C57BL/6 mice were adopted at 1-2 weeks and used for experiments at age 6 to 8 weeks. Animal studies were approved by the Animal Care and Use Committee of the Institute of Laboratory Animal Resources, Seoul National University.

BMNCs were prepared by flushing femurs and tibias of pathogen-free, 6- to 8-week-old mice without enzymatic digestion. They were lysed with BD Pharm Lyse buffer (BD Biosciences, San Jose, CA, US) to remove red blood cells (RBCs), washed, and resuspended in appropriate media for further analysis.

The trans-well migration assay was performed as described elsewhere [25]. Briefly, unless otherwise indicated, RBC-lysed BMNCs from C57BL/6 mice were resuspended in assay media (RPMI containing 0.5% bovine serum albumin) and equilibrated for 30 min at 37℃. Six hundred-fifty microliters of assay media in the presence or absence of CXCL12 (50-300 ng/mL, PeproTech, Rocky Hill, NJ, USA) was added to the lower chambers of a Trans-well 24-well plate (Costar Corning, Cambridge, MA, USA). hBD-2 was purchased from Prospec (East Brunswick, NJ, USA). Aliquots of normal or 30 min hBD-2-exposed cell suspension (1×10⁶ cells/100 µL) were loaded onto the upper chambers with 5 µm-pore filters, which were incubated for 3 hours at 37℃, 95% humidity, and 5% CO₂. Cells from the lower chambers were scored using FACS analysis for migration of BMNCs to test reagents. The results are presented as a migration index, i.e., fold increase of cell numbers migrated toward the lower well compared to control media. In some experiments, migrated cells harvested from the lower chambers were plated in semisolid clonogenic assays supplemented with cytokines and were stimulated to grow colony-forming units-granulocyte-macrophage (CFU-GM) and colony-forming units-macrophage (CFU-M) as described elsewhere [25].

Red blood cells were lysed with BD Pharm Lyse buffer. Nucleated cells were subsequently washed twice and used for CFU-GM and/or CFU-M colonies. BMNCs were resuspended in human methylcellulose base media provided by the manufacturer (R&D Systems, Minneapolis, MN, USA) supplemented with 25 ng/mL recombinant murine granulocyte macrophage colony-stimulating factor (mGM-CSF) and 10 ng/mL recombinant murine interleukin-3 (mIL-3) for CFU-GM, 10 ng/mL recombinant murine macrophage stimulating factor (mM-CSF) for CFU-M. Cultures were incubated for 7 days, at which time they were scored for the number of CFU-GM colonies under an inverted microscope.

Western blot analysis was performed on extracts prepared from mouse EL4 cell lines that had been kept in RPMI medium containing 0.5% bovine serum albumin (BSA) to render the cells quiescent. Cells were stimulated with either a low or high concentration of CXCL12 (50 ng/mL or 300 ng/mL) in the presence or absence of hBD-2 for 90 sec at 37℃ and then lysed for 10 min on ice in M-Per lysing buffer (Pierce, Rockford, IL, USA) containing protease and phosphatase inhibitors (Sigma, St. Louis, MO, USA). Extracted proteins were separated on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred to a nitrocellulose membrane (Hybond ECL; Amersham Life Sciences, Little Chalfont, United Kingdom). Phosphorylation of 44/42 MAPK protein was detected by protein immunoblotting using mouse monoclonal 44/42 phospho-specific MAPK antibody (New England Biolabs, Beverly, MA, USA), with horseradish peroxidase conjugated goat anti-mouse immunoglobulin G (Santa Cruz Biotechnology, Santa Cruz, CA, USA) as secondary antibody. Equal loading in the lanes was evaluated by stripping the blots and reprobing them with the appropriate monoclonal or polyclonal antibodies. The membranes were developed with an ECL reagent (Amersham Life Sciences), dried, and exposed to film (HyperFilm; Amersham Life Sciences).

Wild-type BM stromal cells were grown to confluence, and normal or 30 min hBD-2-exposed BMNCs were subsequently plated over BM-derived stroma feeder layers established in well plates. Five minutes later, plates were gently shaken, and non-adherent cells were discarded. Subsequently, the cultures were trypsinized and stimulated to grow CFU-GM colonies as mentioned earlier. The number of colonies was scored under an inverted microscope.

