Expression of functional interleukin-3 receptors on Hodgkin and Reed-Sternberg cells

Abstract

The human interleukin-3 receptor (IL-3R) is a heterodimeric complex consisting of an IL-3-specific alpha chain (IL-3Ralpha) and a common beta chain (beta(c)), this latter shared with the receptors for granulocyte-macrophage colony-stimulating factor and IL-5. Despite extensive research on cytokine circuitries regulating proliferation and survival of tumor cells in Hodgkin's disease (HD) the functional expression of IL-3Rs in this pathobiological entity has not yet been investigated. In the present study, we demonstrate that the great majority (>90%) of malignant Hodgkin and Reed-Sternberg cells of classic HD (19 of 19 analyzed cases) express IL-3Ralpha by immunostaining of frozen sections and cell suspensions from involved lymph nodes. Accordingly, HD cell lines (L428, KMH2, HDLM2, L1236) expressed the alpha and beta chains of IL-3R both at the mRNA and protein level, with a molecular size of IL-3Ralpha identical (70 kd) to that expressed by human myeloid cells. Exogenous IL-3 promoted the growth of cultured Hodgkin and Reed-Sternberg cells, such effect being potentiated by IL-9 co-stimulation, and was able to partially rescue tumor cells from apoptosis induced by serum deprivation. This data suggests an involvement of IL-3/IL-3R interactions in the cellular growth of HD through paracrine mechanisms.

Figure 1.

Expression of IL-3Rα in reactive lymph node tissues. Expression of IL-3Rα is detected on the endothelium of a high endothelial venule and on surrounding mononuclear cells. Frozen section of a reactive lymph node stained with anti-IL-3Rα mAb. APAAP immunostaining, hematoxylin counterstain. Original magnification, ×250.

Figure 2.

Expression of IL-3Rα on H-RS cells of cHD. Serial frozen sections of a lymph node involved by cHD (A and B) are presented to show that H-RS cells, detected by their typical marker CD30 (A), express IL-3Rα (B). In a frozen section from another lymph node involved by cHD (C) a H-RS cell expressing IL-3Rα displays a cytoplasmic labeling of weak intensity. In the inset, another H-RS cell expressing IL-3Rα with a cytoplasmic labeling of a higher intensity is reported. APAAP immunostaining, hematoxylin counterstain. Arrows indicate morphologically recognizable IL-3Rα-expressing H-RS cells. Original magnifications: ×250 (A, B); ×630 (C).

Figure 3.

Expression of IL-3Rα on H-RS cells. Cytospin preparations from lymph nodes involved by cHD. A: H-RS cells expressing IL-3Rα display a membrane staining associated with cytoplasmic labeling. B: In this case, staining of H-RS cells is mainly membranous. APAAP immunostaining, hematoxylin counterstain. Original magnification, ×630.

Figure 4.

Expression of mRNAs encoding for α and β chains of the IL-3 receptor and IL-3 in HD-derived cell lines. Total RNA was isolated from L428, L540, KMH2, HDLM2, and L1236 cells, reverse-transcribed, and amplified with primers pairs specific for the α (IL-3Rα) and the common β chain of the human receptor for IL-3, and the human IL-3 as detailed in Materials and Methods. Sources of negative (−) and positive (+) control RNAs, are specified in Materials and Methods. Amplified products were resolved on 1.5% agarose gel and visualized by ethidium bromide and ultraviolet light. In all cases, the same cDNA bulks were also amplified with primers specific for the housekeeping gene β-actin. MW, 100-bp molecular weight marker; the double-intensity band corresponds to 600 bp.

Figure 5.

Expression of surface IL-3Rα and β chains on HD-derived cell lines. Flow cytometry profiles were generated by incubating cells with PE-conjugated anti-IL-3Rα mAb 7G3 (closed histograms) and with the biotin-conjugated anti-β chain mAb 3D7, followed by PE-conjugated streptavidin (shaded histograms). Dotted- and continuous-line histograms represent staining profiles obtained with irrelevant isotype-matched PE-conjugated and biotin-conjugated control Igs, respectively. The x and y axes indicate the logarithm of relative red fluorescence intensity and the relative cell counts, respectively.

