ml).

Figure 2. Effect of memIL-13Rα2- or sIL-13Rα2-specific siRNA on the expression of IL-13Rα2 mRNA and protein in BC3H1 cells

(A) Surface expression of IL-13Rα2 determined by flow cytometry. Shaded area: unstained; Solid thin line: stained with normal goat IgG; Solid thick line: stained with goat anti-mouse IL-13Rα2 polyclonal antibody. (B) sIL-13Rα2 in culture medium. (C) Expression of memIL-13Rα2. (D) Expression of sIL-13Rα2. The data are shown as mean±SD of means from three independent experiments with relative concentration or expression normalized to control siRNA IL-13 plus TNFα group of each experiment. Mem: memIL-13Rα2-specific; Sol: sIL-13Rα2-specific.

Figure 3. Effect of memIL-13Rα2-specific siRNA on the expression of IL-13Rα2 mRNA and protein in U87 and HaCaT cells

(A) IL-13Rα2 in culture medium and cell lysate from U87 cells. (B) IL-13Rα2 in culture medium and cell lysate from HaCaT cells. (C) mRNA expression of IL-13Rα2 in U87 cells. (D) mRNA expression of IL-13Rα2 in HaCaT cells. The data are shown as mean±SD of means from three independent experiments with relative concentration normalized to control siRNA group (U87) or control siRNA IL-4 group (HaCaT) of each experiment.

Figure 4. Effect of memIL-13Rα2-specific siRNA on the expression of IL-13Rα2 mRNA and protein in HPASMC cells

(A) Level of IL-13Rα2 in culture medium and cell lysate. (B) Surface expression of IL-13Rα2 by flow cytometry. Shaded area: unstained; Solid thin line: stained with normal goat IgG; Solid thick line: stained with goat anti-human IL-13Rα2 polyclonal antibody. (C) mRNA expression of IL-13Rα2. The data are shown as mean±SD of means from three independent experiments with relative mean channel fluorescence or concentration normalized to control siRNA group of each experiment.

Figure 5. Effect of GM6001 or MMP-8 Inhibitor on production of sIL-13Rα2 from U87 cells

(A) sIL-13Rα2 in culture medium after treatment. (B) Total IL-13Rα2 in cell lysate after treatment. (C) sIL-13Rα2 in culture medium before treatment. (D) sIL-13Rα2 in culture medium after treatment. (E) Total IL-13Rα2 in cell lysate after treatment. (F) sIL-13Rα2 in culture medium before treatment. The cells were treated with GM6001, GM6001 Negative Control, MMP-8 Inhibitor or MMP-8 Inhibitor Negative Control (10μM or 20μM) for 24h. NC: Negative Control. The data are shown as mean±SD of means from three independent experiments with relative concentration normalized to DMSO group of each experiment.

Figure 6. Evolutionarily conserved regions (ECRs) in human, rhesus monkey and mouse IL-13Rα2 genes

Exon 9 of the human gene (top) contains the transmembrane domain. The level of nucleotide identity to human gene is shown by layer height from 50% to 100%. The ECRs in rhesus monkey and mouse genes are indicated by pink rectangles.