Mobilization studies in complement-deficient mice reveal that optimal AMD3100 mobilization of hematopoietic stem cells depends on complement cascade activation by AMD3100-stimulated granulocytes

Abstract

We reported that complement cascade (CC) becomes activated in bone marrow (BM) during mobilization of hematopoietic stem/progenitor cells (HSPCs) induced by granulocyte colony-stimulating factor (G-CSF) and C5 cleavage has an important function in optimal egress of HSPCs. In this work, we explored whether CC is involved in mobilization of HSPCs induced by the CXCR4 antagonist, AMD3100. To address this question, we performed mobilization studies in mice that display a defect in the activation of the proximal steps of CC (Rag(-/-), severe combined immune deficient (SCID), C2.Cfb(-/-)) as well as in mice that do not activate the distal steps of CC (C5(-/-)). We noticed that proximal CC activation-deficient mice (above C5 level), in contrast to distal step CC activation-deficient C5(-/-) ones, mobilize normally in response to AMD3100 administration. We hypothesized that this discrepancy in mobilization could be explained by AMD3100-activating C5 in Rag(-/-), SCID, and C2.Cfb(-/-) animals in a non-canonical mechanism involving activated granulocytes. To support this, granulocytes (i) first egress from BM and (ii) secrete several proteases that cleave/activate C5 in response to AMD3100. We conclude that AMD3100-directed mobilization of HSPCs, similarly to G-CSF-induced mobilization, depends on activation of CC; however, in contrast to G-CSF, AMD3100 activates the distal steps of CC directly at the C5 level. Overall, these data support that C5 cleavage fragments and distal steps of CC activation are required for optimal mobilization of HSPCs.

Figure 1. Normal AMD3100 mobilization of HSPCs in Ig-deficient mice

Rag^−/−, SCID, as well as age- and sex-matched control (C57BL/6 and Balb/c) mice were mobilized with AMD3100 (5 mg/kg s.c.) for 1 h. Number of circulating SKL CD34⁻/μL (A and C) and CFU-GM progenitors (B and D) per microliter of PB are shown. Data show representative results from three independent experiments with 6 animals/group.

Figure 2. Impaired G-CSF- and zymosan- but normal AMD3100 mobilization of HSPCs in C2.Cfb-deficient mice

C2.Cfb^−/− mice as well as age- and sex-matched C57BL/6 mice were mobilized for 6 days with G-CSF (250 μg/kg s.c. per day; n=5 mice per group; upper panel) or 1 h with zymosan (0.5 mg/kg i.v.; n=5 mice per group; middle panel) or for 1 h with AMD3100 (5 mg/kg s.c.) n=5 mice per group (lower panel). Number of circulating leukocytes (A), CFU-GM progenitors (B), and SKL CD34⁻ cells (C) per microliter of PB are shown. * P<.05 as compared with C5^+/+ mice. Data show representative results from three independent experiments.

Figure 3. Impaired AMD3100 mobilization of HSPCs in C5-deficient mice

C5^−/− as well as age- and sex-matched C5^+/+ mice were mobilized with AMD3100 (5 mg/kg s.c.) for 1 h. Number of circulating leukocytes (A), CFU-GM progenitors (B), and SKL cells (C) per microliter of PB are shown. * P<.05 as compared with C5^+/+ mice. Data show representative results from three independent experiments with 6 animals/group.

Figure 4. AMD3100 mobilization activates C5 cleavage and leukocytes precede egress on HSPCs during AMD3100 mobilization

The concentration of C3 and C5 cleavage fragments-C3a/_desArgC3a and C5a/_desArgC5a, respectively, was measured by ELISA. Zymosan, but not AMD3100 or PBS administration activates CC at the C3 level (A) * P<.01. In contrast, CC is activated at the C5 level by both zymosan and AMD3100; however, zymosan seems to be a much more potent C5 activator (B) * P<.01. Panel C -Kinetics of mobilization of leukocytes (WBC) (a), neutrophils (NE) (b), monocytes (Mo) (c), and CFU-GM progenitors (d) in mice mobilized 0–90 min after administration of AMD3100 (5 mg/kg s.c.). Data are pooled from two independent experiments with 6 animals/experimental point. * P<.0001.

Figure 4. AMD3100 mobilization activates C5 cleavage and leukocytes precede egress on HSPCs during AMD3100 mobilization

The concentration of C3 and C5 cleavage fragments-C3a/_desArgC3a and C5a/_desArgC5a, respectively, was measured by ELISA. Zymosan, but not AMD3100 or PBS administration activates CC at the C3 level (A) * P<.01. In contrast, CC is activated at the C5 level by both zymosan and AMD3100; however, zymosan seems to be a much more potent C5 activator (B) * P<.01. Panel C -Kinetics of mobilization of leukocytes (WBC) (a), neutrophils (NE) (b), monocytes (Mo) (c), and CFU-GM progenitors (d) in mice mobilized 0–90 min after administration of AMD3100 (5 mg/kg s.c.). Data are pooled from two independent experiments with 6 animals/experimental point. * P<.0001.

Figure 5. AMD3100 directly activates Gr-1⁺ cells

Gr-1⁺ cells were stimulated with AMD3100 for 5, 10, and 15 min. (A) The level of phosphorylation of MAPK42/44 was measured using Western blot. (B) Expression of MMP-9 in culture supernatants of cultured Gr-1⁺ cells stimulated with AMD3100 (1 μM) measured by zymography is shown. (C) Expression of MMP-9 in serum of control mice and mice mobilized by AMD3100 measured by zymography is shown. (D) Messenger RNA expression of poteolytic enzyme in Gr-1⁺ cells stimulated with AMD3100 (1 μM) or PBS a control. Experiments were repeated independently three times with similar results. * P<.0001. Representative data are shown.

Figure 6. BM-derived Gr-1⁺ cells activate/cleave C5

(A) Gr-1⁺ cells sorted from murine BM. (B) The concentration of C5 cleavage fragments C5a/_desArgC5a, respectively, was measured by ELISA. Gr-1⁺ cells and Gr-1⁺ cell stimulated by AMD3100 (indicated concentration) activate CC at the C5 level. * P<.001 compared to normal plasma, ** P<0.001 compared to plasma incubated only with Gr-1⁺ cells. Experiment was repeated independently two times with two sets of Gr-1⁺ sorted cells combined data are shown.

Figure 7. AMD3100-directed HSPC mobilization depends on activation of CC by AMD3100 mobilized/activated granulocytes

We envision three potential steps at which AMD3100 induces HSPC mobilization. First, AMD3100 blocks the interaction of CXCR4⁺ granulocytes and CXCR4⁺ HSPCs with SDF-1 expressed in the BM microenvironment. At the same time, however, AMD3100 stimulates secretion of metalloproteinases in BM from activated granulocytes, which helps to perturb/block another retention axis for HSPCs in BM, i.e., the VLA-4-VCAM-1 interaction (Step I). Next, granulocytes are the first cells to egress from BM and they “pave the way” for HSPCs to cross the BM-endothelial barrier (Ice Breaker Phenomenon; Step II). Finally, proteolytic enzymes released from AMD3100-activated granulocytes in BM sinusoids activate/cleave CC at the distal stages (i.e., C5) resulting in the release of C5a and _desArgC5a, which facilitate the final steps of HSPC egress. Further studies however, require potential involvement of C5b-C9 - membrane attack complex (MAC) and our data indicate that some factors generated in plasma by activated MAC chemoattract HSPCs in a presence of AMD31000 in CXCR4-independent manner (manuscript in preparation) (Step III).