Modulation of Hematopoietic Chemokine Effects In Vitro and In Vivo by DPP-4/CD26

Abstract

Dipeptidyl peptidase 4 (DPP4)/CD26 truncates certain proteins, and this posttranslational modification can influence their activity. Truncated (T) colony-stimulating factors (CSFs) are decreased in potency for stimulating proliferation of hematopoietic progenitor cells (HPCs). T-CXCL12, a modified chemokine, is inactive as an HPC chemotactic, survival, and enhancing factor for replating or ex-vivo expansion of HPCs. Moreover, T-CSFs and T-CXCL12 specifically downmodulates the positively acting effects of their own full-length molecule. Other chemokines have DPP4 truncation sites. In the present study, we evaluated effects of DPP4 inhibition (by Diprotin A) or gene deletion of HPC on chemokine inhibition of multicytokine-stimulated HPC, and on chemokine-enhancing effects on single CSF-stimulated HPC proliferation, as well as effects of DPP4 treatment of a number of chemokines. Myelosuppressive effects of chemokines with, but not without, a DPP4 truncation site were greatly enhanced in inhibitory potency by pretreating target bone marrow (BM) cells with Diprotin A, or by assaying their activity on dpp4/cd26(-/-) BM cells. DPP4 treatment of myelosuppressive chemokines containing a DPP4 truncation site produced a nonmyelosuppressive molecule, but one which had the capacity to block suppression by that unmodified chemokine both in vitro and in vivo. Additionally, DPP4 treatment ablated the single cytokine-stimulated HPC-enhancing activity of CCL3/MIP-1α and CCL4/MIP-1β, and blocked the enhancing activity of each unmodified molecule, in vitro and in vivo. These results highlight the functional posttranslational modulating effects of DPP4 on chemokine activities, and information offering additional biological insight into chemokine regulation of hematopoiesis.

Figures

FIG. 1.

Influence of DPP4 inhibition or cd26/dpp4 deletion on chemokine suppression of mouse BM CFU-GM colony formation in vitro. BM cells from control mice (Bl/6) preincubated with control medium for 1 h followed by washing of cells, control mouse cells preincubated with 5 mM Diprotin A for 1 h and washed, or cd26/dpp4−/− cells were plated at 5 × 104 cells/mL in the presence of rmGM-CSF (10 ng/mL) and rmSCF (50 ng/mL) minus and plus various concentrations of chemokines as indicated. Results are expressed as percent inhibition compared to control medium. For (A), control colony numbers were 98 ± 4, 98 ± 3, and 84 ± 4, respectively, and for (B) were 71 ± 6, 69 ± 5, and 76 ± 2 (mean ±1SD) for Bl/6, Bl/6 with Diprotin A, and cd26−/− cells. *Significant difference, P < 0.05 compared to control medium within each experiment. DPP4, dipeptidyl peptidase 4; BM, bone marrow; CFU, colony-forming unit; GM, granulocyte macrophage; GM-CSF, granulocyte-macrophage colony-stimulating factor; rm, recombinant murine; SCF, stem cell factor; SD, standard deviation.

FIG. 2.

Influence of DPP4 treatment of chemokines on their suppressive activity on control (Bl/6) and cd26−/− mouse BM CFU-GM colony formation in vitro and their effects on the suppressive activity of their own FL chemokine molecule. BM cells were stimulated as in the legend to Fig. 1, and results expressed as mean colonies ±1SD per 5 × 104 cells plated. *P < 0.05 compared to control. T, DPP4-treated molecule after preincubation with soluble DPP4; NT, molecule without DPP4 truncation site that was pretreated with soluble DPP4. FL, full length.

FIG. 3.

Influence of DPP4 treatment of chemokines on the suppressive activity of cd26−/− mouse BM CFU-GM colony formation in vitro. (A and B) designate 2 separate experiments. Cells were plated as stated in Fig. 1, and results expressed as mean colonies ±1SD per 5 × 104 cells plated. *P < 0.05 compared to control. T and NT are as designated in legend to Fig. 2.

FIG. 4.

Influence of s.c. injection of FL, DPP4-treated, and the combination of DPP4-treated plus FL chemokine (each given at a different site) to C57Bl/6 mice on absolute numbers and cycling status of BM hematopoietic progenitor cells. Results are shown as mean numbers ±1SEM for three mice per group with each mouse assessed individually. Experiments for groups (A–C) mice were each done on different days. BM cells were pretreated ± high specific activity tritiated thymidine, washed and plated at 5 × 104 cells/mL in erythropoietin (EPO) (1 U/mL), pokeweed mitogen spleen cell conditioned medium (PWMSCM) (5% v/v), and SCF (50 ng/mL) with 0.1 mM Hemin. *Indicates significant difference, P < 0.05, compared to control, DPP4 truncated (T), or full length (F) plus T chemokines. s.c., subcutaneous; SEM, standard error of the mean.

FIG. 5.

Effects of DPP4-treated CCL3 and CCL4 on in vitro colony formation of GM-CSF-stimulated CFU-GM (A, B) and of M-CSF-stimulated CFU-M (C, D). For (A) and (B), black bars denote BM cells pretreated with control medium before addition of chemokines, and gray bars denote BM cells pulse pretreated with Diprotin A before addition of chemokines. The BM cells for (C, D) were not pretreated with Diprotin A. Results in (A–D) are given as mean ±1SEM and represent two different experiments each. The significance values shown compare results to the control medium (no chemokine; black bars compared to black bars, and gray bars compared to gray bars) for BM cells stimulated by 10 ng/mL GM-CSF (A) or for 10, 1, and 0.1 ng GM-CSF (B), and the significance values compare results to control medium (no chemokine for BM cells stimulated by 100 ng/mL M-CSF (C) or for 100 and 10 ng/mL M-CSF (D). We also used 1 ng M-CSF, but no colonies formed with or without chemokines in this group. M, macrophage; NS, not significant; P > 0.05.

FIG. 6.

Effects of CCL3 in vivo in C57Bl/6 mice (not pretreated) on absolute numbers and cycling status of GM-CSF-responsive BM CFU-GM (A, B) and M-CSF-responsive BM CFU-M (C, D). Mice were injected s.c. with 10 μg CCL3, 10 μg DPP4-treated CCL3, or with a combination of 10 μg CCL3 and separately into another injection sites (s.c.) with DPP4-treated CCL3. Results are shown as mean ± SEM for three mice per group with each mouse assessed separately. Significance values compare CCL3-injected mice with those of control mice (injected with control diluent). F equals non-DDP4-treated full-length CCL3. T, DDP4-treated CCL3.