To test whether hBD-2 priming enhanced homing and engraftment of transplanted HSPCs, we used a short-homing assay to evaluate lodging of HSPCs to the spleen and bone marrow. Normal recipient mice were lethally irradiated (950 cGy) and 24 hours later were transplanted with 5×10⁵ normal or 30 min hBD-2-exposed BMNCs by tail vein injection. At 18 hours or 12 days after BMNC transplantation, spleens were removed and fixed for colony forming units-spleen (CFU-S) count. At the same time, BM were harvested from the femurs and plated in methylcellulose cultures and stimulated to grow CFU-GM colonies. CFU-S was also counted.

Arithmetic means and SD were calculated using Instat 1.14 software (GraphPad, San Diego, CA, USA). Statistical significance was defined as P<0.05. Data were analyzed using an unpaired Student's t-test.

To optimize the trans-well migration assay we tested different doses of CXCL12 using a 5 µm pore sized trans-well. CXCL12 directly chemoattracted not only BMNCs (Figure 1A), but also CFU-GM progenitor cells (Figure 1B) in a dose-dependent manner. However, CXCL12 at a concentration lower than 25 ng/mL did not chemoattract either BMNCs or CFU-GM progentitor cells efficiently. Significant migrations of BMNCs to CXCL12 were shown in concentrations of 50 ng/mL or higher. Although 50 ng/mL of CXCL12 is relatively high compared to its physiological plasma concentration [26], we chose 50 ng/mL of CXCL12 as the standard concentration for our experiments based on the results obtained from our optimization assay.

The antimicrobial cationic peptide hBD-2, which is known to be released from activated human granulocytes, enhanced responsiveness of BMNCs to a CXCL12 gradient. As expected, the migration of hBD-2-exposed BMNCs to a CXCL12 gradient was significantly enhanced compared to that of normal BMNCs (Figures 2A and 2B). This accelerated migration of BMNCs and CFU-GM progenitor cells to a CXCL12 gradient was dependent on the hBD-2 concentration used. However, hBD-2 itself did not show a direct chemotactic effect on BMNCs (Figure 2C) and CFU-GM progenitor cells (Figure 2D). Considering hBD-2 is known to use CCR6 for signal transduction, the hBD-2 priming effect was not a receptor-mediated phenomenon. MIP-3α, which is known as an agonist for CCR6, did not enhance migration of BMNCs to the CXCL12 gradient (data not shown). These results strongly support that enhanced migration of hBD-2-primed cells might be mediated by enhancing incorporation of CXCR4 into lipid rafts as similar to what was shown in C3a primed cells [20,21].

We tested if enhanced CXCR4 incorporation into membrane lipid rafts in the presence of hBD-2 is better connected to downstream signaling machinery and can respond robustly to a CXCL12 gradient, as a previous report showed that the supernatants of leukapheresis products increased p42/44 MAPK phosphorylation mediated by enhancing incorporation of CXCR4 into membrane lipid rafts [20,21].

We noticed that hBD-2 itself does not induce phosphorylation of p42/44 MAPK but enhanced p42/44 MAPK phosphorylation induced by CXCL12 in EL4 cells. CXCL12-induced phosphorylation of p42/44 MAPK was strongly enhanced in hBD-2-primed cells (Figure 3A). This also strongly supports that the priming effect of hBD-2 is not mediated by receptor-ligand interactions.

We questioned the effects of priming agents on other processes essential for the engraftment of HSPCs, as they must adhere to the BM stroma to proliferate and differentiate. To address this question further, we carried out in vitro adhesion experiments with BMNCs. We found that hBD-2-exposed CFU-GM progenitor cells showed increased adhesion (Figure 3B). We suggest that hBD-2, by enhancing responsiveness of HSPCs to a CXCL12 gradient, may directly modulate HSPC homing.

Based on our in vitro results showing enhanced migration and adhesion, we examined whether hBD-2-primed BMNCs engraft faster than normal BMNCs. As summarized in Table 1, hBD-2-primed BMNCs homed better to the BM and spleen of lethally irradiated recipient mice. CFU-GM progenitor cells at 18 hours after BMNC transplantation were increased more than two fold in the femur of hBD-2-primed BMNC transplanted group compared to the normal BMNC transplanted group (data not shown). No CFU-GM or CFU-S was recovered from lethally irradiated control mice that did not receive BMNCs.

Finally, we evaluated the safety of hBD-2 exposure on hematopoietic progenitor cells. BMNCs (5×10⁵/mL) were incubated for 60 min at 37 CO₂ incubator in the absence or presence of hBD-2 at 0.12, 0.25, or 0.50 µg/mL concentrations. These cells were subsequently examined via in vitro colony forming assays for the number of CFU-GM and CFU-M progenitor cells. As expected, hBD-2 exposure did not affect the viability and the clonogeneic potential of HSPCs (Figures 4A and 4B).