Impaired mobilization of hematopoietic stem/progenitor cells in C5-deficient mice supports the pivotal involvement of innate immunity in this process and reveals novel promobilization effects of granulocytes

Abstract

We reported that complement cascade (CC) becomes activated in bone marrow (BM) during granulocyte colony-stimulating factor (G-CSF) mobilization of hematopoietic stem/progenitor cells (HSPCs) and showed that, although third CC component (C3)-deficient mice are easy mobilizers, fifth CC component (C5)-deficient mice mobilize very poorly. To explain this, we postulated that activation/cleavage of CC releases C3a and C5a anaphylatoxins that differently regulate mobilization. Accordingly, C3a, by enhancing responsiveness of HSPCs to decreasing concentrations of stromal-derived growth factor-1 (SDF-1) in BM, prevents mobilization and promotes their BM retention. Therefore, in this study, we focused on the mobilization-enhancing role of C5a. We found that C5a receptor (C5aR) is not expressed on the surface of HSPCs, and that C5a-mediated promobilization effects are mediated by stimulation of granulocytes. Overall, our data support the following model. First C5aR(+) granulocytes are chemoattracted by plasma C5 cleavage fragments, being the first wave of cells leaving BM. This facilitates a subsequent egress of HSPCs. In the next step, after leaving BM, granulocytes undergo degranulation in response to plasma C5a and secrete some cationic peptides (cathelicidin, beta-defensin) that, as shown here for the first time, highly enhance the responsiveness of HSPCs to plasma SDF-1 gradient. In conclusion, our data reveal the underappreciated central role of innate immunity in mobilization, in which C5 cleavage fragments through granulocytes orchestrate this process.

Figure 1. Impaired mobilization of HSPCs in C5-deficient mice

C5^-/- as well as age- and sex-matched C5^+/+ wt mice were mobilized for 6 days with G-CSF (250 μg/kg s.c. per day; n=5 mice per group) (A) or for 1 h with zymosan (0.5 mg/kg i.v.; n=5 mice per group) (B). Number of circulating neutrophils (left), CFU-GM progenitors (middle), and SKL cells (right) per microliter of PB. * P<.05 as compared with wt C5^+/+ mice. Data show representative results from three independent experiments.

Figure 2. TEM analysis of BM tissue

Granulocytes (arrow) in wt C5^+/+ mouse BM show trans-endothelial migration from BM stroma to sinusoids (■) after 6-day G-CSF administration (A-C). In contrast, granulocytes in C5^-/- mouse BM are accumulated around the endothelial layer and retained in the BM stroma without entering into sinusoids (D-F). Original magnification = x 2,000. Representative TEM pictures are shown.

Figure 3. Chemotactic effect of C5a and _desArgC5a on mouse BMNCs

_desArgC5a, but not C5a, directly chemoattract BMNCs in a C5aR-dependent manner, but do not induce migration of CFU-GM progenitors. (A) Migration of whole BMNCs (A) and CFU-GM progenitors (B) in response to C5a and _desArgC5a in the absence or presence of SDF-1. (C) _desArgC5a directly chemoattract Balb/c mice BMNCs (C). In contrast, it has no chemotactic effect on C5aR^-/- mouse BMNCs (D). Values are the fold increases of migrated cells compared to media alone. M, media alone; C3a (1 μg/ml); SDF-1 (50 ng/ml); C5a and _desArgC5a (L, 70 ng/ml; H, 140 ng/ml). * P<0.05 as compared with media alone control; ** P<0.05 as compared with SDF-1 (50 ng/ml) alone. The data shown represent the combine results from three independent experiments carried out in triplicate per group (n=9).