Figure 6.

Immunostaining patterns of HD-derived cell lines with anti-IL-3Rα antibody. A: L428 cell line. B: HDLM2 cell line. Cultured H-RS cells show an intense membrane staining associated with a strong cytoplasmic reactivity or a selective labeling of cell membranes. Cytospin preparations. APAAP immunostaining, hematoxylin counterstain. Original magnifications, ×250.

Figure 7.

Detection of IL-3Rα in HD-derived cell lines by Western blotting. Cell lysates (50 μg/lane) from the different HD-derived cell lines, KG1A cells (positive control), and HL60 cells (negative control) were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The blots were then incubated with 1.5 μg/ml of anti-IL-3Rα mAb and shown by chemiluminescence. The position of molecular weight markers is indicated.

Figure 8.

Growth of HD-derived cell lines by IL-3. A: Cells were cultured in semisolid medium (0.8% methylcellulose) in the presence of increasing concentrations of IL-3. After 14 days of incubation, plates were observed under phase contrast microscopy and aggregates with ≥40 cells were scored as colonies. Results represent the mean ± SEM of eight replicate wells from three different experiments. The data (filled squares) is presented as percent growth relative to spontaneous colony formation (medium only). Filled circles represent clonogenic growth obtained at the maximal IL-3 concentration (50 ng/ml) in the presence of 10 μg/ml of a mouse anti-human IL-3 mAb. B: Cooperative effect of IL-3 and IL-9 on the clonogenic growth of cultured H-RS cells. Clonogenic assays were performed in the presence of medium alone (open bars), IL-3 (5.0 ng/ml; filled bars), IL-9 (5.0 ng/ml; shaded bars), and their combination (striped bars). Results represent the mean ± SEM of eight replicate wells from three different experiments. The data are presented as percent growth relative to spontaneous colony formation (medium only). C: Cells from HD-derived cell lines (2.0 × 10⁴/ml) were cultured in 96-well flat-bottomed microplates in the presence of IL-3 (50 ng/ml). After 72 hours cells were pulsed with 1 μCi/well ³H-thymidine for the final 12 hours of culture, harvested onto glass fiber membranes, and counted in a liquid scintillator β-counter. Data are presented as percent ± SEM of ³H-thymidine uptake in the presence of IL-3 respective to control (medium alone). D: L1236 cells (2.0 × 10⁴/ml) were cultured as in C in the presence of increasing concentrations (0 to 50 ng/ml) of IL-3. Results are expressed as cpm ± SEM. Data in C and D are means of triplicate cultures of four independent experiments.

Figure 9.

Effects of IL-3 on apoptosis of HD-derived cell lines. A: Exponentially growing HD-derived (KMH2, HDLM2, L1236) cell lines were cultured in IMDM containing 2% FCS in the absence (filled bars) and presence (shaded bars) of IL-3 (50 ng/ml). Percentages of apoptotic cells were assessed by flow cytometry analysis of cells stained with the APO2.7 mAb (top) and Annexin V-FITC (bottom) after 48 hours and 72 hours of culture, respectively. Asterisks refer to experimental points in which a statistical significance (P < 0.05) was reached. B: Representative flow cytometry dot plots showing changes in APO2.7 staining of L1236 cells. Cells were cultured for 48 hours in 2% FCS in the absence (medium) and presence of IL-3 (50 ng/ml) and double-stained with PE-conjugated APO2.7 mAb and FITC-conjugated anti-CD30 mAb BerH2.

Figure 10.

Effects of IL-3 on CD95/Fas-mediated apoptosis of the HD-derived cell line HDLM2. HDLM2 cells were cultured for the indicated time points in complete medium containing 5.0% FCS in the presence of the agonistic anti-CD95 mAb CH11 (100 ng/ml; open circles), anti-CD95 mAb CH11 plus IL-3 (50 ng/ml; filled squares), an irrelevant isotype-matched (IgM) mouse mAb (CNT; open squares), and IL-3 alone (50 ng/ml; filled circles). Percentages of apoptotic cells were assessed by staining cells with the APO2.7 mAb (top) and Annexin V-FITC (bottom) and analyzed by flow cytometry.