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Comparative Study
. 2006 Jan 15;107(2):835-40.
doi: 10.1182/blood-2005-07-2705. Epub 2005 Sep 22.

Beta-glucan enhances complement-mediated hematopoietic recovery after bone marrow injury

Daniel E Cramer  1 , Daniel J Allendorf, Jarek T Baran, Richard Hansen, Jose Marroquin, Bing Li, Janina Ratajczak, Mariusz Z Ratajczak, Jun Yan
Affiliations
Comparative Study

Beta-glucan enhances complement-mediated hematopoietic recovery after bone marrow injury

Daniel E Cramer et al. Blood. .

Abstract

Myelotoxic injury in the bone marrow (BM) as a consequence of total body irradiation (TBI) or granulocyte colony-stimulating factor (G-CSF) mobilization results in the deposition of iC3b on BM stroma (stroma-iC3b). In the present study, we have examined how stroma-iC3b interacts with hematopoietic progenitor cells (HPCs) and the role of complement (C) and complement receptor 3 (CR3) in BM injury/repair. We demonstrate here that stroma-iC3b tethers HPCs via the inserted (I) domain of HPC complement receptor 3 (CR3, CD11b/CD18, Mac-1). Following irradiation, stroma-iC3b was observed in the presence of purified IgM and normal mouse serum (NMS), but not serum from Rag-2(-/-) mice, implicating a role for antibody (Ab) and the classic pathway of C activation. Furthermore, a novel role for soluble yeast beta-glucan, a ligand for the CR3 lectin-like domain (LLD), in the priming of CR3(+) HPC is suggested. Soluble yeast beta-glucan could enhance the proliferation of tethered HPCs, promote leukocyte recovery following sublethal irradiation, and increase the survival of lethally irradiated animals following allogeneic HPC transplantation in a CR3-dependent manner. Taken together, these observations suggest a novel role for C, CR3, and beta-glucan in the restoration of hematopoiesis following injury.

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Figures

Figure 1.
Figure 1.
The deposition of iC3b on injured BM stroma requires IgM. Macrophage-depleted BM stroma cultures were irradiated with 1750 cGy from a cesium-137 source. The gray regions in all panels represent irradiated BM stroma in the absence of serum that were stained with the respective secondary Abs as controls for specificity. (A) Irradiated BM stromal cells were incubated with fresh C57BL/6 NMS (solid line) or heat-inactivated NMS (dotted line) and stained with affinity-purified FITC-conjugated anti-C3 Ab. (B) Fresh Rag-2-/- serum (dotted line), purified IgM (dashed line), or heat-inactivated NMS (solid line) were incubated with irradiated stromal cells and then incubated with Rag-2-/- serum as a C source. The cells were then stained with anti-C3Ab to detect iC3b deposition. (C) Following incubation with NMS, irradiated BM stromal cells were stained with goat anti-mouse IgM (solid line) or anti-mouse IgG (not shown). The data are representative of 1 of 3 separate experiments.
Figure 2.
Figure 2.
A CR3+ population of HPCs tethered to stroma-iC3b and can be observed to proliferate in response to β-glucan in a CR3-dependent manner. (A) Sorted Sca1+, c-Kit+, lin- HPCs were stained with anti-CD11b mAb and analyzed by flow cytometry. Filled curve represents isotype control for anti-CD11b; open curve shows HPCs gated for anti-CD11b staining. (B) A short-term cobblestone assay using WT (▪) or CR3-/- HPCs (□) was analyzed after 12 days of culture in the presence of stroma-iC3b in response to β-glucan (10 μg/mL) compared with their counterparts incubated with stroma-iC3b only. The CFU was also compared between WT and CR3-/- HPCs in the absence of serum (no serum) and β-glucan. (C-D) Representative micrographs captured from WT HPCs grown in the presence of serum and serum and β-glucan, respectively. Arrows indicate colonies. (E) Long-term culture-initiating culture (LTC-IC), established 35 days after coculture of WT (▪) or CR3-/- HPCs (□) and stroma in the presence of stroma-iC3b and β-glucan and analyzed 35 days later in methylcellulose culture compared with their counterparts incubated with stroma-iC3b only. Data in panels B and E represent the mean ± SEM number of CFUs observed in triplicate wells in experiments that were repeated twice (*P < .05 and **P < .005). HPCs were sorted from 3 WT or CR3-/- mice and then plated with stromal cells. The data shown here are 1 representative experiment of 3 separate experiments (total n = 9 in each group).
Figure 3.
Figure 3.
Daily administration of oral WGP β-glucan significantly enhances the recovery of peripheral blood leukocytes following sublethal irradiation in a CR3-dependent manner. Groups of 6 WT (—) or CR3-/- (- - -) were sublethally irradiated with 500 cGy of TBI from a cesium-137 source, and the leukocyte nadir was observed 5 days following irradiation. One day prior to irradiation, the mice were divided to receive no treatment (•), 0.1 mL oral phosphate-buffered saline (PBS; ▪), or 0.1 mL oral WGP β-glucan (0.8 mg/mL; ▾). Following irradiation, mice were treated as indicated daily for 3 weeks, during which time peripheral blood was collected by retro-orbital venipuncture at the indicated intervals for analysis by manual counting by investigators who were blinded to the treatment arms. WT, but not CR3-/-, mice receiving oral WGPs were observed to have a significantly accelerated recovery of peripheral blood leukocytes 7 and 11 days following irradiation compared with their counterparts receiving PBS (*P < .05; ***P < .001). All animals had normal peripheral blood leukocyte counts 3 weeks following irradiation. The data shown here are 1 representative experiment (6 mice in each group) of 3 separate experiments.
Figure 4.
Figure 4.
Oral WGP β-glucan significantly enhances the survival of lethally irradiated mice that are rescued with an allogeneic WT, but not CR3-/-, HPC transplantation. Two thousand WT or CR3-/- C57BL/6 (H-2Kb) HPCs were transplanted into lethally irradiated (950 cGy) C3H/HeJ (H-2Kk) recipients. One group of recipients (n = 23) received daily oral WGP β-glucan (80 mg/kg) on days 1 to 10 after transplantation, and another group (n = 19) received saline. Groups of 10 recipients receiving CR3-/- HPCs were treated similarly. (A) The treatment of recipient mice with oral WGP β-glucan (▾) resulted in a significant increase in survival 35 days after transplantation in mice receiving WT (—), but not CR3-/- (- - -), HPCs (▪ indicates mice receiving HPCs but not WGP β-glucan). In addition, recipients receiving WT HPCs and oral WGP β-glucan were observed to have significantly enhanced survival with respect to recipients receiving only WT HPCs (**P < .005; ***P < .001). (B) Representative experiment of multilineage engraftment from a total of 7 mice. The data are 1 representative experiment of 2 separate experiments.
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