Figure 4. C5aR expression on mouse BMNCs

(A) RT-PCR analysis of C5aR mRNA expression on purified mouse BMNCs (M, size marker; SKL, Sca-1⁺/c-Kit⁺/Lin^- cells; Gr-1, granulocytes; CD14, monocytes; D.W., H₂O instead of cDNA). The experiment was repeated twice on two different batches of sorted cells with similar results. (B) FACS analysis of C5aR expression on mouse BMNCs. C5aR was expressed on mouse BMNCs (6~9%) including granulocytes and monocytes, but not on SKL cells. The experiment was repeated three times on cells from three independent donors with similar results. Representative staining is shown.

Figure 5. Cationic peptides released from granulocytes enhance responsiveness of HSPCs to SDF-1 gradient

CM, prepared by incubating Gr-1⁺ cells (2×10⁶/ml) for 1 h with C5 cleavage fragments (CC, no stimulation; CA, stimulated with C5a; CB, stimulated with _desArgC5a) was added into lower wells (final concentration 50%) to evaluate the priming effect of granules released from granulocytes. CM of Balb/c mice granulocytes, stimulated with C5a but not _desArgC5a, enhanced responsiveness of C5aR^-/- BMNCs (A) and CFU-GM progenitor cells (B) to SDF-1 gradient. In contrast, CM of C5aR^-/- mice granulocytes did not enhance responsiveness of C5aR^-/- BMNCs (C) or CFU-GM progenitor cells (D). Recombinant cationic peptides LL-37 and hBD-2 strongly enhanced migration of BMNCs (E) and CFU-GM progenitor cells (F) in response to SDF-1 gradient. Values are the fold increases of migrated cells compared to media alone. M, media alone; SDF-1 (L), 50 ng/ml; SDF-1 (H), 300 ng/ml; C3a, 1 μg/ml. * P<0.05 as compared with SDF-1 (50 ng/ml) alone. The data shown represent the combine results from four independent experiments carried out in triplicate per group (n=12).

Figure 6. Priming effect of cationic peptides is dependent on enhanced incorporation of CXCR4 into lipid rafts

(A) Western blot analysis of the localization of CXCR4 in various fractions of cell membranes. Membranes enriched in lipid rafts (fractions 3-5) and depleted of lipid rafts (fractions 9-11). Human pre-B cell line, Nalm-6, was stimulated with LL-37 (5 μg/ml) or hBD-2 (250 ng/ml) or was not stimulated (control). CXCR4 was detected in these membrane fractions by Western blot along with Lyn, a marker of lipid rafts. Experiments were performed three times with similar results. (B) LL-37-induced enhancement of THP-1 cell migration to SDF-1 gradient was inhibited by 1 h pretreatment with MβCD (1.0 or 2.5 mM). * P<0.05 as compared with migration of control cells in the absence of LL-37 (2.5 μg/ml). Lipid raft formation was analShown are representative of three independent experiment carried out in triplicate per group. The data shown represent the combine results from four independent experiments carried out in triplicate per group (n=12).

Figure 7. C5 cleavage fragments play an important role in granulocyte egress and HSPCs mobilization from BM

There are three levels at which C5 cleavage fragments (C5a and _desArgC5a) may affect granulocytes and thus affect HSPC mobilization. First, stimulation of granulocytes in the BM microenvironment by C5a and _desArgC5a enhances secretion of proteolytic enzymes, which perturb HSPCs retention signals (e.g., SDF-1-CXCR4 and VLA-4-VCAM-1 interactions) (Step I). Second, serum _desArgC5a chemoattractants induce egress of HSPCs from BM into PB. Migrating from BM into PB, granulocytes rich in metalloproteinases “pave the way” for HSPCs, which follow granulocytes and migrate through the endothelial barrier (“ice breaker phenomenon”) (Step II). Finally, after egress from BM, granulocytes are stimulated in BM vessels by C5a and release several cationic peptides, some of which (e.g., cathelicidin, β-defensin-2) enhance/prime responsiveness of HSPCs to serum levels of SDF-1 (Step III). Finally, we cannot exclude the potential direct effect of C5 cleavage fragments on permeabilization of the endothelium, which will require further studies.