A recombinant single-chain IL-7/HGFbeta hybrid cytokine induces juxtacrine interactions of the IL-7 and HGF (c-Met) receptors and stimulates the proliferation of CFU-S12, CLPs, and pre-pro-B cells

Abstract

A novel recombinant interleukin-7/hepatocyte growth factor beta-chain (IL-7/HGFbeta) hybrid cytokine was constructed as a single chain (sc) composed of IL-7 and HGFbeta connected by a flexible linker. Unlike recombinant (r) IL-7, which stimulated pro-B cells and pre-B cells only, scIL-7/HGFbeta stimulated the proliferation of pre-pro-B cells, common lymphoid progenitors (CLPs), and colony-forming unit (CFU)-S12 in cultures of IL-7-/- mouse BM cells. When injected in vivo, 3- to 4-fold more splenic B-lineage cells appeared in recipients of bone marrow (BM) cells from the scIL-7/HGFbeta-stimulated cultures than from rIL-7-stimulated cultures. Moreover, on a per-cell basis, scIL-7/HGFbeta culture-generated cells produced 16- to 20-fold more BM and splenic B-lineage cells than did normal BM cells. Antibody blocking, receptor phosphorylation, and confocal microscopy demonstrated that scIL-7/HGFbeta signals though both the IL-7 and HGF (c-Met) receptors, which form IL-7R/c-Met complexes on the surface of CLPs and pre-pro-B cells. In addition, the IL-7Ralpha chain, gammac chain, and c-Met were coisolated from purified CLPs and pre-pro-B cells on scIL-7/HGFbeta affinity gels, indicating that they are major components of the IL-7/HGFbeta receptor. Hence, the present results demonstrate that the IL-7/HGFbeta hybrid cytokine efficiently and selectively stimulates the most primitive B-lineage precursors in BM by inducing juxtacrine interactions between the IL-7 and c-Met receptors.

Figure 1.

Cloning strategy for ligation of the IL-7/HGFβ coding sequences into baculovirus transfer vector pAcGP67A. The gp67 secretion sequence, IL-7, linker, and HGFβ DNA are constructed by overlapping PCR as described in “Materials and methods.”

Figure 2.

Stimulation of mouse bone marrow cells by rIL-7 and/or scIL-7/HGFβ in vitro. Freshly harvested BM cells from IL-7^-/- mice were cultured in RPMI-1640 containing 2-ME in the presence of 10 ng/mL rIL-7 or 30 ng/mL scIL-7/HGFβ, or both. Nonadherent cells were harvested at day 17 and analyzed by flow immunocytometry. Top row shows representative histograms of B220⁺ and B220^- cells in each culture. The vertical standards indicate the peaks (or theoretical peak; dashed line) of florescence intensity and are used to eliminate most of the overlap regions between the peaks. Middle row shows the contour plots for CD43 and HSA of the B220^- and B220⁺ cells to the left and right of the peaks in the top row. The various fractions of developing B-lineage cells and their relative proportions in the B220^- and B220⁺ cell subsets are indicated for each quadrant. Bottom row shows the relative proportions of fractions Ao (CLPs), A₁ (early pre-pro-B cells) and A2 (late pre-pro-B cells).

Figure 3.

Incorporation of BrdU by culture-generated BM lymphoid cells stimulated or cross-stimulated in vitro with rIL-7 or scIL-7/HGFβ. BM cells from IL-7^-/- mice were cultured in the presence of rIL-7 (10 ng/mL) or scIL-7/HGFβ (30 ng/mL) for 19 days. The cells were washed, cytokine-starved for 5 hours, stimulated with the homologous or heterologous cytokine for 3 hours, pulsed with BrdU, and stained with combinations of antibodies to B220, HSA, AA4.1, CD43, CD4, and BrdU. (A,C) Distribution early B-lineage subsets in each culture system. (B,D) Percentage of BrdU⁺ cells in each fraction of B-lineage cells. (A-B) ▪ indicates IL-7/HGFβ-generated cells stimulated with IL-7/HGFβ; , IL-7-generated cells stimulated with IL-7. (C-D) ▪ indicates IL-7/HGFβ-generated cells stimulated with IL-7; , IL-7-generated cells stimulated with IL-7/HGFβ. Means of duplicate samples are shown. Data are from 1 representative experiment of 2.

Figure 4.

Ability of antibodies to the IL-7R and/or c-Met to inhibit the stimulation of mouse bone marrow cells by rIL-7 or scIL-7/HGFβ. Culture-generated BM cells (4 × 10⁵ cells/well) from IL-7^-/- mice were incubated for 3 days in the presence of rIL-7 (10 μg/mL) or scIL-7/HGFβ (30 μg/mL) to which antibodies against IL-7Rα, γc, and/or c-Met (10 μg/mL) were added. Incorporation of [methyl-3H] thymidine (mean counts per minute [CPM] ± SD) was determined after a 12-hour pulse. ^*P < .05 between antibody-treated and untreated values (similar results were obtained with isotype controls). ^**P < .05 versus value for anti-IL-7Rα or anti-HGFR alone. One representative experiment of 4 is shown.

Figure 5.

Expression of the IL-7R and/or c-Met on culture-generated and normal pre-pro-B cells and pro-B cells. Images of IL-7R (green) and c-Met (red) examined by confocal microscopy show colocalization (yellow) patching and capping of IL-7R and c-Met on scIL-7/HGFβ culture-generated and enriched pre-pro-B cells from normal BM. IL-7 culture-generated cells expressed the IL-7R only. Some cytoplasmic staining is also seen in the latter cells, which were stained after the smears had been fixed. The top panels are isotype controls. Scale bars equal 10 μm.

Figure 6.

Analysis of the purified IL-7/HGFβ receptor proteins. The IL-7/HGFβ receptor complex was isolated on a scIL-7/HGFβ affinity gel from purified culture-generated CLP/pre-pro-B cells. The eluates were subjected to SDS-PAGE under reducing (R) or nonreducing (NR) conditions, and Western blotting was done with antibodies to IL-7Rα, γc, or c-Met.

Figure 7.

Ability of rIL-7 or scIL-7/HGFβ to activate Jak3 and/or c-Met in mouse B-lineage bone marrow cells. B-lineage cells generated in cultures of IL-7^-/- mouse BM cells supplemented with rIL-7 or scIL-7/HGFβ were harvested, placed in cytokine-free medium for 5 hours, and then stimulated with the homologous cytokine for 10 or 30 minutes. The supernatants from lysed cells were immunoprecipitated with anti-Jak3 or antiphosphotyrosine antibody and subjected to SDS-PAGE and Western blotting using the indicated antibodies. Arrows indicate phospho-c-Met or phospho-Jak3.