Altered responsiveness to chemokines due to targeted disruption of SHIP

Abstract

SHIP has been implicated in negative signaling in a number of hematopoietic cell types and is postulated to downregulate phosphatidylinositol-3-kinase- (PI-3K-) initiated events in diverse receptor signaling pathways. Because PI-3K is implicated in chemokine signaling, we investigated whether SHIP plays any role in cellular responses to chemokines. We found that a number of immature and mature hematopoietic cells from SHIP-deficient mice manifested enhanced directional migration (chemotaxis) in response to the chemokines stromal cell-derived factor-1 (SDF-1) and B-lymphocyte chemoattractant (BLC). SHIP(-/-) cells were also more active in calcium influx and actin polymerization in response to SDF-1. However, colony formation by SHIP-deficient hematopoietic progenitor cell (HPCs) was not inhibited by 13 myelosuppressive chemokines that normally inhibit proliferation of HPCs. These altered biologic activities of chemokines on SHIP-deficient cells are not caused by simple modulation of chemokine receptor expression in SHIP-deficient mice, implicating SHIP in the modulation of chemokine-induced signaling and downstream effects.

Figure 1

Bone marrow HPC, macrophages and B cells from SHIP-deficient mice have enhanced chemotaxis to SDF-1. (a) Bone marrow cells were examined for their ability to transmigrate from the upper chamber toward SDF-1 at indicated concentrations in the lower chamber. After chemotaxis, myeloid progenitors in input and the lower chamber were assayed by methylcellulose colony assay. HPC (CFU-GM) migration was normalized for the number of input colony-forming HPCs to obtain HPC migration rate (% of input ± SD). Chemotaxis of CD11b/Mac-1⁺ F4/80⁺ bone marrow macrophages (b) and bone marrow B220⁺ B cells (c) in response to SDF-1. Data are expressed as the mean (± SD) of percent cell migration obtained from triplicated points. Results are each 1 representative of 5 independent experiments with consistent results. *Significant differences were observed between the wild-type and SHIP-deficient cells (P < 0.03).

Figure 2

SHIP-deficient hematopoietic cells in thymus and spleen migrate better than wild-type cells to SDF-1 or BLC, but not to CKβ-11. (a) Numbers of cells migrating to the lower chamber containing SDF-1 at indicated concentrations were expressed as percentage of input cells in the upper chamber at the start time of chemotaxis. (b) CKβ-11 at indicated concentrations (ng/mL), or BLC at 5 μg/mL was used for chemotaxis of splenic B cells. Data are expressed as the mean (± difference) of percent cell migration obtained from duplicated points. Results are 1 representative of 4–5 independent experiments. *Significant differences were observed between the wild-type and SHIP-deficient cells (P < 0.05).

Figure 3

Enhanced migration to SDF-1 is due to enhanced chemotactic, but not chemokinetic, migration. Bone marrow progenitors, CFU-GM (a) and splenic B cells (b) were examined for their chemotactic responsiveness to various concentration gradients of SDF-1 (ng/mL) in the upper and/or lower chamber in a combinatorial manner. Representative data from 1 of 3 independent experiments is shown in a for CFU-GM, and the combined data of 3 independent experiments is shown for B cells in b (average ± SD). If SD bars are not shown, they were very small.

Figure 4

Enhanced biochemical response to CXC chemokine SDF-1. (a) Calcium flux response to SDF-1 in spleen lymphocytes. Optimal doses of SDF-1 (100 nM) and ionomycin (0.5 μM) were used to activate spleen lymphocytes. The x-axis and y-axis, respectively, represent time in seconds and intracellular calcium concentrations shown as ratio of fluorescence. SDF-1–dependent actin polymerization in splenocytes (b) and thymocytes (c). SDF-1 at indicated concentrations was used to induce actin polymerization. Peak levels of F-actin at 30 seconds after activation were monitored by FACscan after FITC-phalloidin staining of polymerized actin. Results shown are representative of 3 experiments each.

Figure 5

SHIP-deficient HPCs are resistant to the suppressive activity of chemokines but are sensitive to that of TNF-α and IFN-γ. Bone marrow cells from wild-type and SHIP-deficient mice were plated into methylcellulose culture medium (see Methods) in the presence of PBS, 100 ng/mL chemokines, or 10 ng/mL TNF-α and IFN-γ. Colony formation of HPCs (CFU-GM, BFU-E, CFUGEMM) was assessed after 7 days of incubation. Results shown are the average (mean ± SD) for 4 separate experiments using wild-type cells and 5 separate experiments using SHIP-deficient cells.

Figure 6

Chemokine receptor expression in leukocytes from SHIP-deficient (–) and wild-type (+) mice. Freshly isolated bone marrow cells (BM), spleen mononuclear cells (Spln), thymocytes (thym), and B220⁺ B cells (B cells; purity > 95%) from wild-type and SHIP-deficient mice were analyzed for expression of chemokine receptor mRNA by RNase protection assay. In addition to the chemokine receptors, L32 and GAPDH were included as internal controls. Expression of CC chemokine receptors is shown in a, and that of CXC chemokine receptors is shown in b. These results are representative of 3–4 reproducible experiments.