by Mineo Iwata, Lynn Graf, Norihiro Awaya, and Beverly Torok-Storb Functional interleukin-7 receptors (IL-7Rs) are expressed by marrow stromal cells: binding of IL-7 increases levels of IL-6 mRNA and secreted protein by Mineo Iwata, Lynn Graf, Norihiro Awaya, and Beverly Torok-Storb Blood Volume 100(4):1318-1325 August 15, 2002 ©2002 by American Society of Hematology

Microarray data.(A) Comparative analysis of microarray data of HS-27a and HS-5 cell lines. Microarray data.(A) Comparative analysis of microarray data of HS-27a and HS-5 cell lines. Gene sequences that were determined to have significantly different (P < .05) levels of expression in HS-27a cells compared to HS-5 cells are represented by a dot. Gene expression levels in HS-27a cells are shown on the x-axis. The y-axis indicates gene expression of HS-27a divided by HS-5. Therefore, genes expressed highly in HS-27a compared to the Universal control are at the far right in both the upper and lower quadrants. Genes that are also expressed to a greater degree in HS-27a compared to HS-5 are in the upper right quadrant. Two open circles in the upper right quadrant designate IL-7R. The dotted line indicates 10-fold higher gene expression for HS-27a than HS-5. (B) Microarray data on cytokine receptors in HS-27a and HS-5. Gene expression of 22 cytokine receptors from HS-27a (solid bars) and HS-5 (open bars) is shown as the ratio of hybridization signal from each stromal cell line to the hybridization signal from the Universal control RNA. Each bar represents the average ± SD for 4 samples. Stars indicate cytokine receptors whose gene expression is statistically different between HS-27a and HS-5 (*P < .05, **P < .005, and ***P < .0001). Mineo Iwata et al. Blood 2002;100:1318-1325 ©2002 by American Society of Hematology

Northern blot analysis of IL-7R gene expression Northern blot analysis of IL-7R gene expression.Total RNA (20 μg/lane) from HS-27a, HS-5, or PBMCs was separated on an RNA agarose gel and blotted onto a nylon membrane. Northern blot analysis of IL-7R gene expression.Total RNA (20 μg/lane) from HS-27a, HS-5, or PBMCs was separated on an RNA agarose gel and blotted onto a nylon membrane. The membrane was probed with a P32-labeled IL-7R probe (A). The DNA sequence of the probe covers 1098 base pairs from the 3′ end and was confirmed to hybridize to human IL-7R. The bound probe was stripped off, and the membrane was reprobed with a P32-labeled actin probe (B). The data indicate a strong signal for IL-7R sequences in RNA isolated from HS-27a and PBMCs but not HS-5 cells. Mineo Iwata et al. Blood 2002;100:1318-1325 ©2002 by American Society of Hematology

Immunoblot analysis of IL-7R in HS-27a, LTCs, and PBMCs Immunoblot analysis of IL-7R in HS-27a, LTCs, and PBMCs.(A) Total protein extracts of HS-27a, LTCs, and PBMCs were analyzed for IL-7R by Western blot. Immunoblot analysis of IL-7R in HS-27a, LTCs, and PBMCs.(A) Total protein extracts of HS-27a, LTCs, and PBMCs were analyzed for IL-7R by Western blot. The 30-kd form, which bound to the anti–IL-7R-IC antibodies, was detected in both HS-27a and primary stromal cell extracts (LTCs) but was almost absent in the PBMC extract. Comparable results were obtained from 4 different cell preparations. (B) Subcellular fractions (26 μg/lane) of HS-27a cells were analyzed by Western blot. Lane 1 represents the cytosol fraction; lane 2, the membrane fraction; and lane 3, the insoluble fraction. The full-length form of IL-7R (90 kd) was found in the membrane fraction, and the 30-kd form was exclusively found in the insoluble fraction of HS-27a cells. This is in keeping with the immunocytochemistry data that show staining localized to the nucleus. Mineo Iwata et al. Blood 2002;100:1318-1325 ©2002 by American Society of Hematology

Immunofluorescence localization of intracellular IL-7R Immunofluorescence localization of intracellular IL-7R.HS-27a cells were fixed with methanol and incubated with the anti–IL7R-IC antibodies (A) or control immunoglobulins (B). Immunofluorescence localization of intracellular IL-7R.HS-27a cells were fixed with methanol and incubated with the anti–IL7R-IC antibodies (A) or control immunoglobulins (B). The bound antibodies were detected with Cy5-conjugated secondary antibodies (red). Nuclei of the cells were counterstained with DAPI (blue), and 2-color fluorescence images were captured with a deconvolution fluorescence microscope. The images of a nucleus stained with the anti–IL7R-IC antibodies are reconstructed into a 3-dimensional image and rotated by 45° 4 times as shown in panel C. This clearly shows localization of the intracellular receptor in the nucleus. Original magnification × 400. Mineo Iwata et al. Blood 2002;100:1318-1325 ©2002 by American Society of Hematology

