Transforming growth factor-{beta}1 modulates responses of CD34+ cord blood cells to stromal cell-derived factor-1/CXCL12

Abstract

Disruption of stromal cell-derived factor-1 (SDF-1/CXCL12 [CXC chemokine ligand 12]) interaction leads to mobilization of stem/progenitor cells from bone marrow to circulation. However, prolonged exposure of CD34+ cells to SDF-1 desensitizes them to SDF-1. So how do cells remain responsive to SDF-1 in vivo when they are continuously exposed to SDF-1? We hypothesized that one or more mechanisms mediated by cytokines exist that could modulate SDF-1 responsiveness of CD34+ cells and the desensitization process. We considered transforming growth factor-beta1 (TGF-beta1) a possible candidate, since TGF-beta1 has effects on CD34+ cells and is produced by stromal cells, which provide niches for maintenance and proliferation of stem/progenitor cells. TGF-beta1 significantly restored SDF-1-induced chemotaxis and sustained adhesion responses in cord blood CD34+ cells preexposed to SDF-1. Effects of TGF-beta1 were dependent on the dose and duration of TGF-beta1 pretreatment. Phosphorylation of extracellular signal-regulated kinase 1 (Erk1)/Erk2 was implicated in TGF-beta1 modulation of migratory and adhesion responses to SDF-1. Our results indicate that low levels of TGF-beta1 can modulate SDF-1 responsiveness of CD34+ cells and thus may facilitate SDF-1-mediated retention and nurturing of stem/progenitor cells in bone marrow.

Figure 1.

CD34 and CXCR4 expression on freshly isolated versus cytokine-expanded CD34⁺-enriched cord blood cells. (A) Representative dot blot of CD34 and CXCR4 expression on freshly isolated CD34⁺ cells and CD34⁺ cells expanded in a cocktail of cytokines (see “Materials and methods”) for 4 days. Freshly isolated and expanded CD34⁺ cells were stained using CD34-FITC, CXCR4-APC monoclonal antibodies (mAbs) and analyzed by multivariant flow cytometry. Gates were set on the basis of cell staining with matched-isotype control mAbs. Similar results were obtained in 8 other experiments performed independently. (B) Representative dot blot showing expression of CD34 and CXCR4 on ex vivo–expanded CD34 cells after 24 hours of culture in cytokine cocktail alone (medium) or along with TGF-β1 (0.5 ng/mL), SDF-1 (200 ng/mL), or the combination of SDF-1 (200 ng/mL) + TGF-β1 (0.5 ng/mL). CXCR4 expression on CD34⁺ cells is presented as mean ± SD of 3 independent experiments. MFI indicates mean fluorescence intensity.

Figure 2.

Desensitization of CXCR4. Nonexpanded CD34⁺ cells were pretreated with SDF-1 (200 ng/mL) for 1 hour, 20 hours, or kept in medium alone. Cells were washed and either immediately loaded with Fluo-3AM or kept in medium for 1 hour or 4 hours (in the absence of SDF-1) and then loaded with Fluo-3AM. The time noted in parentheses in the insert key box denotes the time for which SDF-1–pretreated cells were kept in medium prior to SDF-1 stimulation. Baseline fluorescence was first recorded and calcium flux following SDF-1 (200 ng/mL) stimulation (indicated by ↑) was analyzed by FACScan. Increase in fluorescence intensity following SDF-1 addition (compared with baseline), represents the calcium flux in cells and is shown on the y-axis of the plot. Data represent mean ± SEM of 3 independent experiments. *P < .05 and **P < .05 compared with cells pretreated in with SDF-1 for 1 hour and 20 hours, respectively.

Figure 3.

