Report

Discovery of a Cytokine and Its Receptor by Functional Screening of the Extracellular Proteome

See allHide authors and affiliations

Science  09 May 2008:
Vol. 320, Issue 5877, pp. 807-811
DOI: 10.1126/science.1154370

You are currently viewing the figures only.

View Full Text

Log in to view the full text

Log in through your institution

Log in through your institution

  1. Fig. 1.

    Identification of IL-34 using a functional screening approach. (A) Comparison of functional selectivity of IL-15, IGF-II, FGF3, and IL-34. Using P values for a Wilcoxon rank sum test, activities (–logP) for several proteins against their respective sorted assays are shown. The assays were sorted from the lowest to the highest P value, producing a protein-dependent list of sorted assays. Assay descriptions are shown in table S4. (B) Analysis of functional selectivity of secreted proteins. Functional selectivity of 1910 secreted proteins in the screening set was analyzed using the maximal differential activity s and selectivity index k.

  2. Fig. 2.

    Activities and cell-binding selectivity of purified IL-34 protein. (A) Dose-response curve of purified IL-34 protein in a monocyte viability assay. Recombinant IL-34 protein was purified from conditioned medium of 293T cells stably transfected with IL-34 cDNA. Relative viability (survival and/or proliferation) of primary human monocytes induced by the purified IL-34 protein was determined by the cell viability (CellTiter-Glo) assay (Promega). In this assay, the amount of ATP released from lysed cells was determined based on the luminescence signal [relative light unit (RLU)] generated from reaction with luciferin in the presence of luciferase. (B) Specific binding of IL-34 to human monocytes in PBMC. PBMC incubated with unlabeled IL-34 or bovine serum albumin (BSA) as competitors at 200-fold molar excess were incubated with biotinylated IL-34 protein and stained with streptavidin-allophycocyanin (APC). The cells also were labeled with fluorescein isothiocyanate (FITC)–conjugated antibodies against lineage markers, CD14 (monocytes), CD3 (T cells), and CD56 (NK cells) and analyzed by flow cytometry.

  3. Fig. 3.

    Identification of the receptor for IL-34 by screening of the ECDs of transmembrane proteins. IL-34 protein was individually incubated with conditioned medium of 293T cells transfected with one of the cDNAs encoding ECDs of transmembrane proteins. Human primary monocytes were then incubated with the treated IL-34 protein and analyzed by the cell viability assay. Results from a screen of the collection of 858 ECDs are shown as the standard deviation from the median (sigma from median). Purple circles (inhibitors) indicate clones for which activity was detected with greater than twofold standard deviation of the assay plate for more than one test. Cells without IL-34 treatment were used as controls (blue circles) on each assay plate. The activities of the ECD clones that inhibited IL-34 activity on monocytes in primary and secondary screens are shown in table S6. CSF-1R ECD V5-His Tag, CSF1R ECD with a V5-His epitope tag.

  4. Fig. 4.

    IL-34 functions as a specific and independent ligand of CSF-1 receptor, stimulates CSF-1R–dependent phosphorylation of ERK1/2, and promotes CFU-M colony formation. (A) IL-34 binding to CSF-1R on human monocytic THP-1 cells. Biotinylated IL-34 protein was incubated with conditioned media of 293T cells transfected with cDNA encoding CSF-1R ECD (i), GM-CSFR ECD (ii), or vector. THP-1 cells were then incubated with the treated IL-34 protein, labeled with streptavidin-APC, and analyzed by flow cytometry. THP-1 cells preincubated with BSA (iii), unlabeled IL-34 (iv), CSF-1 (v), neutralizing antibody to CSF-1R (Anti-CSF-1R Ab) and control mouse immunoglobulin G1 (mIgG1) (vi), or buffer control [phosphate-buffered saline (PBS)] were labeled with biotinylated IL-34, stained with streptavidin-APC, and analyzed by flow cytometry. (B) IL-34 function on monocytes is independent of CSF-1. Human monocytes were incubated with IL-34 or CSF-1 that was heat-inactivated (100°C for 5 min) or treated with rabbit polyclonal antibodies to IL-34 (rabbit anti-IL-34 Ab), a mouse antibody to CSF-1 [mouse anti-CSF-1 Ab (R&D Systems, no. MAB216], or control IgG [rabbit IgG (Millipore) and mouse IgG2a (R&D Systems, no. MAB003)] and assayed for viability by CellTiter-Glo assay (Promega). Relative viability is shown as luminescence (RLU). (C) Inhibition of IL-34 activity on ERK1/2 phosphorylation by a CSF-1R–specific inhibitor. Human primary monocytes were incubated with medium control, 5 nM IL-34, or 5 nM CSF-1 at 37°C for 3 min in the presence or absence of 60 nM CSF-1R–specific inhibitor, GW2580 (11). The monocyte cell lysate was analyzed by Western blotting using an antibody to phospho-ERK1/2 (p-ERK) (Santa Cruz Biotechnology, Inc., no. sc-7383). The Western blot was then stripped and probed with antibodies to ERK (Santa Cruz Biotechnology, Inc., no. sc-94) for detection of the total ERK. The results were reproducible in three experiments. (D) IL-34 promotes CFU-M colony formation from human bone marrow. Colony formation assays were performed by StemCell Technologies, Inc. as described (14, 15), using CSF-1 (R&D Systems, Inc.) or IL-34, at indicated concentrations. Representative data (n = 3 replicates for each concentration and treatment) from one of three experiments are shown. Images of the CFU-M colonies and of the May-Grunwald-Giemsa–stained cells are shown in fig. S9.

Stay Connected to Science