IL-7 binds to HS-27a cells.Cells in suspension were incubated in PBS containing BTN–IL-7, BTN-Ctrl, or no protein (FITC only), followed by incubation with FITC-avidin. IL-7 binds to HS-27a cells.Cells in suspension were incubated in PBS containing BTN–IL-7, BTN-Ctrl, or no protein (FITC only), followed by incubation with FITC-avidin. Live cells were gated, as shown in panel A, and analyzed by flow cytometry (B). Neg indicates the position of unstained control cells. (C) Cells were incubated under adherent conditions with BTN–IL-7, the mixture of BTN–IL-7 plus blocking anti–IL-7 antibodies (BTN–IL-7+Ab), or BTN-Ctrl. The cells were incubated with FITC-avidin and washed, and the fluorescence was measured by a fluorescence plate reader. Each bar represents the average ± SD for 4 samples. Nuclei of the cells incubated with BTN–IL-7 or the mixture of BTN–IL-7 plus blocking anti–IL-7 were counterstained with Hoechst dye, and staining of FITC (green) and Hoechst (blue) was visualized by fluorescence microscopy in panel D or E, respectively. Original magnification × 600. Mineo Iwata et al. Blood 2002;100:1318-1325 ©2002 by American Society of Hematology

Recombinant IL-7 enhanced tyrosine phosphorylation of proteins in HS-27a.In panels A and B, tyrosine phosphorylated proteins were analyzed by immunoblots using an antiphosphotyrosine antibody. Recombinant IL-7 enhanced tyrosine phosphorylation of proteins in HS-27a.In panels A and B, tyrosine phosphorylated proteins were analyzed by immunoblots using an antiphosphotyrosine antibody. (A) HS-27a cells were cultured for 5 minutes in standard media (lane 1), or media containing 2 ng/mL IL-1β (lane 2), 10 ng/mL IL-7 (lane 3), or 100 ng/mL IL-7 (lane 4). (B) Cells were cultured in the presence of 100 ng/mL IL-7 for various times as shown. Arrows indicate positions of the tyrosine phosphorylated proteins, which increased after exposure to recombinant IL-7. MW shows a lane of molecular weight marker proteins. (C) Tyrosine-phosphorylated proteins were immunoprecipitated with the biotinylated recombinant RC20 antiphosphotyrosine antibody and immunoblotted with the anti–IL7R-IC antibodies. Cells were cultured in the presence (lanes 3 and 4) or absence (lanes 1 and 2) of 100 ng/mL IL-7 for 1 hour. Total proteins of the cells were extracted under denaturing conditions and immunoprecipitated with (lanes 1 and 3) or without (lanes 2 and4) the RC20 antibody. Mock immunoprecipitation was conducted with the RC20 antibody but without cellular extracts (lane 5). Mineo Iwata et al. Blood 2002;100:1318-1325 ©2002 by American Society of Hematology

Cytokine production of IL-7–stimulated HS-27a cells Cytokine production of IL-7–stimulated HS-27a cells.(A) Concentrations of the cytokines IL-6, SDF-1α, IL-1β, and macrophage inhibitory protein 1-α (MIP-1α) in conditioned media were determined after 6 days of culture without (open bars) or with 100 ng/mL IL... Cytokine production of IL-7–stimulated HS-27a cells.(A) Concentrations of the cytokines IL-6, SDF-1α, IL-1β, and macrophage inhibitory protein 1-α (MIP-1α) in conditioned media were determined after 6 days of culture without (open bars) or with 100 ng/mL IL-7 (hatched bars) by ELISA. Each bar represents the average ± SD for 4 samples. (B) Northern blot analysis to measure the IL-6 (upper gel) and actin (lower gel) mRNA levels in IL-7 stimulated (+) or control (−) HS-27a cells at different time points. Each lane contained total RNA (10 μg/lane) blotted onto a nylon membrane. The membrane was probed with a P32-labeled IL-6 probe (upper gel). The bound probe was stripped off, and the membrane was reprobed with a P32-labeled actin probe (lower gel). (C) The blots were quantitatively scanned, and the ratio of IL-6 RNA to actin RNA was calculated. The IL-7–stimulated and control cells are represented by solid and dashed lines, respectively. Mineo Iwata et al. Blood 2002;100:1318-1325 ©2002 by American Society of Hematology