Effect of TGF-β1 on chemotactic response to SDF-1 of CD34⁺ cells preexposed to SDF-1. (A) CB CD34⁺ cells were expanded for 4 days as described in “Materials and methods.” Expanded CD34⁺ cells were first pretreated with indicated doses of SDF-1 for 24 hours, and then after washing the cells their chemotactic activity toward SDF-1 (200 ng/mL) was determined. *P < .05 compared with cells cultured in cytokine cocktail. (B) CD34⁺ cells were cultured in a cytokine cocktail (SCF, TPO, and Flt-3 ligand) alone, or along with TGF-β1, SDF-1, or SDF-1 and TGF-β1 for 24 hours. Cells were washed, and their chemotactic activity to SDF-1 (200 ng/mL) was determined in migration assay. (C) CD34⁺ cells were pretreated with SDF-1 (200 ng/mL) in the presence of various doses of TGF-β1 for 24 hours, and their chemotactic response to SDF-1 (200 ng/mL) activity was determined. *P < .05 compared with cells pretreated with SDF-1. (D) Cytokine-expanded CD34⁺ cells were coincubated with SDF-1 and TGF-β1 or SDF-1 alone for the indicated time periods, and SDF-1 (200 ng/mL) directed chemotactic activity of the cells was assessed. *P < .05 compared with cells pretreated with SDF-1 alone. (E) Freshly isolated CD34⁺ cells were cultured in medium alone or along with TGF-β1, SDF-1, or SDF-1 and TGF-β1 for 20 hours. Cells were washed, and their chemotactic activity to SDF-1 (200 ng/mL) was determined in migration assay. (A-D) Data are presented as mean ± SD of 4 separate experiments. (E) Data are presented as mean ± SEM for 3 separate experiments. Of 3 experiments shown, 2 were conducted by pooling cells from different CB collections (after pretreatments), and in 1 experiment cells from a single CB collection were used. *P < .05 compared with cells cultured in medium alone and **P < .05 compared with SDF-1–pretreated cells.

Figure 4.

Migration potential of CD38^lo/CD34⁺⁺ cells and myeloid progenitors pretreated with TGF-β1 and SDF-1. (A) Ex vivo–expanded CD34⁺ cells cultured in cytokine cocktail alone (medium) or containing TGF-β1 (0.5 ng/mL), SDF-1 (200 ng/mL), or SDF-1 (200 ng/mL) along with TGF-β1 (0.5 ng/mL) for 24 hours were assayed for their chemotactic activity toward SDF-1 (200 ng/mL). The proportion of CD38^lo/CD34⁺⁺ cells that migrated was determined by staining aliquots of input and migrated cells with CD34-FITC and CD38-APC and isotype-matched control antibodies. Data are presented as mean ± SD of 3 independent experiments. (B) Freshly isolated CD34⁺ cells cultured in medium alone or containing TGF-β1 (0.5 ng/mL), SDF-1 (200 ng/mL), or SDF-1 (200 ng/mL) along with TGF-β1 (0.5 ng/mL) for 20 hours were assayed for their chemotactic activity toward SDF-1 (200 ng/mL). The proportion of CD38^lo/CD34⁺⁺ cells that migrated was determined by staining aliquots of input and migrated cells with CD34-FITC and CD38-APC and isotype-matched control antibodies. Data are presented as mean ± SEM of 3 independent experiments. (C) Cytokine-expanded and (D) nonexpanded, myeloid in input (upper chamber), and migrated cells (lower chamber) were assayed by methylcellulose colony assay. The fold increase in progenitor cell migration for SDF-1 and SDF-1 + TGF-β1 pretreatment was normalized with respect to the efficiency of progenitor migration in untreated CD34⁺ cells. (C) Data are represented as mean ± SD of 3 independent experiments, and (D) data are represented as mean ± SEM of 3 independent experiments. *P < .05 compared with cells cultured in medium and **P < .05 compared with cells pretreated with SDF-1.

Figure 5.

SDF-1–induced actin polymerization and calcium (Ca²⁺) flux in CD34⁺ cells. (A) Ex vivo–expanded CD34⁺ cells and (B) nonexpanded CD34⁺ cells were pretreated with TGF-β1 (0.5 ng/mL), SDF-1 (200 ng/mL), SDF-1 (200 ng/mL) + TGF-β1 (0.5 ng/mL), or cytokine cocktail alone (medium) for 20 to 24 hours. Cells were then washed and stimulated with 200 ng/mL SDF-1 for the indicated times to induce actin polymerization. Cells were stained with FITC-phalloidin and then subjected to flow cytometry. Data represent the mean ± SD of 3 independent experiments. *P < .05 compared with CD34⁺ cells pretreated with medium, TGF-1, or SDF-1 + TGF-β1. SDF-1–mediated calcium flux in (C) ex vivo–expanded CD34⁺ cells and (D) nonexpanded CD34⁺ cells pretreated with medium alone, TGF-β1 (0.5 ng/mL), SDF-1 (200 ng/mL), or TGF-β1 (0.5 ng/mL) along with SDF-1 (200 ng/mL). Cells were loaded with Fluo-3AM. Baseline fluorescence was first recorded and then calcium flux following SDF-1 (200 ng/mL) stimulation (indicated by ↑) was analyzed by FACScan. Increase in fluorescence intensity following SDF-1 addition (compared with baseline) represented the calcium flux in the cells and is shown on the y-axis of the plot. Data represent the mean ± SEM of 3 independent experiments. *P < .05 compared with cells pretreated in cytokine cocktail alone or along with TGF-β1.

