IL-13Ralpha2 is a glioma-restricted receptor for interleukin-13

Abstract

We have found that binding sites for interleukin-13 (IL-13) are overexpressed in a vast majority of high-grade astrocytomas (HGAs). These binding sites for IL-13 are distinct from the physiological receptor in that it does not bind IL-4. We also demonstrated that IL-13 receptor alpha 2 protein chain (IL-13Ralpha2), an IL-4-independent receptor for IL-13, is abundant among HGAs, but not in normal organs. To examine if IL-13Ralpha2 is the tumor-associated site for IL-13, we stably transfected normal Chinese hamster ovary (CHO) cells and glioma G-26 cells to express either human (h) or murine (m) IL-13Ralpha2. CHO-hIL-13Ralpha2(+) cells and G-26-h/mIL-13Ralpha2(+) cells, and not CHO and G-26 parental or mock-transfected cells, specifically bound IL-13 in an IL-4-independent manner. The IL-13Ralpha2(+) cells also became highly susceptible to the killing by an IL-13-based cytotoxic fusion protein. In loss of function studies, a HGA cell line, SNB-19, was transfected with antisense (as) hIL-13Ralpha2. as-SNB-19-hIL-13Ralpha2(+) cells lost their natural affinity towards IL-13 and became resistant to IL-13-based cytotoxins. The fact, that IL-13Ralpha2-positive cells bind IL-13 independent of IL-4, become susceptible to IL-13 cytotoxins, and cells deprived of IL-13Ralpha2 receptor lose these features, demonstrates that IL-13Ralpha2 is the brain tumor-associated receptor for IL-13.

Figure 1

(A) hIL-13Rα2 immunoreactivity in a GBM tumor specimen. Nonimmunized serum was used on a contiguous section of the same tumor as a negative control. Tissues were also stained for nuclear presence using DAPI. (B) Cytotoxicity of high-grade astrocytoma cells (SW-1088, U-373 MG, U-87 MG), low-grade astrocytoma cells (H-4), and epidermoid tumor cells (A-431) using 100 ng/ml IL-13.E13K-PE38QQR determined in a colorimetric cell proliferation assay. Vertical lines represent SD. (C) Reactivity of high-grade astrocytoma cells (U-87 MG, U-373 MG, SW-1088), low-grade astrocytoma cells (H-4), and epidermoid tumor cell (A-431) to anti-hIL-13Rα2 serum in a cell ELISA assay. Vertical lines represent SD.

Figure 2

(A) Northern blot of CHO and G-26 cells transfected with hIL-13Rα2 or mIL-13Rα2 and controls. Parental CHO cells (lane 1), mock-transfected CHO (lane 2), CHO-hIL-13Rα2(+) clone 5 (lane 3), parental G-26 cells (lane 4), mock-transfected G-26 (lane 5), G-26-hIL-13Rα2(+) clone 2 (lane 6), and G-26-mIL-13Rα2(+) clone 17 (lane 7) are shown. The size of transcripts is given in kilobases (kb). (B) RT-PCR of hIL-13α2 or mIL-13Rα2 in G-26 and CHO controls and transfected cells. The order of analyzed cells is the same as in (A). The size of PCR-amplified fragments is given in kilobases (kb).

Figure 3

(A) Autoradiography of CHO-hIL-13Rα2(+) clone 5, G-26-hIL-13Rα2(+) clone 2, G-26-mIL-13Rα2(+) clone 17, and control cells using labeled IL-13.E13K. Parental CHO cells (column 1), mock-transfected CHO (column 2), CHO-hIL-13Rα2(+) clone 5 (column 3), parental G-26 cells (column 4), mock-transfected G-26 (column 5), G-26-hIL-13Rα2(+) clone 2 (column 6), and G-26-mIL-13Rα2(+) clone 17 (column 7). The assay was performed in the absence of blocker or in the presence of an excess of unlabeled IL-13.E13K or IL-4. Each experiment was repeated at least four times and the results shown are a representative sample of one of the experiments. (B) Histogram of peak densities of ¹²⁵I IL-13.E13K binding in the absence or presence of IL-13.E13K and IL-4 in CHO-hIL-13Rα2(+) clone 5, G-26-hIL-13Rα2(+) clone 2, G-26-mIL-13Rα2(+) clone 17, and control cells. Column designations are the same as in (A). Measurements were taken from scanned X-rays shown in (A).

Figure 4

Cytotoxicity using IL-13.E13K-PE38QQR determined in a colorimetric cell proliferation assay. Vertical bars represent SD. (A) CHO parental cells, CHO mock-transfected, and CHO-hIL-13Rα2(+) clones 2 and 5. (B) G-26 parental cells, G-26 mock-transfected, and G-26-hIL-13Rα2(+) clones 2 and 5. (C-E) Neutralization assays in which the cell killing by IL-13.E13K-PE38QQR in IL-13Rα2(+) clones is blocked in the presence of an excess of IL-13.E13K, but not IL-4, in CHO and G-26 cells. (F) G-48a and SNB-19 HGA cells and neutralization of IL-13.E13K-PE38QQR cytotoxicity in the presence of an excess of IL-13.E13K, but not IL-4.

Figure 5

(A) Northern blot of IL-13Rα2 and actin in SNB-19 mock-transfected, and as-SNB-19-hIL-13Rα2(+) clones 1 and 6. (B) IL-13Rα2 immunoreactivity in SNB-19 cells transfected with as-IL-13Rα2 (clones 1 and 6) and with vector only. DAPI nuclear staining was also performed. (C) Flow cytometry performed on SNB-19 parental cells and as-SNB-19-IL-13Rα2(+) clone 6 cells. The bottom graph juxtaposes the distribution of the two experiments. Mean values of each distribution are listed in the area under the peaks. Experiment was repeated three times.

Figure 6

(A) Autoradiography and histogram of peak densities of IL-13 binding sites in SNB-19 parental cells, SNB-19 mock-transfected, and in as-SNB-19-hIL-13Rα2(+) clones treated with labeled IL-13.E13K. Each experiment was repeated at least four times and the results shown are a representative sample of the experiments. (B) Cytotoxicity assay using IL-13.E13K-PE38QQR cytotoxin or wild-type PE in SNB-19 parental cells, SNB-19 mock-transfected, and in as-SNB-19-hIL-13Rα2(+) clones 1 and 6. Vertical bars represent SD.

Figure 7

Schemata of possible therapeutic interventions by using IL-13Rα2 as a molecularly defined target.