Figure 6.

Improved chemotactic activity of cells pretreated with SDF-1 and TGF-β1 is associated with enhanced Erk1/Erk2 phosphorylation in response to SDF-1. (A) Ex vivo–expanded CD34⁺ cells were cultured in cytokine cocktail alone (medium) or along with TGF-β1, SDF-1, or SDF-1 and TGF-β1 for 24 hours. Cells were harvested, washed, and either left unstimulated or stimulated with 200 ng/mL SDF-1 for 3 minutes. Cells were solubilized, and extracts were subjected to immunoblotting by using anti–phospho Erk1/Erk2 or anti–phospho-Akt (Ser473) antibodies. After stripping and saturating nonspecific protein binding, we reprobed the same blots with anti–Erk1/Erk2 or anti-Akt antibody. Blots were developed by a chemiluminescence reaction and exposed to radiographic film. A representative experiment of 1 of 3 experiments performed is shown. (B) Effect of MEK inhibitor, PD98059, on migration activity of CD34⁺ cells pretreated with cytokine cocktail along with SDF-1 (200 ng/mL) or SDF-1 (200 ng/mL) + TGF-β1 (0.5 ng/mL) SDF-1 for 24 hours. Cells were tested for their migration response to SDF-1 (200 ng/mL). Data represent the mean ± SD of 4 independent experiments.

Figure 7.

SDF-1 induces adhesion of CD34⁺ cells to fibronectin. (A) Ex vivo–expanded CD34⁺ cells were pretreated with cytokine cocktail alone (medium) or containing TGF-β1 (0.5 ng/mL), SDF-1 (200 ng/mL), or SDF-1 (200 ng/mL) + TGF-β1 (0.5 ng/mL) for 24 hours. The cells were washed and then plated on fibronectin (10 μg/mL) coated wells and incubated for 30 minutes. Cells were either left unstimulated (□) or were stimulated with SDF-1 (200 ng/mL) for the full 30 minutes (▪) or only for the last 2 minutes () of the adhesion assay. Data represent the mean ± SD of 3 independent experiments. *P < .05 compared with adhesion response in absence of SDF-1 stimulation (basal adhesion) for respective pretreatment and **P < .05 compared with cells pretreated with SDF-1 alone. (B) CD34⁺ cells pretreated under various conditions were preincubated with anti–VLA-4 and anti–VLA-5 antibodies and assayed in adhesion assay. The cells were plated in fibronectin-coated plates and stimulated with SDF-1 for 30 minutes. Data represent the mean ± SD of 3 independent experiments. *P < .05 compared with adhesion response in the absence of antibodies. (C) Nonexpanded CD34⁺-enriched cord blood cells were cultured in medium (containing 20% FCS) alone or along with TGF-β1 (0.5 ng/mL), SDF-1 (200 ng/mL), or SDF-1 (200 ng/mL) and TGF-β1 (0.5 ng/mL) for 24 hours. These cells were then assayed for their ability to bind to fibronectin in the absence or presence of 200 ng/mL SDF-1 for 2 minutes (transient response) and 30 minutes (sustained response). Nonadherent cells were removed, and adherent cells were quantified as described in “Materials and methods.” Data represent the mean ± SEM of 3 independent experiments. *P < .05 compared with adhesion response in absence of SDF-1 stimulation (basal adhesion) for respective pretreatment. (D) Fresh CD34⁺ cells pretreated under various conditions for 24 hours were preincubated with anti–VLA-4 antibody and assayed in adhesion assay as described in “Materials and methods.” Data represent mean ± SEM of 1 experiment. *P < .05 compared with adhesion response in absence of antibodies.